Research ArticleGlutathione Peroxidase-1 Suppresses the Unfolded ProteinResponse upon Cigarette Smoke Exposure
Patrick Geraghty12 Nathalie Baumlin3 Matthias A Salathe3
Robert F Foronjy12 and Jeanine M DrsquoArmiento4
1Division of Pulmonary amp Critical Care Medicine Department of MedicineState University of New York Downstate Medical Center Brooklyn NY USA2Department of Cell Biology State University of New York Downstate Medical Center Brooklyn NY USA3Division of Pulmonary Allergy Critical Care and Sleep Medicine University of Miami Miami FL USA4Center for Pulmonary Disease Department of Anesthesiology College of Physicians and SurgeonsColumbia University New York NY USA
Correspondence should be addressed to Patrick Geraghty patrickgeraghtydownstateedu
Received 18 August 2016 Revised 19 October 2016 Accepted 31 October 2016
Academic Editor Karen Ridge
Copyright copy 2016 Patrick Geraghty et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Oxidative stress provokes endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) in the lungs of chronicobstructive pulmonary (COPD) subjects The antioxidant glutathione peroxidase-1 (GPx-1) counters oxidative stress induced bycigarette smoke exposure Here we investigate whether GPx-1 expression deters the UPR following exposure to cigarette smokeExpression of ER stress markers was investigated in fully differentiated normal human bronchial epithelial (NHBE) cells isolatedfrom nonsmoking smoking and COPD donors and redifferentiated at the air liquid interface NHBE cells from COPD donorsexpressed heightenedATF4 XBP1 GRP78 GRP94 EDEM1 andCHOP compared to cells fromnonsmoking donorsThese changescoincided with reduced GPx-1 expression Reintroduction of GPx-1 into NHBE cells isolated fromCOPD donors reduced the UPRTo determine whether the loss of GPx-1 expression has a direct impact on these ER stress markers during smoke exposure Gpx-1minusminus mice were exposed to cigarette smoke for 1 year Loss of Gpx-1 expression enhanced cigarette smoke-induced ER stress andapoptosis Equally induction of ER stress with tunicamycin enhanced antioxidant expression in mouse precision-cut lung slicesSmoke inhalation also exacerbated the UPR response during respiratory syncytial virus infection Therefore ER stress may be anantioxidant-related pathophysiological event in COPD
1 Introduction
Chronic obstructive pulmonary disease (COPD) is the thirdleading cause of death in the US [1] with cigarette smokingbeing themost important environmental risk factor Cigarettesmoke inhalation alters the expression profile of oxidants andantioxidants in the lungs and produces an enormous oxidantburden [2] Antioxidant enzymes counter this oxidative stress[2] and deter lung inflammation responses by targetingmultiple signaling pathways [3] Detoxifying reactive oxy-gen species (ROS) is a therapeutic strategy to limit tissuedamage in cigarette smoke-induced diseases [3] Recentlycigarette smoke-mediated oxidative stress was shown to
induce endoplasmic reticulum (ER) stress [4] However theability of antioxidants to counter ER stress has not been fullycharacterized
It is well established that cigarette smoke induces ERstress which activates the unfolded protein response (UPR)[5ndash9] However COPD is a complex heterogeneous diseaseand the significance and intensity of the UPR during thedisease is unknown The UPR is a complex stress responseprogram that modulates multiple cellular responses and sur-vival via regulation of protein synthesis folding and degra-dation [10]Threemajor pathways of theUPRhave been char-acterized (i) PKR-like ER kinase (PERK)eIF2120572activatingtranscription factor (ATF) 4CHOP (ii) inositol-requiring
Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 9461289 16 pageshttpdxdoiorg10115520169461289
2 Mediators of Inflammation
enzyme 1 (IRE1)X-box binding protein 1 (XBP1) and the (iii)ATF6 pathway [11] These pathways regulate ER chaperoneresponses and reduce protein translation following cellularstress [12] However persistent ER stress results in significantexpression of the proapoptotic gene CEBP homologousprotein (CHOP) resulting in cell death [11] We previouslydemonstrated that smoke exposure triggers aminor ER stressresponse in primary cells and rodent animal models [5]However a secondary insult may be required to provoke asignificant smoke-induced UPR
Our group demonstrated that overexpression of glu-tathione peroxidase- (GPx-) 1 a member of the selenoproteinfamily prevents cigarette smoke-induced air space enlarge-ment in mice [13] GPx-1 is the most abundant GPx isoformin eukaryotic cells and deficiency of this enzyme can lead toendothelial dysfunction [14] and apoptosis [15] GPx-1 defi-ciency has also been implemented as a contributor toatherosclerosis [16]Gpx-1 deficient mice exposed to cigarettesmoke are more susceptible to cigarette smoke-induced lunginflammation and emphysema [13 17] Oxidative stressinduces ER stress [4] and increased expression of ER stressmarkers is observed in the lungs of smokers [8] GPx-1expression however is reduced in COPD lungs [13]Thus wespeculate that GPx-1 could modulate ER stress responseslinked to the pathogenesis of COPD
In view of the potential association between GPx-1 andcigarette smoke-mediatedUPR we explored whether the lossof GPx-1 expression enhanced the UPR that contributes tolung cell injury and death Using normal human bronchialepithelial (NHBE) cells from nonsmokers smokers andCOPD subjects we found that ER stress markers weresignificantly elevated in cells isolated from COPD subjectsand this increase coincided with reduced GPx-1 expressionReintroducing GPx-1 into these cells blunted the UPR Todetermine if GPx-1 depletion in the lung directly enhancesER stress Gpx-1minusminus mice were exposed to cigarette smoke for1 year Interestingly the loss of GPx-1 expression activatedall three branches of the UPR PERKeIF2120572ATF4CHOPIRE1XBP1 and ATF6 This UPR coincided with elevatedlung cell death in Gpx-1minusminus mice following smoke exposureInterestingly precision-cut lung slices (PCLS) frommice hadelevated GPx proteins following induction of ER stressThesefindings indicate that the altered GPx-1 expression in COPDlungs contributes to heightened ER stress In addition earlyinduction of ER stress induces an antioxidant response tocounter oxidative stress thereby limiting the UPR
2 Materials and Methods
21 Human Primary Airway Cells NHBE cells from non-smokers smokers and COPD patients were isolated fromhuman lungs Lungs were obtained from organ donors whoselungs were rejected for transplant (see Table 1 for demograph-ics) Consent for research was obtained by the Life AllianceOrgan Recovery Agency of the University of Miami All con-sents were IRB-approved and conformed to the Declarationof Helsinki For lungs from donors with COPD the diagnosiswas listed in the chart before the death of the donor and we
confirmed the macropathological presence of emphysema inthese lungs All COPD subjects had a significant smokinghistory NHBE cells isolated from nonsmokers smokers andCOPD subjects were dedifferentiated through expansion andredifferentiated at an air liquid interface (ALI) on 24mmT-clear filters (Costar Corning Corning NY USA) at37∘C 5 CO2 as previously described [18] Cells werecollected for protein and RNA analysis CD45 and CD11Cexpressions were analyzed which determined a low level ofinflammatory-cell contamination and confirmed NHBE cellpurity Fully differentiated NHBE cells from nonsmokerswere also exposed to cigarette smoke using a Vitrocell VC-10smoking robot (Vitrocell Systems GMBH Waldkirch Ger-many) Four cigarettes were smoked according to ISO stan-dard 3308 six puffs per cigarette with a 35mL volume per puffand a waiting time between each puff of 60 seconds NHBEcells were exposed every second day on three separate daysto 4 cigarettes RNA was extracted from the NHBE cells forquantitative PCR (qPCR) analysis A subset of NHBE cellsfrom COPD subjects was protein transfected 2120583g humanGPx-1 protein or human albumin (both from Signal Aldrich)usingPierce transfection reagent (ThermoFisher Scientific) aspreviously described [13 19] RNA and protein were collect24 hours later Human RSV strain A2 (ATCC ManassasVA VR-1540) was infected in NHBE cells as previouslydescribed [20] RNA and protein were collect 24 hours later
22 Animal Models Gpx-1minusminus mice were bred in C57BL6 timesCBAJ background Eight-week-old wild type and Gpx-1minusminusmice were used for all experiments All mice weremaintainedin a specific pathogen-free facility at Columbia UniversityBoth male and female mice 8-week-old were used at theinitiation point for all experiments and each experimentalparameter had at least 10 animals per group Mice wereexposed to cigarette smoke in a chamber (Teague EnterprisesDavis CA USA) for four hours a day five days per week ata total particulate matter concentration of 80mgm3 Smokeexposurewas continued for 1 yearTheUniversity ofKentuckyreference research cigarettes 3R4F (Lexington KY USA)were used to generate cigarette smoke Another group of wildtype animals was infected with 1times 106 pfu of RSV following 6-month exposure to room air or cigarette smokeThe instituteapproved all experiments for Animal Care and Use Commit-tee ofColumbiaUniversityThis studywas performed in strictaccordance with the recommendations in the Guide for the
Mediators of Inflammation 3
Table 2 TaqMan probe details for gene expression analysis
Care andUse of LaboratoryAnimals of theNational Institutesof Health and Institutional Animal Care and Use Committee(IACUC) guidelines
23 Precision-Cut Lung Slices (PCLS) Mouse precision-cutlung slices (PCLS) were prepared as previously described[21 22] Briefly mice were euthanized the trachea wascannulated and the animal was exsanguinated by cutting thejugular vein The lungs were filled through the cannulawith 15mL low melting-point agarose solution (15 finalconcentration of agarose in PBS) Lungs were placed on icefor 15 minutes to solidify the agarose Lobes were separatedand tissue cores were prepared of the individual lobesafter which the lobes were sliced at a thickness of 300 120583musing a Krumdieck tissue slicer (Alabama Research andDevelopment Munford AL USA) in Earlersquos balanced saltsolution (Sigma Aldrich) Tissue slices were incubated inDulbeccorsquos modified eaglersquos mediumnutrient mixture F-12 HAM solution (Sigma Aldrich) at 37∘C in a humidatmosphere under 5 CO295 air To remove agarose andcell debris slices were washed every 30 minutes for 2 hoursPCLS were incubated in DMEM supplemented with peni-cillin (100UmL) and streptomycin (100 120583gmL) (Gibco byLife Technologies) Slices were cultured at 37∘C in a humid-ified atmosphere under 5 CO295 air in 6-well tissueculture plates using 3 slices per well Slices were treated with1 120583M tunicamycin (Sigma Aldrich) for 24 hours To assessthe viability of the PCLS subjected to tunicamycin lactate
dehydrogenase (LDH) released from the PCLS into theincubation mediumwas analyzed Maximal LDH release wasdetermined by lysing 3 slices with 1 Triton X-100 for 30minutes at 37∘C LDH release was determined using an assayform Sigma Aldrich
24 qPCR Analysis Total RNA was isolated from cells ormouse lung tissue using the Qiagen RNeasy Mini Kit asdescribed by manufactures Gene transcript levels of mouseand human specific CHOP ATF4 XBP1 GRP78 GRP94EDEM1 GPx-1 GPx-2 GPx-3 GPx-4 IL-6 and ACTB werequantified by real-time PCR with the use of an Bio-RadCFX384 real-time system (Bio-Rad) TaqManGene Expres-sion Assays were purchased from Applied Biosystems (seeTable 2 for details) Data is represented as relative quan-tification (RQ) corrected to ACTB XBP1 splicing was alsoexamined in NHBE cells using the following primers 51015840-TTACGA GAG AAA ACT CAT GGC-31015840 and 51015840-GGG TCC AAGTTGTCCAGAATGC-31015840 XBP1 PCRproductswere resolvedand run on a 25 agarose gel [23] 289 and 286 base pairamplicons were generated from unspliced and spliced XBP1respectively Percent of XBP1 slicing was examined by densit-ometry analysis of the unspliced (XBP1u) and spliced (XBP1s)amplicons of XBP1 using Bio-Rad Laboratories Image Labsoftware (version 40 build 16)
25 Immunoblotting Cell monolayers were removed byscrapping in cold phosphate-buffered saline and resuspended
4 Mediators of Inflammation
in 100 120583L of protein lysis buffer (50mM HEPES pH 75150mM NaCl 1 Triton X-100 1 glycerol 1mM EDTA10mM NaF 2 120583gmL leupeptin 1 120583gmL pepstatin A 10mMNa3VO4 and 1mM phenylmethylsulfonyl fluoride) and20120583g of protein was separated on 12 SDS-polyacrylamidegels and transferred to nitrocellulose membranes Rab-bit antibodies against CHOP (Cell Signaling 5554) ERdegradation-enhancing 120572-mannosidase-like (EDEM) (SantaCruz Biotechnology sc-27389) pho-eIF2120572 (Ser51) (Cell Sig-naling 9721) eIF2120572 (Cell Signaling 9722) pho-PERK(Thr980) (Cell Signaling 3179) PERK (Cell Signaling5683) XBP-1 (Cell Signaling 12782) IRE1120572 (Cell Signaling3294) BiPGRP78 (Cell Signaling 3183) GRP94 (Cell Sig-naling 2104) ATF4 (Cell Signaling 11815) ATF6 (Abcamab11909) GPx-1 (Cell Signaling 3206) GPx-2 (Abcamab140130) GPx-3 (Abcam ab27325) GPx-4 (Cell Signal-ing 2104) and 120573-actin (Cell Signaling 4970) were detectedwith enhanced chemiluminescence reagents (Pierce) Chemi-luminescence detection was performed using the Bio-RadLaboratories Molecular Imager ChemiDoc XRS+ imagingsystem Densitometry was performed on each target andrepresented as a ratio of pixel intensity compared to totalprotein or 120573-actin using Bio-Rad Laboratories Image Labsoftware (version 40 build 16)
26 Statistical Analysis Data are expressed as dot plotswith the means plusmn SEM highlighted Differences betweentwo groups were compared by Studentrsquos 119905 test (two-tailed)Experiments with more than 2 groups were analyzed by 2-way ANOVA with Tukeyrsquos post hoc test analysis 119901 values forsignificance were set at 005 and all significant changes werenoted with lowast All analysis was performed using GraphPadPrism Software (Version 60h for Mac OS X)
3 Results
31 NHBE Cells Isolated from COPD Donors Express MoreER Stress Markers than Cells from Smokers Our group previ-ously demonstrated that undifferentiated NHBE cells have anincreased trend in ER stress upon exposure to cigarette smokeextract (CSE) [5] To examine whether fully differentiatedNHBE cells cultured at the air liquid interface (ALI) have anUPR to smoke NHBE cells from nonsmokers were exposedto 0 (room air RA) or repeat exposure to four cigarettes (CS)using a Vitrocell VC-10 smoking robot (Figures 1(a)-1(b))Repeat exposureswere performed tomaximize smoke stimuliwithout inducing apoptosis determined by LDH releaseassays (Figure 1(a)) Gene expression of IL-6 was utilizedas a positive control for sufficient exposure to smoke [20](Figure 1(a)) CD45 andCD11C expressionswere analyzed butdetection was below significant amplification levels therebyconfirming low levels of inflammatory-cell contamination(data not shown) Gene expression levels of ATF4 XBP1GRP78 GRP94 EDEM1 and CHOP were examined Thesetargets are readouts for the three major pathways of theUPR No ER stress marker was significantly altered followingsmoke exposure (Figure 1(b)) as we previously describedin submerged cultured NHBE cells [5] However when
comparing the same ER stressmarkers inNHBE cells isolatedfrom nonsmokers smokers and COPD donors expressionsof ATF4 XBP1 GRP78 GRP94 EDEM1 and CHOP were allincreased in cells isolated from COPD subjects (Figure 1(c))EDEM1 gene expression was significantly enhanced in cellsisolated from smokers (Figure 1(c)) There were increasedtrend changes for ER stress markers in cells from smokersProtein analysis also confirmed increased expression ofATF4 IRE1120572 GRP78 GRP94 EDEM and CHOP in cellsisolated from COPD subjects (Figure 2(a)) Equally elevatedphosphorylation of eIF2 and PERKwas observed only in cellsisolated from COPD donors (Figure 2(a))
XBP1 coordinates the adaptive UPR by playing a vitalrole in maintaining the ER function Gene expression resultsshowed that NHBE cells isolated from COPD subjects hadenhanced XBP1 mRNA splicing compared to cells fromnonsmokers and smokers (Figure 2(b)) demonstrating activeXBP1 signaling Protein analysis also confirmed increasedexpression of XBP1 in cells isolated from COPD donors(Figure 2(b)) Overall the disease-state predisposes NHBEcells to enhanced ER stress On the other hand acute smokeexposure has only a minor impact on ER stress
32 GPx-1 Regulates CHOP Expression in NHBE Cells Isolatedfrom COPD Donors Since oxidative stress induces ER stress[4] GPx-1 expression is reduced in the COPD lungs [13] andGPx-1 deficiency increases susceptibility to cigarette smoke-induced emphysema [13 17] we examined whether GPx-1 expression was altered in fully differentiated NHBE cellsisolated from nonsmokers smokers and COPD donors(Figure 3) GPx-1 expression was unchanged in NHBE cellsfrom nonsmokers when exposed to cigarette smoke (Fig-ure 3(a)) NHBE cells from nonsmokers and smokersexpressed comparable levels of GPx-1 (Figures 3(b)-3(c))However cells isolated fromCOPD subjects had significantlyreducedGPx-1 expression confirmed by q-PCR (Figure 3(b))and immunoblots (Figure 3(c)) Therefore the disease-statepredisposes NHBE cells to subdued GPx-1 expression
To determine whether restoring GPx-1 levels in NHBEcells fromCOPD subjects would reverse heightened UPR weprotein-transfected GPx-1 protein into NHBE cells fromCOPD subjects Albumin was transfected as a negative con-trol Transfection of GPx-1 significantly reduced CHOP gene(Figure 3(d)) and protein (Figure 3(e)) expression in NHBEcells from COPD subjects Therefore GPx-1 is a potentregulator of the UPR in the lungs
33 Viral Exacerbations of the Lung Enhance the UPR Todetermine whether a second environmental exposure couldalter the UPR in our models we infected NHBE cells andmice with respiratory syncytial virus (RSV) Viral infectionshave been implicated in the pathogenesis of COPD exacer-bations [24 25] and also trigger the UPR [26] RSV infectedreduced GPx-1 expression and significantly enhanced CHOPexpression in NHBE cells from all subject groups (Figures4(a)-4(b)) Similar changes to CHOP and GPx-1 expressionwere observed in the lungs ofmice infectedwithRSV (Figures4(c)-4(d)) Importantly prior exposure to cigarette smoke
Mediators of Inflammation 5
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Figure 1 NHBE cells isolated from COPD donors have enhanced ER stress responses compared to nonsmokers and smokers (a) Fullydifferentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) or cigarette smoke (CS) from 4 cigarettes everysecond day (3 exposures) using a Vitrocell VC-10 smoking robot LDH release into media and IL-6 gene expression were examined (b)Gene expression of ATF4 XBP1 GRP78 GRP94 EDEM1 and CHOP was examined (c) Fully differentiated NHBE cells from nonsmoker(NS) smoker (SM) and COPD (COPD) individuals (119899 = 6 donors per group) were examined for gene expression of ATF4 XBP1 GRP78GRP94 EDEM1 and CHOP Dot plots are represented as relative quantification (RQ) compared to ACTB expression and shown as the meanplusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
6 Mediators of Inflammation
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Mediators of Inflammation 7
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Figure 2 NHBE cells isolated fromCOPDdonors have enhanced ER stress protein responses compared to nonsmokers and smokers Proteinwas collected from fully differentiated NHBE cells from nonsmokers (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors pergroup) (a) Protein expression of ATF6 ATF4 GRP94 GRP78 EDEM CHOP and 120573-actin was examined by immunoblots Phosphorylationof elF2 and PERK was also determined (b) XBP1 splicing was examined on XBP1 amplified cDNA from NHBE cells from nonsmoker (NS)smokers (S) and COPD (COPD) individuals Protein expression of XBP1 and 120573-actin was examined by immunoblots (a)-(b) For each blotor gel every lane represents an individual cell donor Densitometry analysis was performed of XBP1s from DNA gels and other targets byanalyzing immunoblots XBP1 slicing was scored as percent of XBP1s of total XBP1 Dot plots are represented as densitometry units (DU) ofpixel intensity expressed as a ratio to120573-actin or total elF2 and PERKData are shown asmeanplusmn SEMwhere eachmeasurement was performedon 3 independent days on 6 donorsgroup lowast denotes 119901 value lt 005 when comparing both treatments connected by a line determined by2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
enhanced the UPR in animals also infected with RSVcompared to infected animals exposed to room air (RA)(Figure 4(d)) Therefore the lungs of smokers and COPDsubjects are likely to be more sensitive to viral infectioninduced ER stress which may impact disease progression
34 Gpx-1minusminus Mice Have Heightened ER Stress and Apoptosisfollowing Cigarette Smoke Exposure To determine whetherthe loss of GPx-1 expression directly influences ER stressfollowing inhalation of cigarette smoke in vivo we examinedER stress markers in Gpx-1minusminus mice and their wild type litter-mates exposed to cigarette smoke for 1 year We previouslydemonstrated that loss of Gpx-1 expression in mice resultsin enhanced air space enlargement and inflammation fol-lowing long-term cigarette smoke exposure [13] Expressionlevels of Atf4 Xbp1 Grp78 Grp94 Edem1 and Chop wereexamined in Gpx-1minusminus and wild type mice Long-term expo-sure to cigarette smoke did not significantly enhance ERstress marker expression in the lungs of wild type mice(Figure 5(a))We previously observed similar findings in wildtype mice [5] However Gpx-1minusminus mice exposed to cigarettesmoke had enhanced gene expression of Atf4 Xbp1 Grp78Grp94 Edem1 and Chop (Figure 5(a)) Equally loss of Gpx-1 expression resulted in elevated lung tissue protein levels ofATF4 XBP1 GRP78 GRP94 EDEM and CHOP followingsmoke exposure (Figure 5(b)) Densitometry analysis con-firmed significant increases in lung levels of ATF4 XBP1GRP78GRP94 EDEM andCHOP inGpx-1minusminusmice exposedto cigarette smoke (Figure 5(b))
Prolonged activation of CHOP by ER stress can result incellular apoptosis Increased structural and immune cellapoptosis is also observed in COPD lungs [27]Therefore weexamined whetherGpx-1minusminus mice exposed to cigarette smokehad elevated apoptosis Gpx-1minusminus mice exposed to cigarettesmoke had enhanced lung cell apoptosis observed byTUNEL caspase-3 cleavage and lactate dehydrogenase(LDH) release assays (Figure 6) Gpx-1minusminus mice exposed tocigarette smoke exhibited the highest frequency of TUNELpositive cells (Figure 6(a)) Enhanced caspase-3 cleavage wasobserved in Gpx-1minusminus mice exposed to cigarette smoke (Fig-ure 6(b)) Additionally elevated levels of LDHwere observedin the BALF of Gpx-1minusminus mice exposed to cigarette smokecompared to the other groups (Figure 6(c)) which indicatesenhanced cell membrane damage in the lung Thereforeenhanced apoptosis in the lungs could contribute to lungremodelling and failure to clear apoptotic cells could con-tribute to lung inflammation
35 Triggering the UPR Enhances GPx-1 GPx-2 and GPx-4Expression in Mouse Precision-Cut Lung Slices In Gpx-1 andGpx-2 double knockout mice apoptotic cells are increased inileal crypts [28] suggesting that GPx proteins regulate apop-tosis However the effect of ER stress on GPx proteins hasnot been directly investigated To determine the effect of ERstress on GPx-1 expression mouse precision-cut lung slices(PCLS) were exposed to the ER stress inducer tunicamycinfor 24 hoursThe concentration of tunicamycin tested did notinduce LDH release from the PCLS (Figure 7(a)) indicating
8 Mediators of Inflammation
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Figure 3 Reintroducing GPx-1 into NHBE cells isolated fromCOPD donors subdues the UPR (a) Gene expression ofGPx-1was determinedin fully differentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) and cigarette smoke (CS from 4 cigarettesevery second day (3 exposures)) using a Vitrocell VC-10 smoking robot (b) RNA and (c) protein was analyzed for GPx-1 expression fromfully differentiated NHBE cells from nonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) (d) NHBEcells isolated from COPD subjects were transfected with albumin or GPx-1 protein and CHOP expression was determined by qPCR (e)Immunoblots and corresponding densitometry analysis for CHOP Gpx-1 and 120573-actin from NHBE cells from COPD subjects followingalbumin or GPx-1 protein transfection In each immunoblot every lane represents an individual cell donor Data are shown as mean plusmn SEMwhere each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
no induction of apoptosis Tunicamycin induced all threebranches of the UPR and CHOP expression was observedin PCLS (Figures 7(b)-7(c)) Tunicamycin enhanced gene(Figure 7(b)) and protein (Figure 7(c)) expression of ATF4XBP1 ATF6 and CHOP in PCLS Interestingly tunicamycininduced GPx-1 GPx-2 and GPx-4 gene (Figure 7(d)) andprotein (Figure 7(e)) expression in PCLSTherefore an acuteER stress induces the expression of antioxidants to counterfurther oxidant and ER stress
4 Discussion
Cigarette smoking is the most relevant environmental riskfactor associated with the development of COPD Howeversmoke inhalation studies are problematic as long-term smoke
exposure is required to trigger disease formation in animalmodels and a secondary event may be required to mimic thehuman disease-state Here we observed that the loss of GPx-1 expression enhances cigarette smoke-induced ER stressGPx-1 regulates the UPR following smoke exposure and wefound that the expression of GPx-1 itself was triggered by anacute ER stress stimulus NHBE cells isolated from COPDdonors expressed significantly less GPx-1 which coincideswith elevated UPR Reintroducing GPx-1 protein into NHBEcells isolated from COPD donors reduced the UPR RSVinfection contributes to loss of lung GPx-1 expression whichis exaggerated in lungs exposed to smoke and coincides withelevated UPR Gpx-1minusminus mice exhibited greater UPR andsubsequent enhanced apoptosis following long-term cigarettesmoke exposure Interestingly triggering an acute ER stress in
Mediators of Inflammation 9
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05
10
15
GPx-
1 RQ
0
5
10
Chop
RQ
1 3 5 7 90(dpi)
1 3 5 7 90(dpi)
lowast
lowast
lowast
lowast
lowast
(c)
120573-Actin
CHOP
Room air Smoke
42
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
Mouse lungs
9dpi
(d)
Figure 4 RSV infection enhances the UPR in the lung (a) GPx-1 and CHOP gene expression were determined in NHBE cells isolated fromnonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) infected with mock or RSV and analyzed byqPCR (b) Protein expression of GPx-1 ATF6 ATF4 CHOP and 120573-actin was examined by immunoblots (c) Wild type mice were exposedto cigarette smoke or room air for six months and subsequently infected with 1 times 106 pfu of RSV Animals were euthanized at 0 1 3 5 7 and9 days after infection (dpi) and Gpx-1 and Chop expression were determined by qPCR (d) Protein expression of GPx-1 ATF6 ATF4 CHOPand 120573-actin was examined by immunoblots Data are shown as mean plusmn SEM where each measurement was performed on 3 independentdays lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line or the same infection day determined by Studentrsquos119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lungs of mice induces a potent antioxidant responseThisantioxidant response is diminished in the COPD lungs [29]which may explain the heightened UPR observed in NHBEcells isolated from COPD subjects The exact role of thisheightened UPR on the progression of COPD still remainsto be fully determined However we have established thatloss of GPx-1 in vivo leads to a marked increased in all threebranches of the UPR (see Figure 8 for proposed signalingscheme) and this coincides with enhanced apoptosis and lungtissue destruction in mice [13] Our results suggest that GPx-1 significantly regulates the UPR in COPD and enhancing
GPx-1 expression may be feasible means of offsetting theUPR and lung injury responses that drive the onset andprogression of this disease
Multiple studies utilizing the Gpx-1minusminus and transgenicmice demonstrated the protective role of GPx-1 in counteringoxidative injury and cell death mediated by ROS [13 30]GPX-1 activity also affects protein kinase phosphorylation[31] and oxidant-mediated activation of NF-120581B [32] In thiscurrent study GPx-1 was significantly reduced in NHBE cellsisolated from COPD subjects compared to nonsmokers andsmokers Others have reported that the alteration of GPx-1
Figure 5 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) Lung gene expression of Chop Atf4 Edem1 Grp78 Grp94 and Xbp1 was examined (b)Immunoblots were performed of whole lung protein for CHOP ATF4 EDEM GRP78 GRP94 and XBP1 Dot plots are represented as (a)relative quantification (RQ) compared to ACTB expression or (b) densitometry units (DU) of pixel intensity expressed as a ratio to 120573-actinEvery lane represents an individual mouse Data are shown as mean plusmn SEM where each measurement was performed on 3 independent dayson 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line determined by 2-way ANOVA withTukeyrsquos post hoc test (gt2 groups)
expression does not affect the mRNA or activity expressionof other selenoproteins [33] which suggests no compensationexpression of other selenoproteins following loss of GPx-1 expression Currently the mechanism by which cigarettesmoke regulates GPx-1 expression is not fully elucidated butconsidering our GPx-1 reintroduction data further analysis
of GPx-1 regulation is critical GPx-1 expression and activityhave been reported to be regulated by Nrf2 [34] the tran-scription factor TFAP2C [35] CpG methylation of the GPx-1promoter [35] Bcr-AblmTOR [36] selenium [37] estrogen[38] adenosine [39] Sec-insertion sequence (SECIS) factors[40] EGFR [41] and homocysteine [42] Specifically within
Mediators of Inflammation 11
Room airCigarette smoke
Wildtype
lowastlowast
00
02
04
06
08
10
TUN
EL p
ositi
ve (
)
Gpx-1minusminus
(a)
Caspase-3
42
19
Wild type
35 Pro
Cleaved
RA SM RA SM1 2 3 4 5 6 7 8
120573-Actin
Gpx-1minusminus
(b)
Wild type
lowastlowast lowast
lowastlowast
00
01
02
03
04LD
H re
leas
e (ab
s 350
nm)
0
2
4
6
8
10
Clea
ved
casp
ase D
U
Wildtype
Gpx-1minusminus Gpx-1minusminus
(c)
Figure 6 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) TUNEL analysis was performed on lung tissue from each mouse group (b) Enhancedlung tissue caspase-3 cleavage coincided with (c) elevated LDH into BALF ofGpx-1minusminus exposed to cigarette smoke Every lane in (b) representsan individual mouse and densitometry units (DU) of pixel intensity expressed as a ratio to total caspase-3 levels Dot plots are representedas mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 whencomparing both treatments connected by a line determined by 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lung during smoke exposure Singh et al show elevatedGPx-1 expression in the lungs following one-month cigarettesmoke exposure that was regulated by Nrf2 [34] HoweverNrf2 expression is lost in COPD subjects suggesting that thissecondary event could result in reduced GPx-1 expressionand heightened ER stress Loss of Nrf2 in mice resultsin enhanced susceptibility to cigarette smoke [43 44] andelastase [45] induced emphysema in mice However genomicstudies in Nrf2minusminus mouse samples suggest that Nrf2 mayregulate other GPx genes but not GPx-1 [46] Whether RSVinfection alters regulation of GPx-1 expression in a similarmanner to chronic smoke is unknown Interestingly theUPR upon RSV infection can counter viral proliferation [26]Further studies on the regulation of GPx-1 in smoke exposureand COPD and the significance of ER stress in the lungs arerequired This will be a major area for our future work
Since GPx-1 expression regulates all three branches of theUPR GPx-1 may affect a common mediator of the UPR oreach branch individually Dissociation of GRP78BiP uponER stress is required for all three branches of the UPR GPx-1expression directly regulated the gene expression of GRP78However whetherGPx-1 impacts onGRP78dissociation dur-ing ER stress is unknown Nrf2 interacts directly with PERK[47] and may play a major role in GPx-1 expression therebyregulating the UPR The ER stress inducer thapsigargininduces Nrf2 protein production in 16-HBE cells [47] whichsuggests that the UPR induces a Nrf2 response to reverseER stress We observe a similar effect on GPx-1 expressionin PCLS following tunicamycin treatment Equally XBP1regulates several antioxidants including catalase SOD1 andthioredoxinTRX1 [48]However XBP1 does not regulateGPxproteins [48] but XBP1 expression is regulated by GPx-1 The
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
enzyme 1 (IRE1)X-box binding protein 1 (XBP1) and the (iii)ATF6 pathway [11] These pathways regulate ER chaperoneresponses and reduce protein translation following cellularstress [12] However persistent ER stress results in significantexpression of the proapoptotic gene CEBP homologousprotein (CHOP) resulting in cell death [11] We previouslydemonstrated that smoke exposure triggers aminor ER stressresponse in primary cells and rodent animal models [5]However a secondary insult may be required to provoke asignificant smoke-induced UPR
Our group demonstrated that overexpression of glu-tathione peroxidase- (GPx-) 1 a member of the selenoproteinfamily prevents cigarette smoke-induced air space enlarge-ment in mice [13] GPx-1 is the most abundant GPx isoformin eukaryotic cells and deficiency of this enzyme can lead toendothelial dysfunction [14] and apoptosis [15] GPx-1 defi-ciency has also been implemented as a contributor toatherosclerosis [16]Gpx-1 deficient mice exposed to cigarettesmoke are more susceptible to cigarette smoke-induced lunginflammation and emphysema [13 17] Oxidative stressinduces ER stress [4] and increased expression of ER stressmarkers is observed in the lungs of smokers [8] GPx-1expression however is reduced in COPD lungs [13]Thus wespeculate that GPx-1 could modulate ER stress responseslinked to the pathogenesis of COPD
In view of the potential association between GPx-1 andcigarette smoke-mediatedUPR we explored whether the lossof GPx-1 expression enhanced the UPR that contributes tolung cell injury and death Using normal human bronchialepithelial (NHBE) cells from nonsmokers smokers andCOPD subjects we found that ER stress markers weresignificantly elevated in cells isolated from COPD subjectsand this increase coincided with reduced GPx-1 expressionReintroducing GPx-1 into these cells blunted the UPR Todetermine if GPx-1 depletion in the lung directly enhancesER stress Gpx-1minusminus mice were exposed to cigarette smoke for1 year Interestingly the loss of GPx-1 expression activatedall three branches of the UPR PERKeIF2120572ATF4CHOPIRE1XBP1 and ATF6 This UPR coincided with elevatedlung cell death in Gpx-1minusminus mice following smoke exposureInterestingly precision-cut lung slices (PCLS) frommice hadelevated GPx proteins following induction of ER stressThesefindings indicate that the altered GPx-1 expression in COPDlungs contributes to heightened ER stress In addition earlyinduction of ER stress induces an antioxidant response tocounter oxidative stress thereby limiting the UPR
2 Materials and Methods
21 Human Primary Airway Cells NHBE cells from non-smokers smokers and COPD patients were isolated fromhuman lungs Lungs were obtained from organ donors whoselungs were rejected for transplant (see Table 1 for demograph-ics) Consent for research was obtained by the Life AllianceOrgan Recovery Agency of the University of Miami All con-sents were IRB-approved and conformed to the Declarationof Helsinki For lungs from donors with COPD the diagnosiswas listed in the chart before the death of the donor and we
confirmed the macropathological presence of emphysema inthese lungs All COPD subjects had a significant smokinghistory NHBE cells isolated from nonsmokers smokers andCOPD subjects were dedifferentiated through expansion andredifferentiated at an air liquid interface (ALI) on 24mmT-clear filters (Costar Corning Corning NY USA) at37∘C 5 CO2 as previously described [18] Cells werecollected for protein and RNA analysis CD45 and CD11Cexpressions were analyzed which determined a low level ofinflammatory-cell contamination and confirmed NHBE cellpurity Fully differentiated NHBE cells from nonsmokerswere also exposed to cigarette smoke using a Vitrocell VC-10smoking robot (Vitrocell Systems GMBH Waldkirch Ger-many) Four cigarettes were smoked according to ISO stan-dard 3308 six puffs per cigarette with a 35mL volume per puffand a waiting time between each puff of 60 seconds NHBEcells were exposed every second day on three separate daysto 4 cigarettes RNA was extracted from the NHBE cells forquantitative PCR (qPCR) analysis A subset of NHBE cellsfrom COPD subjects was protein transfected 2120583g humanGPx-1 protein or human albumin (both from Signal Aldrich)usingPierce transfection reagent (ThermoFisher Scientific) aspreviously described [13 19] RNA and protein were collect24 hours later Human RSV strain A2 (ATCC ManassasVA VR-1540) was infected in NHBE cells as previouslydescribed [20] RNA and protein were collect 24 hours later
22 Animal Models Gpx-1minusminus mice were bred in C57BL6 timesCBAJ background Eight-week-old wild type and Gpx-1minusminusmice were used for all experiments All mice weremaintainedin a specific pathogen-free facility at Columbia UniversityBoth male and female mice 8-week-old were used at theinitiation point for all experiments and each experimentalparameter had at least 10 animals per group Mice wereexposed to cigarette smoke in a chamber (Teague EnterprisesDavis CA USA) for four hours a day five days per week ata total particulate matter concentration of 80mgm3 Smokeexposurewas continued for 1 yearTheUniversity ofKentuckyreference research cigarettes 3R4F (Lexington KY USA)were used to generate cigarette smoke Another group of wildtype animals was infected with 1times 106 pfu of RSV following 6-month exposure to room air or cigarette smokeThe instituteapproved all experiments for Animal Care and Use Commit-tee ofColumbiaUniversityThis studywas performed in strictaccordance with the recommendations in the Guide for the
Mediators of Inflammation 3
Table 2 TaqMan probe details for gene expression analysis
Care andUse of LaboratoryAnimals of theNational Institutesof Health and Institutional Animal Care and Use Committee(IACUC) guidelines
23 Precision-Cut Lung Slices (PCLS) Mouse precision-cutlung slices (PCLS) were prepared as previously described[21 22] Briefly mice were euthanized the trachea wascannulated and the animal was exsanguinated by cutting thejugular vein The lungs were filled through the cannulawith 15mL low melting-point agarose solution (15 finalconcentration of agarose in PBS) Lungs were placed on icefor 15 minutes to solidify the agarose Lobes were separatedand tissue cores were prepared of the individual lobesafter which the lobes were sliced at a thickness of 300 120583musing a Krumdieck tissue slicer (Alabama Research andDevelopment Munford AL USA) in Earlersquos balanced saltsolution (Sigma Aldrich) Tissue slices were incubated inDulbeccorsquos modified eaglersquos mediumnutrient mixture F-12 HAM solution (Sigma Aldrich) at 37∘C in a humidatmosphere under 5 CO295 air To remove agarose andcell debris slices were washed every 30 minutes for 2 hoursPCLS were incubated in DMEM supplemented with peni-cillin (100UmL) and streptomycin (100 120583gmL) (Gibco byLife Technologies) Slices were cultured at 37∘C in a humid-ified atmosphere under 5 CO295 air in 6-well tissueculture plates using 3 slices per well Slices were treated with1 120583M tunicamycin (Sigma Aldrich) for 24 hours To assessthe viability of the PCLS subjected to tunicamycin lactate
dehydrogenase (LDH) released from the PCLS into theincubation mediumwas analyzed Maximal LDH release wasdetermined by lysing 3 slices with 1 Triton X-100 for 30minutes at 37∘C LDH release was determined using an assayform Sigma Aldrich
24 qPCR Analysis Total RNA was isolated from cells ormouse lung tissue using the Qiagen RNeasy Mini Kit asdescribed by manufactures Gene transcript levels of mouseand human specific CHOP ATF4 XBP1 GRP78 GRP94EDEM1 GPx-1 GPx-2 GPx-3 GPx-4 IL-6 and ACTB werequantified by real-time PCR with the use of an Bio-RadCFX384 real-time system (Bio-Rad) TaqManGene Expres-sion Assays were purchased from Applied Biosystems (seeTable 2 for details) Data is represented as relative quan-tification (RQ) corrected to ACTB XBP1 splicing was alsoexamined in NHBE cells using the following primers 51015840-TTACGA GAG AAA ACT CAT GGC-31015840 and 51015840-GGG TCC AAGTTGTCCAGAATGC-31015840 XBP1 PCRproductswere resolvedand run on a 25 agarose gel [23] 289 and 286 base pairamplicons were generated from unspliced and spliced XBP1respectively Percent of XBP1 slicing was examined by densit-ometry analysis of the unspliced (XBP1u) and spliced (XBP1s)amplicons of XBP1 using Bio-Rad Laboratories Image Labsoftware (version 40 build 16)
25 Immunoblotting Cell monolayers were removed byscrapping in cold phosphate-buffered saline and resuspended
4 Mediators of Inflammation
in 100 120583L of protein lysis buffer (50mM HEPES pH 75150mM NaCl 1 Triton X-100 1 glycerol 1mM EDTA10mM NaF 2 120583gmL leupeptin 1 120583gmL pepstatin A 10mMNa3VO4 and 1mM phenylmethylsulfonyl fluoride) and20120583g of protein was separated on 12 SDS-polyacrylamidegels and transferred to nitrocellulose membranes Rab-bit antibodies against CHOP (Cell Signaling 5554) ERdegradation-enhancing 120572-mannosidase-like (EDEM) (SantaCruz Biotechnology sc-27389) pho-eIF2120572 (Ser51) (Cell Sig-naling 9721) eIF2120572 (Cell Signaling 9722) pho-PERK(Thr980) (Cell Signaling 3179) PERK (Cell Signaling5683) XBP-1 (Cell Signaling 12782) IRE1120572 (Cell Signaling3294) BiPGRP78 (Cell Signaling 3183) GRP94 (Cell Sig-naling 2104) ATF4 (Cell Signaling 11815) ATF6 (Abcamab11909) GPx-1 (Cell Signaling 3206) GPx-2 (Abcamab140130) GPx-3 (Abcam ab27325) GPx-4 (Cell Signal-ing 2104) and 120573-actin (Cell Signaling 4970) were detectedwith enhanced chemiluminescence reagents (Pierce) Chemi-luminescence detection was performed using the Bio-RadLaboratories Molecular Imager ChemiDoc XRS+ imagingsystem Densitometry was performed on each target andrepresented as a ratio of pixel intensity compared to totalprotein or 120573-actin using Bio-Rad Laboratories Image Labsoftware (version 40 build 16)
26 Statistical Analysis Data are expressed as dot plotswith the means plusmn SEM highlighted Differences betweentwo groups were compared by Studentrsquos 119905 test (two-tailed)Experiments with more than 2 groups were analyzed by 2-way ANOVA with Tukeyrsquos post hoc test analysis 119901 values forsignificance were set at 005 and all significant changes werenoted with lowast All analysis was performed using GraphPadPrism Software (Version 60h for Mac OS X)
3 Results
31 NHBE Cells Isolated from COPD Donors Express MoreER Stress Markers than Cells from Smokers Our group previ-ously demonstrated that undifferentiated NHBE cells have anincreased trend in ER stress upon exposure to cigarette smokeextract (CSE) [5] To examine whether fully differentiatedNHBE cells cultured at the air liquid interface (ALI) have anUPR to smoke NHBE cells from nonsmokers were exposedto 0 (room air RA) or repeat exposure to four cigarettes (CS)using a Vitrocell VC-10 smoking robot (Figures 1(a)-1(b))Repeat exposureswere performed tomaximize smoke stimuliwithout inducing apoptosis determined by LDH releaseassays (Figure 1(a)) Gene expression of IL-6 was utilizedas a positive control for sufficient exposure to smoke [20](Figure 1(a)) CD45 andCD11C expressionswere analyzed butdetection was below significant amplification levels therebyconfirming low levels of inflammatory-cell contamination(data not shown) Gene expression levels of ATF4 XBP1GRP78 GRP94 EDEM1 and CHOP were examined Thesetargets are readouts for the three major pathways of theUPR No ER stress marker was significantly altered followingsmoke exposure (Figure 1(b)) as we previously describedin submerged cultured NHBE cells [5] However when
comparing the same ER stressmarkers inNHBE cells isolatedfrom nonsmokers smokers and COPD donors expressionsof ATF4 XBP1 GRP78 GRP94 EDEM1 and CHOP were allincreased in cells isolated from COPD subjects (Figure 1(c))EDEM1 gene expression was significantly enhanced in cellsisolated from smokers (Figure 1(c)) There were increasedtrend changes for ER stress markers in cells from smokersProtein analysis also confirmed increased expression ofATF4 IRE1120572 GRP78 GRP94 EDEM and CHOP in cellsisolated from COPD subjects (Figure 2(a)) Equally elevatedphosphorylation of eIF2 and PERKwas observed only in cellsisolated from COPD donors (Figure 2(a))
XBP1 coordinates the adaptive UPR by playing a vitalrole in maintaining the ER function Gene expression resultsshowed that NHBE cells isolated from COPD subjects hadenhanced XBP1 mRNA splicing compared to cells fromnonsmokers and smokers (Figure 2(b)) demonstrating activeXBP1 signaling Protein analysis also confirmed increasedexpression of XBP1 in cells isolated from COPD donors(Figure 2(b)) Overall the disease-state predisposes NHBEcells to enhanced ER stress On the other hand acute smokeexposure has only a minor impact on ER stress
32 GPx-1 Regulates CHOP Expression in NHBE Cells Isolatedfrom COPD Donors Since oxidative stress induces ER stress[4] GPx-1 expression is reduced in the COPD lungs [13] andGPx-1 deficiency increases susceptibility to cigarette smoke-induced emphysema [13 17] we examined whether GPx-1 expression was altered in fully differentiated NHBE cellsisolated from nonsmokers smokers and COPD donors(Figure 3) GPx-1 expression was unchanged in NHBE cellsfrom nonsmokers when exposed to cigarette smoke (Fig-ure 3(a)) NHBE cells from nonsmokers and smokersexpressed comparable levels of GPx-1 (Figures 3(b)-3(c))However cells isolated fromCOPD subjects had significantlyreducedGPx-1 expression confirmed by q-PCR (Figure 3(b))and immunoblots (Figure 3(c)) Therefore the disease-statepredisposes NHBE cells to subdued GPx-1 expression
To determine whether restoring GPx-1 levels in NHBEcells fromCOPD subjects would reverse heightened UPR weprotein-transfected GPx-1 protein into NHBE cells fromCOPD subjects Albumin was transfected as a negative con-trol Transfection of GPx-1 significantly reduced CHOP gene(Figure 3(d)) and protein (Figure 3(e)) expression in NHBEcells from COPD subjects Therefore GPx-1 is a potentregulator of the UPR in the lungs
33 Viral Exacerbations of the Lung Enhance the UPR Todetermine whether a second environmental exposure couldalter the UPR in our models we infected NHBE cells andmice with respiratory syncytial virus (RSV) Viral infectionshave been implicated in the pathogenesis of COPD exacer-bations [24 25] and also trigger the UPR [26] RSV infectedreduced GPx-1 expression and significantly enhanced CHOPexpression in NHBE cells from all subject groups (Figures4(a)-4(b)) Similar changes to CHOP and GPx-1 expressionwere observed in the lungs ofmice infectedwithRSV (Figures4(c)-4(d)) Importantly prior exposure to cigarette smoke
Mediators of Inflammation 5
lowast
000
002
004
006
008
LDH
rele
ase (
abs
350
nm)
0
1
2
3
4
5
IL-6
RQ
CSRAExposure
CSRAExposure
(a)
CHO
P RQ
ATF4
RQ
GRP7
8 RQ
GRP9
4 RQ
XBP1
RQ
EDEM
1 RQ
00
05
10
15
20
0
1
2
3
4
00
05
10
15
00
05
10
15
20
00
05
10
15
20
25
00
05
10
15
20
25
CSRAExposure
CSRAExposure
CSRAExposure
CSRAExposure
CSRAExposure
CSRAExposure
(b)
CHO
P RQ
ATF4
RQ
GRP7
8 RQ
GRP9
4 RQ
XBP1
RQ
EDEM
1 RQ
lowastlowast lowast
lowast
lowastlowast lowast
lowast
lowast lowastlowastlowast
lowast
0
2
4
6
8
10
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
0
5
10
15
0
5
10
15
COPDSMN
S
Cell source
COPDSMN
S
Cell source
COPDSMN
S
Cell source
COPDSMN
S
Cell source
COPDSMN
S
Cell source
COPDSMN
S
Cell source(c)
Figure 1 NHBE cells isolated from COPD donors have enhanced ER stress responses compared to nonsmokers and smokers (a) Fullydifferentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) or cigarette smoke (CS) from 4 cigarettes everysecond day (3 exposures) using a Vitrocell VC-10 smoking robot LDH release into media and IL-6 gene expression were examined (b)Gene expression of ATF4 XBP1 GRP78 GRP94 EDEM1 and CHOP was examined (c) Fully differentiated NHBE cells from nonsmoker(NS) smoker (SM) and COPD (COPD) individuals (119899 = 6 donors per group) were examined for gene expression of ATF4 XBP1 GRP78GRP94 EDEM1 and CHOP Dot plots are represented as relative quantification (RQ) compared to ACTB expression and shown as the meanplusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
6 Mediators of Inflammation
CHOP
42120573-Actin
27
Cell source
EDEM
49 ATF4
74
GRP94
78 GRP78
100
XBP1
110 IRE1120572
60
p-elF2120572
38 elF2120572
38
p-PERK
170 PERK
170
120573-Actin 120573-Actin
289
263
(kDa)
(bp)
XBP1u
XBP1s
42
(kDa)
(a)
36 ATF6
COPDSMNS1 2 3 4 5 6
Cell source
COPDSMNS
1 2 3 4 5 6
Cell source
COPDSMNS
1 2 3 4 5 6
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
lowast
lowast
lowastlowast
lowastlowast lowast
lowast
lowastlowast
lowastlowast lowast
lowast
lowastlowast
lowastlowast
0
1
2
3
4
p-PE
RK D
U
0
2
4
6
p-el
F2120572
DU
0
1
2
3
ATF6
DU
0
1
2
3
ATF4
DU
00
05
10
15
20
25
IRE1
120572 D
U00
05
10
15
20
25
GRP
94 D
U
0
1
2
3
GRP
78 D
U
00
05
10
15
20
25
EDEM
DU
0
5
10
15
CHO
P D
U
Figure 2 Continued
Mediators of Inflammation 7
XBP1
DU
XBP1
splic
ing
()
COPDSM
(b)N
S
COPDSMN
S
lowastlowast
lowastlowast
0
20
40
60
80
0
1
2
3
Figure 2 NHBE cells isolated fromCOPDdonors have enhanced ER stress protein responses compared to nonsmokers and smokers Proteinwas collected from fully differentiated NHBE cells from nonsmokers (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors pergroup) (a) Protein expression of ATF6 ATF4 GRP94 GRP78 EDEM CHOP and 120573-actin was examined by immunoblots Phosphorylationof elF2 and PERK was also determined (b) XBP1 splicing was examined on XBP1 amplified cDNA from NHBE cells from nonsmoker (NS)smokers (S) and COPD (COPD) individuals Protein expression of XBP1 and 120573-actin was examined by immunoblots (a)-(b) For each blotor gel every lane represents an individual cell donor Densitometry analysis was performed of XBP1s from DNA gels and other targets byanalyzing immunoblots XBP1 slicing was scored as percent of XBP1s of total XBP1 Dot plots are represented as densitometry units (DU) ofpixel intensity expressed as a ratio to120573-actin or total elF2 and PERKData are shown asmeanplusmn SEMwhere eachmeasurement was performedon 3 independent days on 6 donorsgroup lowast denotes 119901 value lt 005 when comparing both treatments connected by a line determined by2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
enhanced the UPR in animals also infected with RSVcompared to infected animals exposed to room air (RA)(Figure 4(d)) Therefore the lungs of smokers and COPDsubjects are likely to be more sensitive to viral infectioninduced ER stress which may impact disease progression
34 Gpx-1minusminus Mice Have Heightened ER Stress and Apoptosisfollowing Cigarette Smoke Exposure To determine whetherthe loss of GPx-1 expression directly influences ER stressfollowing inhalation of cigarette smoke in vivo we examinedER stress markers in Gpx-1minusminus mice and their wild type litter-mates exposed to cigarette smoke for 1 year We previouslydemonstrated that loss of Gpx-1 expression in mice resultsin enhanced air space enlargement and inflammation fol-lowing long-term cigarette smoke exposure [13] Expressionlevels of Atf4 Xbp1 Grp78 Grp94 Edem1 and Chop wereexamined in Gpx-1minusminus and wild type mice Long-term expo-sure to cigarette smoke did not significantly enhance ERstress marker expression in the lungs of wild type mice(Figure 5(a))We previously observed similar findings in wildtype mice [5] However Gpx-1minusminus mice exposed to cigarettesmoke had enhanced gene expression of Atf4 Xbp1 Grp78Grp94 Edem1 and Chop (Figure 5(a)) Equally loss of Gpx-1 expression resulted in elevated lung tissue protein levels ofATF4 XBP1 GRP78 GRP94 EDEM and CHOP followingsmoke exposure (Figure 5(b)) Densitometry analysis con-firmed significant increases in lung levels of ATF4 XBP1GRP78GRP94 EDEM andCHOP inGpx-1minusminusmice exposedto cigarette smoke (Figure 5(b))
Prolonged activation of CHOP by ER stress can result incellular apoptosis Increased structural and immune cellapoptosis is also observed in COPD lungs [27]Therefore weexamined whetherGpx-1minusminus mice exposed to cigarette smokehad elevated apoptosis Gpx-1minusminus mice exposed to cigarettesmoke had enhanced lung cell apoptosis observed byTUNEL caspase-3 cleavage and lactate dehydrogenase(LDH) release assays (Figure 6) Gpx-1minusminus mice exposed tocigarette smoke exhibited the highest frequency of TUNELpositive cells (Figure 6(a)) Enhanced caspase-3 cleavage wasobserved in Gpx-1minusminus mice exposed to cigarette smoke (Fig-ure 6(b)) Additionally elevated levels of LDHwere observedin the BALF of Gpx-1minusminus mice exposed to cigarette smokecompared to the other groups (Figure 6(c)) which indicatesenhanced cell membrane damage in the lung Thereforeenhanced apoptosis in the lungs could contribute to lungremodelling and failure to clear apoptotic cells could con-tribute to lung inflammation
35 Triggering the UPR Enhances GPx-1 GPx-2 and GPx-4Expression in Mouse Precision-Cut Lung Slices In Gpx-1 andGpx-2 double knockout mice apoptotic cells are increased inileal crypts [28] suggesting that GPx proteins regulate apop-tosis However the effect of ER stress on GPx proteins hasnot been directly investigated To determine the effect of ERstress on GPx-1 expression mouse precision-cut lung slices(PCLS) were exposed to the ER stress inducer tunicamycinfor 24 hoursThe concentration of tunicamycin tested did notinduce LDH release from the PCLS (Figure 7(a)) indicating
8 Mediators of Inflammation
GPx-
1 RQ
00
05
10
15
20
CSRAExposure
(a)
GPx-
1 RQ
lowastlowast
00
05
10
15
20
Cell sourceCO
PDSMNS
(b)
GPx-1
42
22
GPx
-1 D
U
lowastlowast
Cell source
COPDSMN
S
120573-Actin
00
05
10
15
20
Cell sourceCOPDSMNS
1 2 3 4 5 6
(c)
lowast
Albumin GPx-1Protein transfected
NHBE COPD cells
00
05
10
15
20
CHO
P RQ
(d)
lowastlowast
GPx-1
42
22
NHBE COPD cells
CHOP27
Albumin GPx-1Protein transfected
Albumin GPx-1Protein transfected
NHBE COPD cells
120573-Actin
Albumin GPx-1Protein transfected
1 2 3 4 5 6
0
1
2
3
4
5
GPx
-1 D
U
00
05
10
15
CHO
P D
U(e)
Figure 3 Reintroducing GPx-1 into NHBE cells isolated fromCOPD donors subdues the UPR (a) Gene expression ofGPx-1was determinedin fully differentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) and cigarette smoke (CS from 4 cigarettesevery second day (3 exposures)) using a Vitrocell VC-10 smoking robot (b) RNA and (c) protein was analyzed for GPx-1 expression fromfully differentiated NHBE cells from nonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) (d) NHBEcells isolated from COPD subjects were transfected with albumin or GPx-1 protein and CHOP expression was determined by qPCR (e)Immunoblots and corresponding densitometry analysis for CHOP Gpx-1 and 120573-actin from NHBE cells from COPD subjects followingalbumin or GPx-1 protein transfection In each immunoblot every lane represents an individual cell donor Data are shown as mean plusmn SEMwhere each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
no induction of apoptosis Tunicamycin induced all threebranches of the UPR and CHOP expression was observedin PCLS (Figures 7(b)-7(c)) Tunicamycin enhanced gene(Figure 7(b)) and protein (Figure 7(c)) expression of ATF4XBP1 ATF6 and CHOP in PCLS Interestingly tunicamycininduced GPx-1 GPx-2 and GPx-4 gene (Figure 7(d)) andprotein (Figure 7(e)) expression in PCLSTherefore an acuteER stress induces the expression of antioxidants to counterfurther oxidant and ER stress
4 Discussion
Cigarette smoking is the most relevant environmental riskfactor associated with the development of COPD Howeversmoke inhalation studies are problematic as long-term smoke
exposure is required to trigger disease formation in animalmodels and a secondary event may be required to mimic thehuman disease-state Here we observed that the loss of GPx-1 expression enhances cigarette smoke-induced ER stressGPx-1 regulates the UPR following smoke exposure and wefound that the expression of GPx-1 itself was triggered by anacute ER stress stimulus NHBE cells isolated from COPDdonors expressed significantly less GPx-1 which coincideswith elevated UPR Reintroducing GPx-1 protein into NHBEcells isolated from COPD donors reduced the UPR RSVinfection contributes to loss of lung GPx-1 expression whichis exaggerated in lungs exposed to smoke and coincides withelevated UPR Gpx-1minusminus mice exhibited greater UPR andsubsequent enhanced apoptosis following long-term cigarettesmoke exposure Interestingly triggering an acute ER stress in
Mediators of Inflammation 9
Cell source
NS SM COPD
MockRSV
Cell source
NS SM COPD
MockRSV
NHBE cells
00
05
10
15
20GP
x-1
RQ
0
2
4
6
8
10
CHO
P RQ
lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)
CHOP
42 120573-Actin
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
NHBE cells
minus + minus + minus + RSV infection
Cell sourceCOPDSMNS
(b)
Mouse lungs
RA + mockRA + RSV
Smoke + mockSmoke + RSV
RA + mockRA + RSV
Smoke + mockSmoke + RSV
00
05
10
15
GPx-
1 RQ
0
5
10
Chop
RQ
1 3 5 7 90(dpi)
1 3 5 7 90(dpi)
lowast
lowast
lowast
lowast
lowast
(c)
120573-Actin
CHOP
Room air Smoke
42
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
Mouse lungs
9dpi
(d)
Figure 4 RSV infection enhances the UPR in the lung (a) GPx-1 and CHOP gene expression were determined in NHBE cells isolated fromnonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) infected with mock or RSV and analyzed byqPCR (b) Protein expression of GPx-1 ATF6 ATF4 CHOP and 120573-actin was examined by immunoblots (c) Wild type mice were exposedto cigarette smoke or room air for six months and subsequently infected with 1 times 106 pfu of RSV Animals were euthanized at 0 1 3 5 7 and9 days after infection (dpi) and Gpx-1 and Chop expression were determined by qPCR (d) Protein expression of GPx-1 ATF6 ATF4 CHOPand 120573-actin was examined by immunoblots Data are shown as mean plusmn SEM where each measurement was performed on 3 independentdays lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line or the same infection day determined by Studentrsquos119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lungs of mice induces a potent antioxidant responseThisantioxidant response is diminished in the COPD lungs [29]which may explain the heightened UPR observed in NHBEcells isolated from COPD subjects The exact role of thisheightened UPR on the progression of COPD still remainsto be fully determined However we have established thatloss of GPx-1 in vivo leads to a marked increased in all threebranches of the UPR (see Figure 8 for proposed signalingscheme) and this coincides with enhanced apoptosis and lungtissue destruction in mice [13] Our results suggest that GPx-1 significantly regulates the UPR in COPD and enhancing
GPx-1 expression may be feasible means of offsetting theUPR and lung injury responses that drive the onset andprogression of this disease
Multiple studies utilizing the Gpx-1minusminus and transgenicmice demonstrated the protective role of GPx-1 in counteringoxidative injury and cell death mediated by ROS [13 30]GPX-1 activity also affects protein kinase phosphorylation[31] and oxidant-mediated activation of NF-120581B [32] In thiscurrent study GPx-1 was significantly reduced in NHBE cellsisolated from COPD subjects compared to nonsmokers andsmokers Others have reported that the alteration of GPx-1
Figure 5 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) Lung gene expression of Chop Atf4 Edem1 Grp78 Grp94 and Xbp1 was examined (b)Immunoblots were performed of whole lung protein for CHOP ATF4 EDEM GRP78 GRP94 and XBP1 Dot plots are represented as (a)relative quantification (RQ) compared to ACTB expression or (b) densitometry units (DU) of pixel intensity expressed as a ratio to 120573-actinEvery lane represents an individual mouse Data are shown as mean plusmn SEM where each measurement was performed on 3 independent dayson 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line determined by 2-way ANOVA withTukeyrsquos post hoc test (gt2 groups)
expression does not affect the mRNA or activity expressionof other selenoproteins [33] which suggests no compensationexpression of other selenoproteins following loss of GPx-1 expression Currently the mechanism by which cigarettesmoke regulates GPx-1 expression is not fully elucidated butconsidering our GPx-1 reintroduction data further analysis
of GPx-1 regulation is critical GPx-1 expression and activityhave been reported to be regulated by Nrf2 [34] the tran-scription factor TFAP2C [35] CpG methylation of the GPx-1promoter [35] Bcr-AblmTOR [36] selenium [37] estrogen[38] adenosine [39] Sec-insertion sequence (SECIS) factors[40] EGFR [41] and homocysteine [42] Specifically within
Mediators of Inflammation 11
Room airCigarette smoke
Wildtype
lowastlowast
00
02
04
06
08
10
TUN
EL p
ositi
ve (
)
Gpx-1minusminus
(a)
Caspase-3
42
19
Wild type
35 Pro
Cleaved
RA SM RA SM1 2 3 4 5 6 7 8
120573-Actin
Gpx-1minusminus
(b)
Wild type
lowastlowast lowast
lowastlowast
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01
02
03
04LD
H re
leas
e (ab
s 350
nm)
0
2
4
6
8
10
Clea
ved
casp
ase D
U
Wildtype
Gpx-1minusminus Gpx-1minusminus
(c)
Figure 6 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) TUNEL analysis was performed on lung tissue from each mouse group (b) Enhancedlung tissue caspase-3 cleavage coincided with (c) elevated LDH into BALF ofGpx-1minusminus exposed to cigarette smoke Every lane in (b) representsan individual mouse and densitometry units (DU) of pixel intensity expressed as a ratio to total caspase-3 levels Dot plots are representedas mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 whencomparing both treatments connected by a line determined by 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lung during smoke exposure Singh et al show elevatedGPx-1 expression in the lungs following one-month cigarettesmoke exposure that was regulated by Nrf2 [34] HoweverNrf2 expression is lost in COPD subjects suggesting that thissecondary event could result in reduced GPx-1 expressionand heightened ER stress Loss of Nrf2 in mice resultsin enhanced susceptibility to cigarette smoke [43 44] andelastase [45] induced emphysema in mice However genomicstudies in Nrf2minusminus mouse samples suggest that Nrf2 mayregulate other GPx genes but not GPx-1 [46] Whether RSVinfection alters regulation of GPx-1 expression in a similarmanner to chronic smoke is unknown Interestingly theUPR upon RSV infection can counter viral proliferation [26]Further studies on the regulation of GPx-1 in smoke exposureand COPD and the significance of ER stress in the lungs arerequired This will be a major area for our future work
Since GPx-1 expression regulates all three branches of theUPR GPx-1 may affect a common mediator of the UPR oreach branch individually Dissociation of GRP78BiP uponER stress is required for all three branches of the UPR GPx-1expression directly regulated the gene expression of GRP78However whetherGPx-1 impacts onGRP78dissociation dur-ing ER stress is unknown Nrf2 interacts directly with PERK[47] and may play a major role in GPx-1 expression therebyregulating the UPR The ER stress inducer thapsigargininduces Nrf2 protein production in 16-HBE cells [47] whichsuggests that the UPR induces a Nrf2 response to reverseER stress We observe a similar effect on GPx-1 expressionin PCLS following tunicamycin treatment Equally XBP1regulates several antioxidants including catalase SOD1 andthioredoxinTRX1 [48]However XBP1 does not regulateGPxproteins [48] but XBP1 expression is regulated by GPx-1 The
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
Care andUse of LaboratoryAnimals of theNational Institutesof Health and Institutional Animal Care and Use Committee(IACUC) guidelines
23 Precision-Cut Lung Slices (PCLS) Mouse precision-cutlung slices (PCLS) were prepared as previously described[21 22] Briefly mice were euthanized the trachea wascannulated and the animal was exsanguinated by cutting thejugular vein The lungs were filled through the cannulawith 15mL low melting-point agarose solution (15 finalconcentration of agarose in PBS) Lungs were placed on icefor 15 minutes to solidify the agarose Lobes were separatedand tissue cores were prepared of the individual lobesafter which the lobes were sliced at a thickness of 300 120583musing a Krumdieck tissue slicer (Alabama Research andDevelopment Munford AL USA) in Earlersquos balanced saltsolution (Sigma Aldrich) Tissue slices were incubated inDulbeccorsquos modified eaglersquos mediumnutrient mixture F-12 HAM solution (Sigma Aldrich) at 37∘C in a humidatmosphere under 5 CO295 air To remove agarose andcell debris slices were washed every 30 minutes for 2 hoursPCLS were incubated in DMEM supplemented with peni-cillin (100UmL) and streptomycin (100 120583gmL) (Gibco byLife Technologies) Slices were cultured at 37∘C in a humid-ified atmosphere under 5 CO295 air in 6-well tissueculture plates using 3 slices per well Slices were treated with1 120583M tunicamycin (Sigma Aldrich) for 24 hours To assessthe viability of the PCLS subjected to tunicamycin lactate
dehydrogenase (LDH) released from the PCLS into theincubation mediumwas analyzed Maximal LDH release wasdetermined by lysing 3 slices with 1 Triton X-100 for 30minutes at 37∘C LDH release was determined using an assayform Sigma Aldrich
24 qPCR Analysis Total RNA was isolated from cells ormouse lung tissue using the Qiagen RNeasy Mini Kit asdescribed by manufactures Gene transcript levels of mouseand human specific CHOP ATF4 XBP1 GRP78 GRP94EDEM1 GPx-1 GPx-2 GPx-3 GPx-4 IL-6 and ACTB werequantified by real-time PCR with the use of an Bio-RadCFX384 real-time system (Bio-Rad) TaqManGene Expres-sion Assays were purchased from Applied Biosystems (seeTable 2 for details) Data is represented as relative quan-tification (RQ) corrected to ACTB XBP1 splicing was alsoexamined in NHBE cells using the following primers 51015840-TTACGA GAG AAA ACT CAT GGC-31015840 and 51015840-GGG TCC AAGTTGTCCAGAATGC-31015840 XBP1 PCRproductswere resolvedand run on a 25 agarose gel [23] 289 and 286 base pairamplicons were generated from unspliced and spliced XBP1respectively Percent of XBP1 slicing was examined by densit-ometry analysis of the unspliced (XBP1u) and spliced (XBP1s)amplicons of XBP1 using Bio-Rad Laboratories Image Labsoftware (version 40 build 16)
25 Immunoblotting Cell monolayers were removed byscrapping in cold phosphate-buffered saline and resuspended
4 Mediators of Inflammation
in 100 120583L of protein lysis buffer (50mM HEPES pH 75150mM NaCl 1 Triton X-100 1 glycerol 1mM EDTA10mM NaF 2 120583gmL leupeptin 1 120583gmL pepstatin A 10mMNa3VO4 and 1mM phenylmethylsulfonyl fluoride) and20120583g of protein was separated on 12 SDS-polyacrylamidegels and transferred to nitrocellulose membranes Rab-bit antibodies against CHOP (Cell Signaling 5554) ERdegradation-enhancing 120572-mannosidase-like (EDEM) (SantaCruz Biotechnology sc-27389) pho-eIF2120572 (Ser51) (Cell Sig-naling 9721) eIF2120572 (Cell Signaling 9722) pho-PERK(Thr980) (Cell Signaling 3179) PERK (Cell Signaling5683) XBP-1 (Cell Signaling 12782) IRE1120572 (Cell Signaling3294) BiPGRP78 (Cell Signaling 3183) GRP94 (Cell Sig-naling 2104) ATF4 (Cell Signaling 11815) ATF6 (Abcamab11909) GPx-1 (Cell Signaling 3206) GPx-2 (Abcamab140130) GPx-3 (Abcam ab27325) GPx-4 (Cell Signal-ing 2104) and 120573-actin (Cell Signaling 4970) were detectedwith enhanced chemiluminescence reagents (Pierce) Chemi-luminescence detection was performed using the Bio-RadLaboratories Molecular Imager ChemiDoc XRS+ imagingsystem Densitometry was performed on each target andrepresented as a ratio of pixel intensity compared to totalprotein or 120573-actin using Bio-Rad Laboratories Image Labsoftware (version 40 build 16)
26 Statistical Analysis Data are expressed as dot plotswith the means plusmn SEM highlighted Differences betweentwo groups were compared by Studentrsquos 119905 test (two-tailed)Experiments with more than 2 groups were analyzed by 2-way ANOVA with Tukeyrsquos post hoc test analysis 119901 values forsignificance were set at 005 and all significant changes werenoted with lowast All analysis was performed using GraphPadPrism Software (Version 60h for Mac OS X)
3 Results
31 NHBE Cells Isolated from COPD Donors Express MoreER Stress Markers than Cells from Smokers Our group previ-ously demonstrated that undifferentiated NHBE cells have anincreased trend in ER stress upon exposure to cigarette smokeextract (CSE) [5] To examine whether fully differentiatedNHBE cells cultured at the air liquid interface (ALI) have anUPR to smoke NHBE cells from nonsmokers were exposedto 0 (room air RA) or repeat exposure to four cigarettes (CS)using a Vitrocell VC-10 smoking robot (Figures 1(a)-1(b))Repeat exposureswere performed tomaximize smoke stimuliwithout inducing apoptosis determined by LDH releaseassays (Figure 1(a)) Gene expression of IL-6 was utilizedas a positive control for sufficient exposure to smoke [20](Figure 1(a)) CD45 andCD11C expressionswere analyzed butdetection was below significant amplification levels therebyconfirming low levels of inflammatory-cell contamination(data not shown) Gene expression levels of ATF4 XBP1GRP78 GRP94 EDEM1 and CHOP were examined Thesetargets are readouts for the three major pathways of theUPR No ER stress marker was significantly altered followingsmoke exposure (Figure 1(b)) as we previously describedin submerged cultured NHBE cells [5] However when
comparing the same ER stressmarkers inNHBE cells isolatedfrom nonsmokers smokers and COPD donors expressionsof ATF4 XBP1 GRP78 GRP94 EDEM1 and CHOP were allincreased in cells isolated from COPD subjects (Figure 1(c))EDEM1 gene expression was significantly enhanced in cellsisolated from smokers (Figure 1(c)) There were increasedtrend changes for ER stress markers in cells from smokersProtein analysis also confirmed increased expression ofATF4 IRE1120572 GRP78 GRP94 EDEM and CHOP in cellsisolated from COPD subjects (Figure 2(a)) Equally elevatedphosphorylation of eIF2 and PERKwas observed only in cellsisolated from COPD donors (Figure 2(a))
XBP1 coordinates the adaptive UPR by playing a vitalrole in maintaining the ER function Gene expression resultsshowed that NHBE cells isolated from COPD subjects hadenhanced XBP1 mRNA splicing compared to cells fromnonsmokers and smokers (Figure 2(b)) demonstrating activeXBP1 signaling Protein analysis also confirmed increasedexpression of XBP1 in cells isolated from COPD donors(Figure 2(b)) Overall the disease-state predisposes NHBEcells to enhanced ER stress On the other hand acute smokeexposure has only a minor impact on ER stress
32 GPx-1 Regulates CHOP Expression in NHBE Cells Isolatedfrom COPD Donors Since oxidative stress induces ER stress[4] GPx-1 expression is reduced in the COPD lungs [13] andGPx-1 deficiency increases susceptibility to cigarette smoke-induced emphysema [13 17] we examined whether GPx-1 expression was altered in fully differentiated NHBE cellsisolated from nonsmokers smokers and COPD donors(Figure 3) GPx-1 expression was unchanged in NHBE cellsfrom nonsmokers when exposed to cigarette smoke (Fig-ure 3(a)) NHBE cells from nonsmokers and smokersexpressed comparable levels of GPx-1 (Figures 3(b)-3(c))However cells isolated fromCOPD subjects had significantlyreducedGPx-1 expression confirmed by q-PCR (Figure 3(b))and immunoblots (Figure 3(c)) Therefore the disease-statepredisposes NHBE cells to subdued GPx-1 expression
To determine whether restoring GPx-1 levels in NHBEcells fromCOPD subjects would reverse heightened UPR weprotein-transfected GPx-1 protein into NHBE cells fromCOPD subjects Albumin was transfected as a negative con-trol Transfection of GPx-1 significantly reduced CHOP gene(Figure 3(d)) and protein (Figure 3(e)) expression in NHBEcells from COPD subjects Therefore GPx-1 is a potentregulator of the UPR in the lungs
33 Viral Exacerbations of the Lung Enhance the UPR Todetermine whether a second environmental exposure couldalter the UPR in our models we infected NHBE cells andmice with respiratory syncytial virus (RSV) Viral infectionshave been implicated in the pathogenesis of COPD exacer-bations [24 25] and also trigger the UPR [26] RSV infectedreduced GPx-1 expression and significantly enhanced CHOPexpression in NHBE cells from all subject groups (Figures4(a)-4(b)) Similar changes to CHOP and GPx-1 expressionwere observed in the lungs ofmice infectedwithRSV (Figures4(c)-4(d)) Importantly prior exposure to cigarette smoke
Mediators of Inflammation 5
lowast
000
002
004
006
008
LDH
rele
ase (
abs
350
nm)
0
1
2
3
4
5
IL-6
RQ
CSRAExposure
CSRAExposure
(a)
CHO
P RQ
ATF4
RQ
GRP7
8 RQ
GRP9
4 RQ
XBP1
RQ
EDEM
1 RQ
00
05
10
15
20
0
1
2
3
4
00
05
10
15
00
05
10
15
20
00
05
10
15
20
25
00
05
10
15
20
25
CSRAExposure
CSRAExposure
CSRAExposure
CSRAExposure
CSRAExposure
CSRAExposure
(b)
CHO
P RQ
ATF4
RQ
GRP7
8 RQ
GRP9
4 RQ
XBP1
RQ
EDEM
1 RQ
lowastlowast lowast
lowast
lowastlowast lowast
lowast
lowast lowastlowastlowast
lowast
0
2
4
6
8
10
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
0
5
10
15
0
5
10
15
COPDSMN
S
Cell source
COPDSMN
S
Cell source
COPDSMN
S
Cell source
COPDSMN
S
Cell source
COPDSMN
S
Cell source
COPDSMN
S
Cell source(c)
Figure 1 NHBE cells isolated from COPD donors have enhanced ER stress responses compared to nonsmokers and smokers (a) Fullydifferentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) or cigarette smoke (CS) from 4 cigarettes everysecond day (3 exposures) using a Vitrocell VC-10 smoking robot LDH release into media and IL-6 gene expression were examined (b)Gene expression of ATF4 XBP1 GRP78 GRP94 EDEM1 and CHOP was examined (c) Fully differentiated NHBE cells from nonsmoker(NS) smoker (SM) and COPD (COPD) individuals (119899 = 6 donors per group) were examined for gene expression of ATF4 XBP1 GRP78GRP94 EDEM1 and CHOP Dot plots are represented as relative quantification (RQ) compared to ACTB expression and shown as the meanplusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
6 Mediators of Inflammation
CHOP
42120573-Actin
27
Cell source
EDEM
49 ATF4
74
GRP94
78 GRP78
100
XBP1
110 IRE1120572
60
p-elF2120572
38 elF2120572
38
p-PERK
170 PERK
170
120573-Actin 120573-Actin
289
263
(kDa)
(bp)
XBP1u
XBP1s
42
(kDa)
(a)
36 ATF6
COPDSMNS1 2 3 4 5 6
Cell source
COPDSMNS
1 2 3 4 5 6
Cell source
COPDSMNS
1 2 3 4 5 6
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
lowast
lowast
lowastlowast
lowastlowast lowast
lowast
lowastlowast
lowastlowast lowast
lowast
lowastlowast
lowastlowast
0
1
2
3
4
p-PE
RK D
U
0
2
4
6
p-el
F2120572
DU
0
1
2
3
ATF6
DU
0
1
2
3
ATF4
DU
00
05
10
15
20
25
IRE1
120572 D
U00
05
10
15
20
25
GRP
94 D
U
0
1
2
3
GRP
78 D
U
00
05
10
15
20
25
EDEM
DU
0
5
10
15
CHO
P D
U
Figure 2 Continued
Mediators of Inflammation 7
XBP1
DU
XBP1
splic
ing
()
COPDSM
(b)N
S
COPDSMN
S
lowastlowast
lowastlowast
0
20
40
60
80
0
1
2
3
Figure 2 NHBE cells isolated fromCOPDdonors have enhanced ER stress protein responses compared to nonsmokers and smokers Proteinwas collected from fully differentiated NHBE cells from nonsmokers (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors pergroup) (a) Protein expression of ATF6 ATF4 GRP94 GRP78 EDEM CHOP and 120573-actin was examined by immunoblots Phosphorylationof elF2 and PERK was also determined (b) XBP1 splicing was examined on XBP1 amplified cDNA from NHBE cells from nonsmoker (NS)smokers (S) and COPD (COPD) individuals Protein expression of XBP1 and 120573-actin was examined by immunoblots (a)-(b) For each blotor gel every lane represents an individual cell donor Densitometry analysis was performed of XBP1s from DNA gels and other targets byanalyzing immunoblots XBP1 slicing was scored as percent of XBP1s of total XBP1 Dot plots are represented as densitometry units (DU) ofpixel intensity expressed as a ratio to120573-actin or total elF2 and PERKData are shown asmeanplusmn SEMwhere eachmeasurement was performedon 3 independent days on 6 donorsgroup lowast denotes 119901 value lt 005 when comparing both treatments connected by a line determined by2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
enhanced the UPR in animals also infected with RSVcompared to infected animals exposed to room air (RA)(Figure 4(d)) Therefore the lungs of smokers and COPDsubjects are likely to be more sensitive to viral infectioninduced ER stress which may impact disease progression
34 Gpx-1minusminus Mice Have Heightened ER Stress and Apoptosisfollowing Cigarette Smoke Exposure To determine whetherthe loss of GPx-1 expression directly influences ER stressfollowing inhalation of cigarette smoke in vivo we examinedER stress markers in Gpx-1minusminus mice and their wild type litter-mates exposed to cigarette smoke for 1 year We previouslydemonstrated that loss of Gpx-1 expression in mice resultsin enhanced air space enlargement and inflammation fol-lowing long-term cigarette smoke exposure [13] Expressionlevels of Atf4 Xbp1 Grp78 Grp94 Edem1 and Chop wereexamined in Gpx-1minusminus and wild type mice Long-term expo-sure to cigarette smoke did not significantly enhance ERstress marker expression in the lungs of wild type mice(Figure 5(a))We previously observed similar findings in wildtype mice [5] However Gpx-1minusminus mice exposed to cigarettesmoke had enhanced gene expression of Atf4 Xbp1 Grp78Grp94 Edem1 and Chop (Figure 5(a)) Equally loss of Gpx-1 expression resulted in elevated lung tissue protein levels ofATF4 XBP1 GRP78 GRP94 EDEM and CHOP followingsmoke exposure (Figure 5(b)) Densitometry analysis con-firmed significant increases in lung levels of ATF4 XBP1GRP78GRP94 EDEM andCHOP inGpx-1minusminusmice exposedto cigarette smoke (Figure 5(b))
Prolonged activation of CHOP by ER stress can result incellular apoptosis Increased structural and immune cellapoptosis is also observed in COPD lungs [27]Therefore weexamined whetherGpx-1minusminus mice exposed to cigarette smokehad elevated apoptosis Gpx-1minusminus mice exposed to cigarettesmoke had enhanced lung cell apoptosis observed byTUNEL caspase-3 cleavage and lactate dehydrogenase(LDH) release assays (Figure 6) Gpx-1minusminus mice exposed tocigarette smoke exhibited the highest frequency of TUNELpositive cells (Figure 6(a)) Enhanced caspase-3 cleavage wasobserved in Gpx-1minusminus mice exposed to cigarette smoke (Fig-ure 6(b)) Additionally elevated levels of LDHwere observedin the BALF of Gpx-1minusminus mice exposed to cigarette smokecompared to the other groups (Figure 6(c)) which indicatesenhanced cell membrane damage in the lung Thereforeenhanced apoptosis in the lungs could contribute to lungremodelling and failure to clear apoptotic cells could con-tribute to lung inflammation
35 Triggering the UPR Enhances GPx-1 GPx-2 and GPx-4Expression in Mouse Precision-Cut Lung Slices In Gpx-1 andGpx-2 double knockout mice apoptotic cells are increased inileal crypts [28] suggesting that GPx proteins regulate apop-tosis However the effect of ER stress on GPx proteins hasnot been directly investigated To determine the effect of ERstress on GPx-1 expression mouse precision-cut lung slices(PCLS) were exposed to the ER stress inducer tunicamycinfor 24 hoursThe concentration of tunicamycin tested did notinduce LDH release from the PCLS (Figure 7(a)) indicating
8 Mediators of Inflammation
GPx-
1 RQ
00
05
10
15
20
CSRAExposure
(a)
GPx-
1 RQ
lowastlowast
00
05
10
15
20
Cell sourceCO
PDSMNS
(b)
GPx-1
42
22
GPx
-1 D
U
lowastlowast
Cell source
COPDSMN
S
120573-Actin
00
05
10
15
20
Cell sourceCOPDSMNS
1 2 3 4 5 6
(c)
lowast
Albumin GPx-1Protein transfected
NHBE COPD cells
00
05
10
15
20
CHO
P RQ
(d)
lowastlowast
GPx-1
42
22
NHBE COPD cells
CHOP27
Albumin GPx-1Protein transfected
Albumin GPx-1Protein transfected
NHBE COPD cells
120573-Actin
Albumin GPx-1Protein transfected
1 2 3 4 5 6
0
1
2
3
4
5
GPx
-1 D
U
00
05
10
15
CHO
P D
U(e)
Figure 3 Reintroducing GPx-1 into NHBE cells isolated fromCOPD donors subdues the UPR (a) Gene expression ofGPx-1was determinedin fully differentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) and cigarette smoke (CS from 4 cigarettesevery second day (3 exposures)) using a Vitrocell VC-10 smoking robot (b) RNA and (c) protein was analyzed for GPx-1 expression fromfully differentiated NHBE cells from nonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) (d) NHBEcells isolated from COPD subjects were transfected with albumin or GPx-1 protein and CHOP expression was determined by qPCR (e)Immunoblots and corresponding densitometry analysis for CHOP Gpx-1 and 120573-actin from NHBE cells from COPD subjects followingalbumin or GPx-1 protein transfection In each immunoblot every lane represents an individual cell donor Data are shown as mean plusmn SEMwhere each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
no induction of apoptosis Tunicamycin induced all threebranches of the UPR and CHOP expression was observedin PCLS (Figures 7(b)-7(c)) Tunicamycin enhanced gene(Figure 7(b)) and protein (Figure 7(c)) expression of ATF4XBP1 ATF6 and CHOP in PCLS Interestingly tunicamycininduced GPx-1 GPx-2 and GPx-4 gene (Figure 7(d)) andprotein (Figure 7(e)) expression in PCLSTherefore an acuteER stress induces the expression of antioxidants to counterfurther oxidant and ER stress
4 Discussion
Cigarette smoking is the most relevant environmental riskfactor associated with the development of COPD Howeversmoke inhalation studies are problematic as long-term smoke
exposure is required to trigger disease formation in animalmodels and a secondary event may be required to mimic thehuman disease-state Here we observed that the loss of GPx-1 expression enhances cigarette smoke-induced ER stressGPx-1 regulates the UPR following smoke exposure and wefound that the expression of GPx-1 itself was triggered by anacute ER stress stimulus NHBE cells isolated from COPDdonors expressed significantly less GPx-1 which coincideswith elevated UPR Reintroducing GPx-1 protein into NHBEcells isolated from COPD donors reduced the UPR RSVinfection contributes to loss of lung GPx-1 expression whichis exaggerated in lungs exposed to smoke and coincides withelevated UPR Gpx-1minusminus mice exhibited greater UPR andsubsequent enhanced apoptosis following long-term cigarettesmoke exposure Interestingly triggering an acute ER stress in
Mediators of Inflammation 9
Cell source
NS SM COPD
MockRSV
Cell source
NS SM COPD
MockRSV
NHBE cells
00
05
10
15
20GP
x-1
RQ
0
2
4
6
8
10
CHO
P RQ
lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)
CHOP
42 120573-Actin
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
NHBE cells
minus + minus + minus + RSV infection
Cell sourceCOPDSMNS
(b)
Mouse lungs
RA + mockRA + RSV
Smoke + mockSmoke + RSV
RA + mockRA + RSV
Smoke + mockSmoke + RSV
00
05
10
15
GPx-
1 RQ
0
5
10
Chop
RQ
1 3 5 7 90(dpi)
1 3 5 7 90(dpi)
lowast
lowast
lowast
lowast
lowast
(c)
120573-Actin
CHOP
Room air Smoke
42
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
Mouse lungs
9dpi
(d)
Figure 4 RSV infection enhances the UPR in the lung (a) GPx-1 and CHOP gene expression were determined in NHBE cells isolated fromnonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) infected with mock or RSV and analyzed byqPCR (b) Protein expression of GPx-1 ATF6 ATF4 CHOP and 120573-actin was examined by immunoblots (c) Wild type mice were exposedto cigarette smoke or room air for six months and subsequently infected with 1 times 106 pfu of RSV Animals were euthanized at 0 1 3 5 7 and9 days after infection (dpi) and Gpx-1 and Chop expression were determined by qPCR (d) Protein expression of GPx-1 ATF6 ATF4 CHOPand 120573-actin was examined by immunoblots Data are shown as mean plusmn SEM where each measurement was performed on 3 independentdays lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line or the same infection day determined by Studentrsquos119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lungs of mice induces a potent antioxidant responseThisantioxidant response is diminished in the COPD lungs [29]which may explain the heightened UPR observed in NHBEcells isolated from COPD subjects The exact role of thisheightened UPR on the progression of COPD still remainsto be fully determined However we have established thatloss of GPx-1 in vivo leads to a marked increased in all threebranches of the UPR (see Figure 8 for proposed signalingscheme) and this coincides with enhanced apoptosis and lungtissue destruction in mice [13] Our results suggest that GPx-1 significantly regulates the UPR in COPD and enhancing
GPx-1 expression may be feasible means of offsetting theUPR and lung injury responses that drive the onset andprogression of this disease
Multiple studies utilizing the Gpx-1minusminus and transgenicmice demonstrated the protective role of GPx-1 in counteringoxidative injury and cell death mediated by ROS [13 30]GPX-1 activity also affects protein kinase phosphorylation[31] and oxidant-mediated activation of NF-120581B [32] In thiscurrent study GPx-1 was significantly reduced in NHBE cellsisolated from COPD subjects compared to nonsmokers andsmokers Others have reported that the alteration of GPx-1
Figure 5 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) Lung gene expression of Chop Atf4 Edem1 Grp78 Grp94 and Xbp1 was examined (b)Immunoblots were performed of whole lung protein for CHOP ATF4 EDEM GRP78 GRP94 and XBP1 Dot plots are represented as (a)relative quantification (RQ) compared to ACTB expression or (b) densitometry units (DU) of pixel intensity expressed as a ratio to 120573-actinEvery lane represents an individual mouse Data are shown as mean plusmn SEM where each measurement was performed on 3 independent dayson 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line determined by 2-way ANOVA withTukeyrsquos post hoc test (gt2 groups)
expression does not affect the mRNA or activity expressionof other selenoproteins [33] which suggests no compensationexpression of other selenoproteins following loss of GPx-1 expression Currently the mechanism by which cigarettesmoke regulates GPx-1 expression is not fully elucidated butconsidering our GPx-1 reintroduction data further analysis
of GPx-1 regulation is critical GPx-1 expression and activityhave been reported to be regulated by Nrf2 [34] the tran-scription factor TFAP2C [35] CpG methylation of the GPx-1promoter [35] Bcr-AblmTOR [36] selenium [37] estrogen[38] adenosine [39] Sec-insertion sequence (SECIS) factors[40] EGFR [41] and homocysteine [42] Specifically within
Mediators of Inflammation 11
Room airCigarette smoke
Wildtype
lowastlowast
00
02
04
06
08
10
TUN
EL p
ositi
ve (
)
Gpx-1minusminus
(a)
Caspase-3
42
19
Wild type
35 Pro
Cleaved
RA SM RA SM1 2 3 4 5 6 7 8
120573-Actin
Gpx-1minusminus
(b)
Wild type
lowastlowast lowast
lowastlowast
00
01
02
03
04LD
H re
leas
e (ab
s 350
nm)
0
2
4
6
8
10
Clea
ved
casp
ase D
U
Wildtype
Gpx-1minusminus Gpx-1minusminus
(c)
Figure 6 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) TUNEL analysis was performed on lung tissue from each mouse group (b) Enhancedlung tissue caspase-3 cleavage coincided with (c) elevated LDH into BALF ofGpx-1minusminus exposed to cigarette smoke Every lane in (b) representsan individual mouse and densitometry units (DU) of pixel intensity expressed as a ratio to total caspase-3 levels Dot plots are representedas mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 whencomparing both treatments connected by a line determined by 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lung during smoke exposure Singh et al show elevatedGPx-1 expression in the lungs following one-month cigarettesmoke exposure that was regulated by Nrf2 [34] HoweverNrf2 expression is lost in COPD subjects suggesting that thissecondary event could result in reduced GPx-1 expressionand heightened ER stress Loss of Nrf2 in mice resultsin enhanced susceptibility to cigarette smoke [43 44] andelastase [45] induced emphysema in mice However genomicstudies in Nrf2minusminus mouse samples suggest that Nrf2 mayregulate other GPx genes but not GPx-1 [46] Whether RSVinfection alters regulation of GPx-1 expression in a similarmanner to chronic smoke is unknown Interestingly theUPR upon RSV infection can counter viral proliferation [26]Further studies on the regulation of GPx-1 in smoke exposureand COPD and the significance of ER stress in the lungs arerequired This will be a major area for our future work
Since GPx-1 expression regulates all three branches of theUPR GPx-1 may affect a common mediator of the UPR oreach branch individually Dissociation of GRP78BiP uponER stress is required for all three branches of the UPR GPx-1expression directly regulated the gene expression of GRP78However whetherGPx-1 impacts onGRP78dissociation dur-ing ER stress is unknown Nrf2 interacts directly with PERK[47] and may play a major role in GPx-1 expression therebyregulating the UPR The ER stress inducer thapsigargininduces Nrf2 protein production in 16-HBE cells [47] whichsuggests that the UPR induces a Nrf2 response to reverseER stress We observe a similar effect on GPx-1 expressionin PCLS following tunicamycin treatment Equally XBP1regulates several antioxidants including catalase SOD1 andthioredoxinTRX1 [48]However XBP1 does not regulateGPxproteins [48] but XBP1 expression is regulated by GPx-1 The
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
in 100 120583L of protein lysis buffer (50mM HEPES pH 75150mM NaCl 1 Triton X-100 1 glycerol 1mM EDTA10mM NaF 2 120583gmL leupeptin 1 120583gmL pepstatin A 10mMNa3VO4 and 1mM phenylmethylsulfonyl fluoride) and20120583g of protein was separated on 12 SDS-polyacrylamidegels and transferred to nitrocellulose membranes Rab-bit antibodies against CHOP (Cell Signaling 5554) ERdegradation-enhancing 120572-mannosidase-like (EDEM) (SantaCruz Biotechnology sc-27389) pho-eIF2120572 (Ser51) (Cell Sig-naling 9721) eIF2120572 (Cell Signaling 9722) pho-PERK(Thr980) (Cell Signaling 3179) PERK (Cell Signaling5683) XBP-1 (Cell Signaling 12782) IRE1120572 (Cell Signaling3294) BiPGRP78 (Cell Signaling 3183) GRP94 (Cell Sig-naling 2104) ATF4 (Cell Signaling 11815) ATF6 (Abcamab11909) GPx-1 (Cell Signaling 3206) GPx-2 (Abcamab140130) GPx-3 (Abcam ab27325) GPx-4 (Cell Signal-ing 2104) and 120573-actin (Cell Signaling 4970) were detectedwith enhanced chemiluminescence reagents (Pierce) Chemi-luminescence detection was performed using the Bio-RadLaboratories Molecular Imager ChemiDoc XRS+ imagingsystem Densitometry was performed on each target andrepresented as a ratio of pixel intensity compared to totalprotein or 120573-actin using Bio-Rad Laboratories Image Labsoftware (version 40 build 16)
26 Statistical Analysis Data are expressed as dot plotswith the means plusmn SEM highlighted Differences betweentwo groups were compared by Studentrsquos 119905 test (two-tailed)Experiments with more than 2 groups were analyzed by 2-way ANOVA with Tukeyrsquos post hoc test analysis 119901 values forsignificance were set at 005 and all significant changes werenoted with lowast All analysis was performed using GraphPadPrism Software (Version 60h for Mac OS X)
3 Results
31 NHBE Cells Isolated from COPD Donors Express MoreER Stress Markers than Cells from Smokers Our group previ-ously demonstrated that undifferentiated NHBE cells have anincreased trend in ER stress upon exposure to cigarette smokeextract (CSE) [5] To examine whether fully differentiatedNHBE cells cultured at the air liquid interface (ALI) have anUPR to smoke NHBE cells from nonsmokers were exposedto 0 (room air RA) or repeat exposure to four cigarettes (CS)using a Vitrocell VC-10 smoking robot (Figures 1(a)-1(b))Repeat exposureswere performed tomaximize smoke stimuliwithout inducing apoptosis determined by LDH releaseassays (Figure 1(a)) Gene expression of IL-6 was utilizedas a positive control for sufficient exposure to smoke [20](Figure 1(a)) CD45 andCD11C expressionswere analyzed butdetection was below significant amplification levels therebyconfirming low levels of inflammatory-cell contamination(data not shown) Gene expression levels of ATF4 XBP1GRP78 GRP94 EDEM1 and CHOP were examined Thesetargets are readouts for the three major pathways of theUPR No ER stress marker was significantly altered followingsmoke exposure (Figure 1(b)) as we previously describedin submerged cultured NHBE cells [5] However when
comparing the same ER stressmarkers inNHBE cells isolatedfrom nonsmokers smokers and COPD donors expressionsof ATF4 XBP1 GRP78 GRP94 EDEM1 and CHOP were allincreased in cells isolated from COPD subjects (Figure 1(c))EDEM1 gene expression was significantly enhanced in cellsisolated from smokers (Figure 1(c)) There were increasedtrend changes for ER stress markers in cells from smokersProtein analysis also confirmed increased expression ofATF4 IRE1120572 GRP78 GRP94 EDEM and CHOP in cellsisolated from COPD subjects (Figure 2(a)) Equally elevatedphosphorylation of eIF2 and PERKwas observed only in cellsisolated from COPD donors (Figure 2(a))
XBP1 coordinates the adaptive UPR by playing a vitalrole in maintaining the ER function Gene expression resultsshowed that NHBE cells isolated from COPD subjects hadenhanced XBP1 mRNA splicing compared to cells fromnonsmokers and smokers (Figure 2(b)) demonstrating activeXBP1 signaling Protein analysis also confirmed increasedexpression of XBP1 in cells isolated from COPD donors(Figure 2(b)) Overall the disease-state predisposes NHBEcells to enhanced ER stress On the other hand acute smokeexposure has only a minor impact on ER stress
32 GPx-1 Regulates CHOP Expression in NHBE Cells Isolatedfrom COPD Donors Since oxidative stress induces ER stress[4] GPx-1 expression is reduced in the COPD lungs [13] andGPx-1 deficiency increases susceptibility to cigarette smoke-induced emphysema [13 17] we examined whether GPx-1 expression was altered in fully differentiated NHBE cellsisolated from nonsmokers smokers and COPD donors(Figure 3) GPx-1 expression was unchanged in NHBE cellsfrom nonsmokers when exposed to cigarette smoke (Fig-ure 3(a)) NHBE cells from nonsmokers and smokersexpressed comparable levels of GPx-1 (Figures 3(b)-3(c))However cells isolated fromCOPD subjects had significantlyreducedGPx-1 expression confirmed by q-PCR (Figure 3(b))and immunoblots (Figure 3(c)) Therefore the disease-statepredisposes NHBE cells to subdued GPx-1 expression
To determine whether restoring GPx-1 levels in NHBEcells fromCOPD subjects would reverse heightened UPR weprotein-transfected GPx-1 protein into NHBE cells fromCOPD subjects Albumin was transfected as a negative con-trol Transfection of GPx-1 significantly reduced CHOP gene(Figure 3(d)) and protein (Figure 3(e)) expression in NHBEcells from COPD subjects Therefore GPx-1 is a potentregulator of the UPR in the lungs
33 Viral Exacerbations of the Lung Enhance the UPR Todetermine whether a second environmental exposure couldalter the UPR in our models we infected NHBE cells andmice with respiratory syncytial virus (RSV) Viral infectionshave been implicated in the pathogenesis of COPD exacer-bations [24 25] and also trigger the UPR [26] RSV infectedreduced GPx-1 expression and significantly enhanced CHOPexpression in NHBE cells from all subject groups (Figures4(a)-4(b)) Similar changes to CHOP and GPx-1 expressionwere observed in the lungs ofmice infectedwithRSV (Figures4(c)-4(d)) Importantly prior exposure to cigarette smoke
Mediators of Inflammation 5
lowast
000
002
004
006
008
LDH
rele
ase (
abs
350
nm)
0
1
2
3
4
5
IL-6
RQ
CSRAExposure
CSRAExposure
(a)
CHO
P RQ
ATF4
RQ
GRP7
8 RQ
GRP9
4 RQ
XBP1
RQ
EDEM
1 RQ
00
05
10
15
20
0
1
2
3
4
00
05
10
15
00
05
10
15
20
00
05
10
15
20
25
00
05
10
15
20
25
CSRAExposure
CSRAExposure
CSRAExposure
CSRAExposure
CSRAExposure
CSRAExposure
(b)
CHO
P RQ
ATF4
RQ
GRP7
8 RQ
GRP9
4 RQ
XBP1
RQ
EDEM
1 RQ
lowastlowast lowast
lowast
lowastlowast lowast
lowast
lowast lowastlowastlowast
lowast
0
2
4
6
8
10
0
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
20
0
5
10
15
0
5
10
15
COPDSMN
S
Cell source
COPDSMN
S
Cell source
COPDSMN
S
Cell source
COPDSMN
S
Cell source
COPDSMN
S
Cell source
COPDSMN
S
Cell source(c)
Figure 1 NHBE cells isolated from COPD donors have enhanced ER stress responses compared to nonsmokers and smokers (a) Fullydifferentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) or cigarette smoke (CS) from 4 cigarettes everysecond day (3 exposures) using a Vitrocell VC-10 smoking robot LDH release into media and IL-6 gene expression were examined (b)Gene expression of ATF4 XBP1 GRP78 GRP94 EDEM1 and CHOP was examined (c) Fully differentiated NHBE cells from nonsmoker(NS) smoker (SM) and COPD (COPD) individuals (119899 = 6 donors per group) were examined for gene expression of ATF4 XBP1 GRP78GRP94 EDEM1 and CHOP Dot plots are represented as relative quantification (RQ) compared to ACTB expression and shown as the meanplusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
6 Mediators of Inflammation
CHOP
42120573-Actin
27
Cell source
EDEM
49 ATF4
74
GRP94
78 GRP78
100
XBP1
110 IRE1120572
60
p-elF2120572
38 elF2120572
38
p-PERK
170 PERK
170
120573-Actin 120573-Actin
289
263
(kDa)
(bp)
XBP1u
XBP1s
42
(kDa)
(a)
36 ATF6
COPDSMNS1 2 3 4 5 6
Cell source
COPDSMNS
1 2 3 4 5 6
Cell source
COPDSMNS
1 2 3 4 5 6
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
lowast
lowast
lowastlowast
lowastlowast lowast
lowast
lowastlowast
lowastlowast lowast
lowast
lowastlowast
lowastlowast
0
1
2
3
4
p-PE
RK D
U
0
2
4
6
p-el
F2120572
DU
0
1
2
3
ATF6
DU
0
1
2
3
ATF4
DU
00
05
10
15
20
25
IRE1
120572 D
U00
05
10
15
20
25
GRP
94 D
U
0
1
2
3
GRP
78 D
U
00
05
10
15
20
25
EDEM
DU
0
5
10
15
CHO
P D
U
Figure 2 Continued
Mediators of Inflammation 7
XBP1
DU
XBP1
splic
ing
()
COPDSM
(b)N
S
COPDSMN
S
lowastlowast
lowastlowast
0
20
40
60
80
0
1
2
3
Figure 2 NHBE cells isolated fromCOPDdonors have enhanced ER stress protein responses compared to nonsmokers and smokers Proteinwas collected from fully differentiated NHBE cells from nonsmokers (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors pergroup) (a) Protein expression of ATF6 ATF4 GRP94 GRP78 EDEM CHOP and 120573-actin was examined by immunoblots Phosphorylationof elF2 and PERK was also determined (b) XBP1 splicing was examined on XBP1 amplified cDNA from NHBE cells from nonsmoker (NS)smokers (S) and COPD (COPD) individuals Protein expression of XBP1 and 120573-actin was examined by immunoblots (a)-(b) For each blotor gel every lane represents an individual cell donor Densitometry analysis was performed of XBP1s from DNA gels and other targets byanalyzing immunoblots XBP1 slicing was scored as percent of XBP1s of total XBP1 Dot plots are represented as densitometry units (DU) ofpixel intensity expressed as a ratio to120573-actin or total elF2 and PERKData are shown asmeanplusmn SEMwhere eachmeasurement was performedon 3 independent days on 6 donorsgroup lowast denotes 119901 value lt 005 when comparing both treatments connected by a line determined by2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
enhanced the UPR in animals also infected with RSVcompared to infected animals exposed to room air (RA)(Figure 4(d)) Therefore the lungs of smokers and COPDsubjects are likely to be more sensitive to viral infectioninduced ER stress which may impact disease progression
34 Gpx-1minusminus Mice Have Heightened ER Stress and Apoptosisfollowing Cigarette Smoke Exposure To determine whetherthe loss of GPx-1 expression directly influences ER stressfollowing inhalation of cigarette smoke in vivo we examinedER stress markers in Gpx-1minusminus mice and their wild type litter-mates exposed to cigarette smoke for 1 year We previouslydemonstrated that loss of Gpx-1 expression in mice resultsin enhanced air space enlargement and inflammation fol-lowing long-term cigarette smoke exposure [13] Expressionlevels of Atf4 Xbp1 Grp78 Grp94 Edem1 and Chop wereexamined in Gpx-1minusminus and wild type mice Long-term expo-sure to cigarette smoke did not significantly enhance ERstress marker expression in the lungs of wild type mice(Figure 5(a))We previously observed similar findings in wildtype mice [5] However Gpx-1minusminus mice exposed to cigarettesmoke had enhanced gene expression of Atf4 Xbp1 Grp78Grp94 Edem1 and Chop (Figure 5(a)) Equally loss of Gpx-1 expression resulted in elevated lung tissue protein levels ofATF4 XBP1 GRP78 GRP94 EDEM and CHOP followingsmoke exposure (Figure 5(b)) Densitometry analysis con-firmed significant increases in lung levels of ATF4 XBP1GRP78GRP94 EDEM andCHOP inGpx-1minusminusmice exposedto cigarette smoke (Figure 5(b))
Prolonged activation of CHOP by ER stress can result incellular apoptosis Increased structural and immune cellapoptosis is also observed in COPD lungs [27]Therefore weexamined whetherGpx-1minusminus mice exposed to cigarette smokehad elevated apoptosis Gpx-1minusminus mice exposed to cigarettesmoke had enhanced lung cell apoptosis observed byTUNEL caspase-3 cleavage and lactate dehydrogenase(LDH) release assays (Figure 6) Gpx-1minusminus mice exposed tocigarette smoke exhibited the highest frequency of TUNELpositive cells (Figure 6(a)) Enhanced caspase-3 cleavage wasobserved in Gpx-1minusminus mice exposed to cigarette smoke (Fig-ure 6(b)) Additionally elevated levels of LDHwere observedin the BALF of Gpx-1minusminus mice exposed to cigarette smokecompared to the other groups (Figure 6(c)) which indicatesenhanced cell membrane damage in the lung Thereforeenhanced apoptosis in the lungs could contribute to lungremodelling and failure to clear apoptotic cells could con-tribute to lung inflammation
35 Triggering the UPR Enhances GPx-1 GPx-2 and GPx-4Expression in Mouse Precision-Cut Lung Slices In Gpx-1 andGpx-2 double knockout mice apoptotic cells are increased inileal crypts [28] suggesting that GPx proteins regulate apop-tosis However the effect of ER stress on GPx proteins hasnot been directly investigated To determine the effect of ERstress on GPx-1 expression mouse precision-cut lung slices(PCLS) were exposed to the ER stress inducer tunicamycinfor 24 hoursThe concentration of tunicamycin tested did notinduce LDH release from the PCLS (Figure 7(a)) indicating
8 Mediators of Inflammation
GPx-
1 RQ
00
05
10
15
20
CSRAExposure
(a)
GPx-
1 RQ
lowastlowast
00
05
10
15
20
Cell sourceCO
PDSMNS
(b)
GPx-1
42
22
GPx
-1 D
U
lowastlowast
Cell source
COPDSMN
S
120573-Actin
00
05
10
15
20
Cell sourceCOPDSMNS
1 2 3 4 5 6
(c)
lowast
Albumin GPx-1Protein transfected
NHBE COPD cells
00
05
10
15
20
CHO
P RQ
(d)
lowastlowast
GPx-1
42
22
NHBE COPD cells
CHOP27
Albumin GPx-1Protein transfected
Albumin GPx-1Protein transfected
NHBE COPD cells
120573-Actin
Albumin GPx-1Protein transfected
1 2 3 4 5 6
0
1
2
3
4
5
GPx
-1 D
U
00
05
10
15
CHO
P D
U(e)
Figure 3 Reintroducing GPx-1 into NHBE cells isolated fromCOPD donors subdues the UPR (a) Gene expression ofGPx-1was determinedin fully differentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) and cigarette smoke (CS from 4 cigarettesevery second day (3 exposures)) using a Vitrocell VC-10 smoking robot (b) RNA and (c) protein was analyzed for GPx-1 expression fromfully differentiated NHBE cells from nonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) (d) NHBEcells isolated from COPD subjects were transfected with albumin or GPx-1 protein and CHOP expression was determined by qPCR (e)Immunoblots and corresponding densitometry analysis for CHOP Gpx-1 and 120573-actin from NHBE cells from COPD subjects followingalbumin or GPx-1 protein transfection In each immunoblot every lane represents an individual cell donor Data are shown as mean plusmn SEMwhere each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
no induction of apoptosis Tunicamycin induced all threebranches of the UPR and CHOP expression was observedin PCLS (Figures 7(b)-7(c)) Tunicamycin enhanced gene(Figure 7(b)) and protein (Figure 7(c)) expression of ATF4XBP1 ATF6 and CHOP in PCLS Interestingly tunicamycininduced GPx-1 GPx-2 and GPx-4 gene (Figure 7(d)) andprotein (Figure 7(e)) expression in PCLSTherefore an acuteER stress induces the expression of antioxidants to counterfurther oxidant and ER stress
4 Discussion
Cigarette smoking is the most relevant environmental riskfactor associated with the development of COPD Howeversmoke inhalation studies are problematic as long-term smoke
exposure is required to trigger disease formation in animalmodels and a secondary event may be required to mimic thehuman disease-state Here we observed that the loss of GPx-1 expression enhances cigarette smoke-induced ER stressGPx-1 regulates the UPR following smoke exposure and wefound that the expression of GPx-1 itself was triggered by anacute ER stress stimulus NHBE cells isolated from COPDdonors expressed significantly less GPx-1 which coincideswith elevated UPR Reintroducing GPx-1 protein into NHBEcells isolated from COPD donors reduced the UPR RSVinfection contributes to loss of lung GPx-1 expression whichis exaggerated in lungs exposed to smoke and coincides withelevated UPR Gpx-1minusminus mice exhibited greater UPR andsubsequent enhanced apoptosis following long-term cigarettesmoke exposure Interestingly triggering an acute ER stress in
Mediators of Inflammation 9
Cell source
NS SM COPD
MockRSV
Cell source
NS SM COPD
MockRSV
NHBE cells
00
05
10
15
20GP
x-1
RQ
0
2
4
6
8
10
CHO
P RQ
lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)
CHOP
42 120573-Actin
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
NHBE cells
minus + minus + minus + RSV infection
Cell sourceCOPDSMNS
(b)
Mouse lungs
RA + mockRA + RSV
Smoke + mockSmoke + RSV
RA + mockRA + RSV
Smoke + mockSmoke + RSV
00
05
10
15
GPx-
1 RQ
0
5
10
Chop
RQ
1 3 5 7 90(dpi)
1 3 5 7 90(dpi)
lowast
lowast
lowast
lowast
lowast
(c)
120573-Actin
CHOP
Room air Smoke
42
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
Mouse lungs
9dpi
(d)
Figure 4 RSV infection enhances the UPR in the lung (a) GPx-1 and CHOP gene expression were determined in NHBE cells isolated fromnonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) infected with mock or RSV and analyzed byqPCR (b) Protein expression of GPx-1 ATF6 ATF4 CHOP and 120573-actin was examined by immunoblots (c) Wild type mice were exposedto cigarette smoke or room air for six months and subsequently infected with 1 times 106 pfu of RSV Animals were euthanized at 0 1 3 5 7 and9 days after infection (dpi) and Gpx-1 and Chop expression were determined by qPCR (d) Protein expression of GPx-1 ATF6 ATF4 CHOPand 120573-actin was examined by immunoblots Data are shown as mean plusmn SEM where each measurement was performed on 3 independentdays lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line or the same infection day determined by Studentrsquos119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lungs of mice induces a potent antioxidant responseThisantioxidant response is diminished in the COPD lungs [29]which may explain the heightened UPR observed in NHBEcells isolated from COPD subjects The exact role of thisheightened UPR on the progression of COPD still remainsto be fully determined However we have established thatloss of GPx-1 in vivo leads to a marked increased in all threebranches of the UPR (see Figure 8 for proposed signalingscheme) and this coincides with enhanced apoptosis and lungtissue destruction in mice [13] Our results suggest that GPx-1 significantly regulates the UPR in COPD and enhancing
GPx-1 expression may be feasible means of offsetting theUPR and lung injury responses that drive the onset andprogression of this disease
Multiple studies utilizing the Gpx-1minusminus and transgenicmice demonstrated the protective role of GPx-1 in counteringoxidative injury and cell death mediated by ROS [13 30]GPX-1 activity also affects protein kinase phosphorylation[31] and oxidant-mediated activation of NF-120581B [32] In thiscurrent study GPx-1 was significantly reduced in NHBE cellsisolated from COPD subjects compared to nonsmokers andsmokers Others have reported that the alteration of GPx-1
Figure 5 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) Lung gene expression of Chop Atf4 Edem1 Grp78 Grp94 and Xbp1 was examined (b)Immunoblots were performed of whole lung protein for CHOP ATF4 EDEM GRP78 GRP94 and XBP1 Dot plots are represented as (a)relative quantification (RQ) compared to ACTB expression or (b) densitometry units (DU) of pixel intensity expressed as a ratio to 120573-actinEvery lane represents an individual mouse Data are shown as mean plusmn SEM where each measurement was performed on 3 independent dayson 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line determined by 2-way ANOVA withTukeyrsquos post hoc test (gt2 groups)
expression does not affect the mRNA or activity expressionof other selenoproteins [33] which suggests no compensationexpression of other selenoproteins following loss of GPx-1 expression Currently the mechanism by which cigarettesmoke regulates GPx-1 expression is not fully elucidated butconsidering our GPx-1 reintroduction data further analysis
of GPx-1 regulation is critical GPx-1 expression and activityhave been reported to be regulated by Nrf2 [34] the tran-scription factor TFAP2C [35] CpG methylation of the GPx-1promoter [35] Bcr-AblmTOR [36] selenium [37] estrogen[38] adenosine [39] Sec-insertion sequence (SECIS) factors[40] EGFR [41] and homocysteine [42] Specifically within
Mediators of Inflammation 11
Room airCigarette smoke
Wildtype
lowastlowast
00
02
04
06
08
10
TUN
EL p
ositi
ve (
)
Gpx-1minusminus
(a)
Caspase-3
42
19
Wild type
35 Pro
Cleaved
RA SM RA SM1 2 3 4 5 6 7 8
120573-Actin
Gpx-1minusminus
(b)
Wild type
lowastlowast lowast
lowastlowast
00
01
02
03
04LD
H re
leas
e (ab
s 350
nm)
0
2
4
6
8
10
Clea
ved
casp
ase D
U
Wildtype
Gpx-1minusminus Gpx-1minusminus
(c)
Figure 6 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) TUNEL analysis was performed on lung tissue from each mouse group (b) Enhancedlung tissue caspase-3 cleavage coincided with (c) elevated LDH into BALF ofGpx-1minusminus exposed to cigarette smoke Every lane in (b) representsan individual mouse and densitometry units (DU) of pixel intensity expressed as a ratio to total caspase-3 levels Dot plots are representedas mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 whencomparing both treatments connected by a line determined by 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lung during smoke exposure Singh et al show elevatedGPx-1 expression in the lungs following one-month cigarettesmoke exposure that was regulated by Nrf2 [34] HoweverNrf2 expression is lost in COPD subjects suggesting that thissecondary event could result in reduced GPx-1 expressionand heightened ER stress Loss of Nrf2 in mice resultsin enhanced susceptibility to cigarette smoke [43 44] andelastase [45] induced emphysema in mice However genomicstudies in Nrf2minusminus mouse samples suggest that Nrf2 mayregulate other GPx genes but not GPx-1 [46] Whether RSVinfection alters regulation of GPx-1 expression in a similarmanner to chronic smoke is unknown Interestingly theUPR upon RSV infection can counter viral proliferation [26]Further studies on the regulation of GPx-1 in smoke exposureand COPD and the significance of ER stress in the lungs arerequired This will be a major area for our future work
Since GPx-1 expression regulates all three branches of theUPR GPx-1 may affect a common mediator of the UPR oreach branch individually Dissociation of GRP78BiP uponER stress is required for all three branches of the UPR GPx-1expression directly regulated the gene expression of GRP78However whetherGPx-1 impacts onGRP78dissociation dur-ing ER stress is unknown Nrf2 interacts directly with PERK[47] and may play a major role in GPx-1 expression therebyregulating the UPR The ER stress inducer thapsigargininduces Nrf2 protein production in 16-HBE cells [47] whichsuggests that the UPR induces a Nrf2 response to reverseER stress We observe a similar effect on GPx-1 expressionin PCLS following tunicamycin treatment Equally XBP1regulates several antioxidants including catalase SOD1 andthioredoxinTRX1 [48]However XBP1 does not regulateGPxproteins [48] but XBP1 expression is regulated by GPx-1 The
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
Figure 1 NHBE cells isolated from COPD donors have enhanced ER stress responses compared to nonsmokers and smokers (a) Fullydifferentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) or cigarette smoke (CS) from 4 cigarettes everysecond day (3 exposures) using a Vitrocell VC-10 smoking robot LDH release into media and IL-6 gene expression were examined (b)Gene expression of ATF4 XBP1 GRP78 GRP94 EDEM1 and CHOP was examined (c) Fully differentiated NHBE cells from nonsmoker(NS) smoker (SM) and COPD (COPD) individuals (119899 = 6 donors per group) were examined for gene expression of ATF4 XBP1 GRP78GRP94 EDEM1 and CHOP Dot plots are represented as relative quantification (RQ) compared to ACTB expression and shown as the meanplusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
6 Mediators of Inflammation
CHOP
42120573-Actin
27
Cell source
EDEM
49 ATF4
74
GRP94
78 GRP78
100
XBP1
110 IRE1120572
60
p-elF2120572
38 elF2120572
38
p-PERK
170 PERK
170
120573-Actin 120573-Actin
289
263
(kDa)
(bp)
XBP1u
XBP1s
42
(kDa)
(a)
36 ATF6
COPDSMNS1 2 3 4 5 6
Cell source
COPDSMNS
1 2 3 4 5 6
Cell source
COPDSMNS
1 2 3 4 5 6
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
COPDSMN
S
lowast
lowast
lowastlowast
lowastlowast lowast
lowast
lowastlowast
lowastlowast lowast
lowast
lowastlowast
lowastlowast
0
1
2
3
4
p-PE
RK D
U
0
2
4
6
p-el
F2120572
DU
0
1
2
3
ATF6
DU
0
1
2
3
ATF4
DU
00
05
10
15
20
25
IRE1
120572 D
U00
05
10
15
20
25
GRP
94 D
U
0
1
2
3
GRP
78 D
U
00
05
10
15
20
25
EDEM
DU
0
5
10
15
CHO
P D
U
Figure 2 Continued
Mediators of Inflammation 7
XBP1
DU
XBP1
splic
ing
()
COPDSM
(b)N
S
COPDSMN
S
lowastlowast
lowastlowast
0
20
40
60
80
0
1
2
3
Figure 2 NHBE cells isolated fromCOPDdonors have enhanced ER stress protein responses compared to nonsmokers and smokers Proteinwas collected from fully differentiated NHBE cells from nonsmokers (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors pergroup) (a) Protein expression of ATF6 ATF4 GRP94 GRP78 EDEM CHOP and 120573-actin was examined by immunoblots Phosphorylationof elF2 and PERK was also determined (b) XBP1 splicing was examined on XBP1 amplified cDNA from NHBE cells from nonsmoker (NS)smokers (S) and COPD (COPD) individuals Protein expression of XBP1 and 120573-actin was examined by immunoblots (a)-(b) For each blotor gel every lane represents an individual cell donor Densitometry analysis was performed of XBP1s from DNA gels and other targets byanalyzing immunoblots XBP1 slicing was scored as percent of XBP1s of total XBP1 Dot plots are represented as densitometry units (DU) ofpixel intensity expressed as a ratio to120573-actin or total elF2 and PERKData are shown asmeanplusmn SEMwhere eachmeasurement was performedon 3 independent days on 6 donorsgroup lowast denotes 119901 value lt 005 when comparing both treatments connected by a line determined by2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
enhanced the UPR in animals also infected with RSVcompared to infected animals exposed to room air (RA)(Figure 4(d)) Therefore the lungs of smokers and COPDsubjects are likely to be more sensitive to viral infectioninduced ER stress which may impact disease progression
34 Gpx-1minusminus Mice Have Heightened ER Stress and Apoptosisfollowing Cigarette Smoke Exposure To determine whetherthe loss of GPx-1 expression directly influences ER stressfollowing inhalation of cigarette smoke in vivo we examinedER stress markers in Gpx-1minusminus mice and their wild type litter-mates exposed to cigarette smoke for 1 year We previouslydemonstrated that loss of Gpx-1 expression in mice resultsin enhanced air space enlargement and inflammation fol-lowing long-term cigarette smoke exposure [13] Expressionlevels of Atf4 Xbp1 Grp78 Grp94 Edem1 and Chop wereexamined in Gpx-1minusminus and wild type mice Long-term expo-sure to cigarette smoke did not significantly enhance ERstress marker expression in the lungs of wild type mice(Figure 5(a))We previously observed similar findings in wildtype mice [5] However Gpx-1minusminus mice exposed to cigarettesmoke had enhanced gene expression of Atf4 Xbp1 Grp78Grp94 Edem1 and Chop (Figure 5(a)) Equally loss of Gpx-1 expression resulted in elevated lung tissue protein levels ofATF4 XBP1 GRP78 GRP94 EDEM and CHOP followingsmoke exposure (Figure 5(b)) Densitometry analysis con-firmed significant increases in lung levels of ATF4 XBP1GRP78GRP94 EDEM andCHOP inGpx-1minusminusmice exposedto cigarette smoke (Figure 5(b))
Prolonged activation of CHOP by ER stress can result incellular apoptosis Increased structural and immune cellapoptosis is also observed in COPD lungs [27]Therefore weexamined whetherGpx-1minusminus mice exposed to cigarette smokehad elevated apoptosis Gpx-1minusminus mice exposed to cigarettesmoke had enhanced lung cell apoptosis observed byTUNEL caspase-3 cleavage and lactate dehydrogenase(LDH) release assays (Figure 6) Gpx-1minusminus mice exposed tocigarette smoke exhibited the highest frequency of TUNELpositive cells (Figure 6(a)) Enhanced caspase-3 cleavage wasobserved in Gpx-1minusminus mice exposed to cigarette smoke (Fig-ure 6(b)) Additionally elevated levels of LDHwere observedin the BALF of Gpx-1minusminus mice exposed to cigarette smokecompared to the other groups (Figure 6(c)) which indicatesenhanced cell membrane damage in the lung Thereforeenhanced apoptosis in the lungs could contribute to lungremodelling and failure to clear apoptotic cells could con-tribute to lung inflammation
35 Triggering the UPR Enhances GPx-1 GPx-2 and GPx-4Expression in Mouse Precision-Cut Lung Slices In Gpx-1 andGpx-2 double knockout mice apoptotic cells are increased inileal crypts [28] suggesting that GPx proteins regulate apop-tosis However the effect of ER stress on GPx proteins hasnot been directly investigated To determine the effect of ERstress on GPx-1 expression mouse precision-cut lung slices(PCLS) were exposed to the ER stress inducer tunicamycinfor 24 hoursThe concentration of tunicamycin tested did notinduce LDH release from the PCLS (Figure 7(a)) indicating
8 Mediators of Inflammation
GPx-
1 RQ
00
05
10
15
20
CSRAExposure
(a)
GPx-
1 RQ
lowastlowast
00
05
10
15
20
Cell sourceCO
PDSMNS
(b)
GPx-1
42
22
GPx
-1 D
U
lowastlowast
Cell source
COPDSMN
S
120573-Actin
00
05
10
15
20
Cell sourceCOPDSMNS
1 2 3 4 5 6
(c)
lowast
Albumin GPx-1Protein transfected
NHBE COPD cells
00
05
10
15
20
CHO
P RQ
(d)
lowastlowast
GPx-1
42
22
NHBE COPD cells
CHOP27
Albumin GPx-1Protein transfected
Albumin GPx-1Protein transfected
NHBE COPD cells
120573-Actin
Albumin GPx-1Protein transfected
1 2 3 4 5 6
0
1
2
3
4
5
GPx
-1 D
U
00
05
10
15
CHO
P D
U(e)
Figure 3 Reintroducing GPx-1 into NHBE cells isolated fromCOPD donors subdues the UPR (a) Gene expression ofGPx-1was determinedin fully differentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) and cigarette smoke (CS from 4 cigarettesevery second day (3 exposures)) using a Vitrocell VC-10 smoking robot (b) RNA and (c) protein was analyzed for GPx-1 expression fromfully differentiated NHBE cells from nonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) (d) NHBEcells isolated from COPD subjects were transfected with albumin or GPx-1 protein and CHOP expression was determined by qPCR (e)Immunoblots and corresponding densitometry analysis for CHOP Gpx-1 and 120573-actin from NHBE cells from COPD subjects followingalbumin or GPx-1 protein transfection In each immunoblot every lane represents an individual cell donor Data are shown as mean plusmn SEMwhere each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
no induction of apoptosis Tunicamycin induced all threebranches of the UPR and CHOP expression was observedin PCLS (Figures 7(b)-7(c)) Tunicamycin enhanced gene(Figure 7(b)) and protein (Figure 7(c)) expression of ATF4XBP1 ATF6 and CHOP in PCLS Interestingly tunicamycininduced GPx-1 GPx-2 and GPx-4 gene (Figure 7(d)) andprotein (Figure 7(e)) expression in PCLSTherefore an acuteER stress induces the expression of antioxidants to counterfurther oxidant and ER stress
4 Discussion
Cigarette smoking is the most relevant environmental riskfactor associated with the development of COPD Howeversmoke inhalation studies are problematic as long-term smoke
exposure is required to trigger disease formation in animalmodels and a secondary event may be required to mimic thehuman disease-state Here we observed that the loss of GPx-1 expression enhances cigarette smoke-induced ER stressGPx-1 regulates the UPR following smoke exposure and wefound that the expression of GPx-1 itself was triggered by anacute ER stress stimulus NHBE cells isolated from COPDdonors expressed significantly less GPx-1 which coincideswith elevated UPR Reintroducing GPx-1 protein into NHBEcells isolated from COPD donors reduced the UPR RSVinfection contributes to loss of lung GPx-1 expression whichis exaggerated in lungs exposed to smoke and coincides withelevated UPR Gpx-1minusminus mice exhibited greater UPR andsubsequent enhanced apoptosis following long-term cigarettesmoke exposure Interestingly triggering an acute ER stress in
Mediators of Inflammation 9
Cell source
NS SM COPD
MockRSV
Cell source
NS SM COPD
MockRSV
NHBE cells
00
05
10
15
20GP
x-1
RQ
0
2
4
6
8
10
CHO
P RQ
lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)
CHOP
42 120573-Actin
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
NHBE cells
minus + minus + minus + RSV infection
Cell sourceCOPDSMNS
(b)
Mouse lungs
RA + mockRA + RSV
Smoke + mockSmoke + RSV
RA + mockRA + RSV
Smoke + mockSmoke + RSV
00
05
10
15
GPx-
1 RQ
0
5
10
Chop
RQ
1 3 5 7 90(dpi)
1 3 5 7 90(dpi)
lowast
lowast
lowast
lowast
lowast
(c)
120573-Actin
CHOP
Room air Smoke
42
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
Mouse lungs
9dpi
(d)
Figure 4 RSV infection enhances the UPR in the lung (a) GPx-1 and CHOP gene expression were determined in NHBE cells isolated fromnonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) infected with mock or RSV and analyzed byqPCR (b) Protein expression of GPx-1 ATF6 ATF4 CHOP and 120573-actin was examined by immunoblots (c) Wild type mice were exposedto cigarette smoke or room air for six months and subsequently infected with 1 times 106 pfu of RSV Animals were euthanized at 0 1 3 5 7 and9 days after infection (dpi) and Gpx-1 and Chop expression were determined by qPCR (d) Protein expression of GPx-1 ATF6 ATF4 CHOPand 120573-actin was examined by immunoblots Data are shown as mean plusmn SEM where each measurement was performed on 3 independentdays lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line or the same infection day determined by Studentrsquos119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lungs of mice induces a potent antioxidant responseThisantioxidant response is diminished in the COPD lungs [29]which may explain the heightened UPR observed in NHBEcells isolated from COPD subjects The exact role of thisheightened UPR on the progression of COPD still remainsto be fully determined However we have established thatloss of GPx-1 in vivo leads to a marked increased in all threebranches of the UPR (see Figure 8 for proposed signalingscheme) and this coincides with enhanced apoptosis and lungtissue destruction in mice [13] Our results suggest that GPx-1 significantly regulates the UPR in COPD and enhancing
GPx-1 expression may be feasible means of offsetting theUPR and lung injury responses that drive the onset andprogression of this disease
Multiple studies utilizing the Gpx-1minusminus and transgenicmice demonstrated the protective role of GPx-1 in counteringoxidative injury and cell death mediated by ROS [13 30]GPX-1 activity also affects protein kinase phosphorylation[31] and oxidant-mediated activation of NF-120581B [32] In thiscurrent study GPx-1 was significantly reduced in NHBE cellsisolated from COPD subjects compared to nonsmokers andsmokers Others have reported that the alteration of GPx-1
Figure 5 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) Lung gene expression of Chop Atf4 Edem1 Grp78 Grp94 and Xbp1 was examined (b)Immunoblots were performed of whole lung protein for CHOP ATF4 EDEM GRP78 GRP94 and XBP1 Dot plots are represented as (a)relative quantification (RQ) compared to ACTB expression or (b) densitometry units (DU) of pixel intensity expressed as a ratio to 120573-actinEvery lane represents an individual mouse Data are shown as mean plusmn SEM where each measurement was performed on 3 independent dayson 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line determined by 2-way ANOVA withTukeyrsquos post hoc test (gt2 groups)
expression does not affect the mRNA or activity expressionof other selenoproteins [33] which suggests no compensationexpression of other selenoproteins following loss of GPx-1 expression Currently the mechanism by which cigarettesmoke regulates GPx-1 expression is not fully elucidated butconsidering our GPx-1 reintroduction data further analysis
of GPx-1 regulation is critical GPx-1 expression and activityhave been reported to be regulated by Nrf2 [34] the tran-scription factor TFAP2C [35] CpG methylation of the GPx-1promoter [35] Bcr-AblmTOR [36] selenium [37] estrogen[38] adenosine [39] Sec-insertion sequence (SECIS) factors[40] EGFR [41] and homocysteine [42] Specifically within
Mediators of Inflammation 11
Room airCigarette smoke
Wildtype
lowastlowast
00
02
04
06
08
10
TUN
EL p
ositi
ve (
)
Gpx-1minusminus
(a)
Caspase-3
42
19
Wild type
35 Pro
Cleaved
RA SM RA SM1 2 3 4 5 6 7 8
120573-Actin
Gpx-1minusminus
(b)
Wild type
lowastlowast lowast
lowastlowast
00
01
02
03
04LD
H re
leas
e (ab
s 350
nm)
0
2
4
6
8
10
Clea
ved
casp
ase D
U
Wildtype
Gpx-1minusminus Gpx-1minusminus
(c)
Figure 6 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) TUNEL analysis was performed on lung tissue from each mouse group (b) Enhancedlung tissue caspase-3 cleavage coincided with (c) elevated LDH into BALF ofGpx-1minusminus exposed to cigarette smoke Every lane in (b) representsan individual mouse and densitometry units (DU) of pixel intensity expressed as a ratio to total caspase-3 levels Dot plots are representedas mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 whencomparing both treatments connected by a line determined by 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lung during smoke exposure Singh et al show elevatedGPx-1 expression in the lungs following one-month cigarettesmoke exposure that was regulated by Nrf2 [34] HoweverNrf2 expression is lost in COPD subjects suggesting that thissecondary event could result in reduced GPx-1 expressionand heightened ER stress Loss of Nrf2 in mice resultsin enhanced susceptibility to cigarette smoke [43 44] andelastase [45] induced emphysema in mice However genomicstudies in Nrf2minusminus mouse samples suggest that Nrf2 mayregulate other GPx genes but not GPx-1 [46] Whether RSVinfection alters regulation of GPx-1 expression in a similarmanner to chronic smoke is unknown Interestingly theUPR upon RSV infection can counter viral proliferation [26]Further studies on the regulation of GPx-1 in smoke exposureand COPD and the significance of ER stress in the lungs arerequired This will be a major area for our future work
Since GPx-1 expression regulates all three branches of theUPR GPx-1 may affect a common mediator of the UPR oreach branch individually Dissociation of GRP78BiP uponER stress is required for all three branches of the UPR GPx-1expression directly regulated the gene expression of GRP78However whetherGPx-1 impacts onGRP78dissociation dur-ing ER stress is unknown Nrf2 interacts directly with PERK[47] and may play a major role in GPx-1 expression therebyregulating the UPR The ER stress inducer thapsigargininduces Nrf2 protein production in 16-HBE cells [47] whichsuggests that the UPR induces a Nrf2 response to reverseER stress We observe a similar effect on GPx-1 expressionin PCLS following tunicamycin treatment Equally XBP1regulates several antioxidants including catalase SOD1 andthioredoxinTRX1 [48]However XBP1 does not regulateGPxproteins [48] but XBP1 expression is regulated by GPx-1 The
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
Figure 2 NHBE cells isolated fromCOPDdonors have enhanced ER stress protein responses compared to nonsmokers and smokers Proteinwas collected from fully differentiated NHBE cells from nonsmokers (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors pergroup) (a) Protein expression of ATF6 ATF4 GRP94 GRP78 EDEM CHOP and 120573-actin was examined by immunoblots Phosphorylationof elF2 and PERK was also determined (b) XBP1 splicing was examined on XBP1 amplified cDNA from NHBE cells from nonsmoker (NS)smokers (S) and COPD (COPD) individuals Protein expression of XBP1 and 120573-actin was examined by immunoblots (a)-(b) For each blotor gel every lane represents an individual cell donor Densitometry analysis was performed of XBP1s from DNA gels and other targets byanalyzing immunoblots XBP1 slicing was scored as percent of XBP1s of total XBP1 Dot plots are represented as densitometry units (DU) ofpixel intensity expressed as a ratio to120573-actin or total elF2 and PERKData are shown asmeanplusmn SEMwhere eachmeasurement was performedon 3 independent days on 6 donorsgroup lowast denotes 119901 value lt 005 when comparing both treatments connected by a line determined by2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
enhanced the UPR in animals also infected with RSVcompared to infected animals exposed to room air (RA)(Figure 4(d)) Therefore the lungs of smokers and COPDsubjects are likely to be more sensitive to viral infectioninduced ER stress which may impact disease progression
34 Gpx-1minusminus Mice Have Heightened ER Stress and Apoptosisfollowing Cigarette Smoke Exposure To determine whetherthe loss of GPx-1 expression directly influences ER stressfollowing inhalation of cigarette smoke in vivo we examinedER stress markers in Gpx-1minusminus mice and their wild type litter-mates exposed to cigarette smoke for 1 year We previouslydemonstrated that loss of Gpx-1 expression in mice resultsin enhanced air space enlargement and inflammation fol-lowing long-term cigarette smoke exposure [13] Expressionlevels of Atf4 Xbp1 Grp78 Grp94 Edem1 and Chop wereexamined in Gpx-1minusminus and wild type mice Long-term expo-sure to cigarette smoke did not significantly enhance ERstress marker expression in the lungs of wild type mice(Figure 5(a))We previously observed similar findings in wildtype mice [5] However Gpx-1minusminus mice exposed to cigarettesmoke had enhanced gene expression of Atf4 Xbp1 Grp78Grp94 Edem1 and Chop (Figure 5(a)) Equally loss of Gpx-1 expression resulted in elevated lung tissue protein levels ofATF4 XBP1 GRP78 GRP94 EDEM and CHOP followingsmoke exposure (Figure 5(b)) Densitometry analysis con-firmed significant increases in lung levels of ATF4 XBP1GRP78GRP94 EDEM andCHOP inGpx-1minusminusmice exposedto cigarette smoke (Figure 5(b))
Prolonged activation of CHOP by ER stress can result incellular apoptosis Increased structural and immune cellapoptosis is also observed in COPD lungs [27]Therefore weexamined whetherGpx-1minusminus mice exposed to cigarette smokehad elevated apoptosis Gpx-1minusminus mice exposed to cigarettesmoke had enhanced lung cell apoptosis observed byTUNEL caspase-3 cleavage and lactate dehydrogenase(LDH) release assays (Figure 6) Gpx-1minusminus mice exposed tocigarette smoke exhibited the highest frequency of TUNELpositive cells (Figure 6(a)) Enhanced caspase-3 cleavage wasobserved in Gpx-1minusminus mice exposed to cigarette smoke (Fig-ure 6(b)) Additionally elevated levels of LDHwere observedin the BALF of Gpx-1minusminus mice exposed to cigarette smokecompared to the other groups (Figure 6(c)) which indicatesenhanced cell membrane damage in the lung Thereforeenhanced apoptosis in the lungs could contribute to lungremodelling and failure to clear apoptotic cells could con-tribute to lung inflammation
35 Triggering the UPR Enhances GPx-1 GPx-2 and GPx-4Expression in Mouse Precision-Cut Lung Slices In Gpx-1 andGpx-2 double knockout mice apoptotic cells are increased inileal crypts [28] suggesting that GPx proteins regulate apop-tosis However the effect of ER stress on GPx proteins hasnot been directly investigated To determine the effect of ERstress on GPx-1 expression mouse precision-cut lung slices(PCLS) were exposed to the ER stress inducer tunicamycinfor 24 hoursThe concentration of tunicamycin tested did notinduce LDH release from the PCLS (Figure 7(a)) indicating
8 Mediators of Inflammation
GPx-
1 RQ
00
05
10
15
20
CSRAExposure
(a)
GPx-
1 RQ
lowastlowast
00
05
10
15
20
Cell sourceCO
PDSMNS
(b)
GPx-1
42
22
GPx
-1 D
U
lowastlowast
Cell source
COPDSMN
S
120573-Actin
00
05
10
15
20
Cell sourceCOPDSMNS
1 2 3 4 5 6
(c)
lowast
Albumin GPx-1Protein transfected
NHBE COPD cells
00
05
10
15
20
CHO
P RQ
(d)
lowastlowast
GPx-1
42
22
NHBE COPD cells
CHOP27
Albumin GPx-1Protein transfected
Albumin GPx-1Protein transfected
NHBE COPD cells
120573-Actin
Albumin GPx-1Protein transfected
1 2 3 4 5 6
0
1
2
3
4
5
GPx
-1 D
U
00
05
10
15
CHO
P D
U(e)
Figure 3 Reintroducing GPx-1 into NHBE cells isolated fromCOPD donors subdues the UPR (a) Gene expression ofGPx-1was determinedin fully differentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) and cigarette smoke (CS from 4 cigarettesevery second day (3 exposures)) using a Vitrocell VC-10 smoking robot (b) RNA and (c) protein was analyzed for GPx-1 expression fromfully differentiated NHBE cells from nonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) (d) NHBEcells isolated from COPD subjects were transfected with albumin or GPx-1 protein and CHOP expression was determined by qPCR (e)Immunoblots and corresponding densitometry analysis for CHOP Gpx-1 and 120573-actin from NHBE cells from COPD subjects followingalbumin or GPx-1 protein transfection In each immunoblot every lane represents an individual cell donor Data are shown as mean plusmn SEMwhere each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
no induction of apoptosis Tunicamycin induced all threebranches of the UPR and CHOP expression was observedin PCLS (Figures 7(b)-7(c)) Tunicamycin enhanced gene(Figure 7(b)) and protein (Figure 7(c)) expression of ATF4XBP1 ATF6 and CHOP in PCLS Interestingly tunicamycininduced GPx-1 GPx-2 and GPx-4 gene (Figure 7(d)) andprotein (Figure 7(e)) expression in PCLSTherefore an acuteER stress induces the expression of antioxidants to counterfurther oxidant and ER stress
4 Discussion
Cigarette smoking is the most relevant environmental riskfactor associated with the development of COPD Howeversmoke inhalation studies are problematic as long-term smoke
exposure is required to trigger disease formation in animalmodels and a secondary event may be required to mimic thehuman disease-state Here we observed that the loss of GPx-1 expression enhances cigarette smoke-induced ER stressGPx-1 regulates the UPR following smoke exposure and wefound that the expression of GPx-1 itself was triggered by anacute ER stress stimulus NHBE cells isolated from COPDdonors expressed significantly less GPx-1 which coincideswith elevated UPR Reintroducing GPx-1 protein into NHBEcells isolated from COPD donors reduced the UPR RSVinfection contributes to loss of lung GPx-1 expression whichis exaggerated in lungs exposed to smoke and coincides withelevated UPR Gpx-1minusminus mice exhibited greater UPR andsubsequent enhanced apoptosis following long-term cigarettesmoke exposure Interestingly triggering an acute ER stress in
Mediators of Inflammation 9
Cell source
NS SM COPD
MockRSV
Cell source
NS SM COPD
MockRSV
NHBE cells
00
05
10
15
20GP
x-1
RQ
0
2
4
6
8
10
CHO
P RQ
lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)
CHOP
42 120573-Actin
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
NHBE cells
minus + minus + minus + RSV infection
Cell sourceCOPDSMNS
(b)
Mouse lungs
RA + mockRA + RSV
Smoke + mockSmoke + RSV
RA + mockRA + RSV
Smoke + mockSmoke + RSV
00
05
10
15
GPx-
1 RQ
0
5
10
Chop
RQ
1 3 5 7 90(dpi)
1 3 5 7 90(dpi)
lowast
lowast
lowast
lowast
lowast
(c)
120573-Actin
CHOP
Room air Smoke
42
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
Mouse lungs
9dpi
(d)
Figure 4 RSV infection enhances the UPR in the lung (a) GPx-1 and CHOP gene expression were determined in NHBE cells isolated fromnonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) infected with mock or RSV and analyzed byqPCR (b) Protein expression of GPx-1 ATF6 ATF4 CHOP and 120573-actin was examined by immunoblots (c) Wild type mice were exposedto cigarette smoke or room air for six months and subsequently infected with 1 times 106 pfu of RSV Animals were euthanized at 0 1 3 5 7 and9 days after infection (dpi) and Gpx-1 and Chop expression were determined by qPCR (d) Protein expression of GPx-1 ATF6 ATF4 CHOPand 120573-actin was examined by immunoblots Data are shown as mean plusmn SEM where each measurement was performed on 3 independentdays lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line or the same infection day determined by Studentrsquos119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lungs of mice induces a potent antioxidant responseThisantioxidant response is diminished in the COPD lungs [29]which may explain the heightened UPR observed in NHBEcells isolated from COPD subjects The exact role of thisheightened UPR on the progression of COPD still remainsto be fully determined However we have established thatloss of GPx-1 in vivo leads to a marked increased in all threebranches of the UPR (see Figure 8 for proposed signalingscheme) and this coincides with enhanced apoptosis and lungtissue destruction in mice [13] Our results suggest that GPx-1 significantly regulates the UPR in COPD and enhancing
GPx-1 expression may be feasible means of offsetting theUPR and lung injury responses that drive the onset andprogression of this disease
Multiple studies utilizing the Gpx-1minusminus and transgenicmice demonstrated the protective role of GPx-1 in counteringoxidative injury and cell death mediated by ROS [13 30]GPX-1 activity also affects protein kinase phosphorylation[31] and oxidant-mediated activation of NF-120581B [32] In thiscurrent study GPx-1 was significantly reduced in NHBE cellsisolated from COPD subjects compared to nonsmokers andsmokers Others have reported that the alteration of GPx-1
Figure 5 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) Lung gene expression of Chop Atf4 Edem1 Grp78 Grp94 and Xbp1 was examined (b)Immunoblots were performed of whole lung protein for CHOP ATF4 EDEM GRP78 GRP94 and XBP1 Dot plots are represented as (a)relative quantification (RQ) compared to ACTB expression or (b) densitometry units (DU) of pixel intensity expressed as a ratio to 120573-actinEvery lane represents an individual mouse Data are shown as mean plusmn SEM where each measurement was performed on 3 independent dayson 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line determined by 2-way ANOVA withTukeyrsquos post hoc test (gt2 groups)
expression does not affect the mRNA or activity expressionof other selenoproteins [33] which suggests no compensationexpression of other selenoproteins following loss of GPx-1 expression Currently the mechanism by which cigarettesmoke regulates GPx-1 expression is not fully elucidated butconsidering our GPx-1 reintroduction data further analysis
of GPx-1 regulation is critical GPx-1 expression and activityhave been reported to be regulated by Nrf2 [34] the tran-scription factor TFAP2C [35] CpG methylation of the GPx-1promoter [35] Bcr-AblmTOR [36] selenium [37] estrogen[38] adenosine [39] Sec-insertion sequence (SECIS) factors[40] EGFR [41] and homocysteine [42] Specifically within
Mediators of Inflammation 11
Room airCigarette smoke
Wildtype
lowastlowast
00
02
04
06
08
10
TUN
EL p
ositi
ve (
)
Gpx-1minusminus
(a)
Caspase-3
42
19
Wild type
35 Pro
Cleaved
RA SM RA SM1 2 3 4 5 6 7 8
120573-Actin
Gpx-1minusminus
(b)
Wild type
lowastlowast lowast
lowastlowast
00
01
02
03
04LD
H re
leas
e (ab
s 350
nm)
0
2
4
6
8
10
Clea
ved
casp
ase D
U
Wildtype
Gpx-1minusminus Gpx-1minusminus
(c)
Figure 6 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) TUNEL analysis was performed on lung tissue from each mouse group (b) Enhancedlung tissue caspase-3 cleavage coincided with (c) elevated LDH into BALF ofGpx-1minusminus exposed to cigarette smoke Every lane in (b) representsan individual mouse and densitometry units (DU) of pixel intensity expressed as a ratio to total caspase-3 levels Dot plots are representedas mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 whencomparing both treatments connected by a line determined by 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lung during smoke exposure Singh et al show elevatedGPx-1 expression in the lungs following one-month cigarettesmoke exposure that was regulated by Nrf2 [34] HoweverNrf2 expression is lost in COPD subjects suggesting that thissecondary event could result in reduced GPx-1 expressionand heightened ER stress Loss of Nrf2 in mice resultsin enhanced susceptibility to cigarette smoke [43 44] andelastase [45] induced emphysema in mice However genomicstudies in Nrf2minusminus mouse samples suggest that Nrf2 mayregulate other GPx genes but not GPx-1 [46] Whether RSVinfection alters regulation of GPx-1 expression in a similarmanner to chronic smoke is unknown Interestingly theUPR upon RSV infection can counter viral proliferation [26]Further studies on the regulation of GPx-1 in smoke exposureand COPD and the significance of ER stress in the lungs arerequired This will be a major area for our future work
Since GPx-1 expression regulates all three branches of theUPR GPx-1 may affect a common mediator of the UPR oreach branch individually Dissociation of GRP78BiP uponER stress is required for all three branches of the UPR GPx-1expression directly regulated the gene expression of GRP78However whetherGPx-1 impacts onGRP78dissociation dur-ing ER stress is unknown Nrf2 interacts directly with PERK[47] and may play a major role in GPx-1 expression therebyregulating the UPR The ER stress inducer thapsigargininduces Nrf2 protein production in 16-HBE cells [47] whichsuggests that the UPR induces a Nrf2 response to reverseER stress We observe a similar effect on GPx-1 expressionin PCLS following tunicamycin treatment Equally XBP1regulates several antioxidants including catalase SOD1 andthioredoxinTRX1 [48]However XBP1 does not regulateGPxproteins [48] but XBP1 expression is regulated by GPx-1 The
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
Figure 2 NHBE cells isolated fromCOPDdonors have enhanced ER stress protein responses compared to nonsmokers and smokers Proteinwas collected from fully differentiated NHBE cells from nonsmokers (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors pergroup) (a) Protein expression of ATF6 ATF4 GRP94 GRP78 EDEM CHOP and 120573-actin was examined by immunoblots Phosphorylationof elF2 and PERK was also determined (b) XBP1 splicing was examined on XBP1 amplified cDNA from NHBE cells from nonsmoker (NS)smokers (S) and COPD (COPD) individuals Protein expression of XBP1 and 120573-actin was examined by immunoblots (a)-(b) For each blotor gel every lane represents an individual cell donor Densitometry analysis was performed of XBP1s from DNA gels and other targets byanalyzing immunoblots XBP1 slicing was scored as percent of XBP1s of total XBP1 Dot plots are represented as densitometry units (DU) ofpixel intensity expressed as a ratio to120573-actin or total elF2 and PERKData are shown asmeanplusmn SEMwhere eachmeasurement was performedon 3 independent days on 6 donorsgroup lowast denotes 119901 value lt 005 when comparing both treatments connected by a line determined by2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
enhanced the UPR in animals also infected with RSVcompared to infected animals exposed to room air (RA)(Figure 4(d)) Therefore the lungs of smokers and COPDsubjects are likely to be more sensitive to viral infectioninduced ER stress which may impact disease progression
34 Gpx-1minusminus Mice Have Heightened ER Stress and Apoptosisfollowing Cigarette Smoke Exposure To determine whetherthe loss of GPx-1 expression directly influences ER stressfollowing inhalation of cigarette smoke in vivo we examinedER stress markers in Gpx-1minusminus mice and their wild type litter-mates exposed to cigarette smoke for 1 year We previouslydemonstrated that loss of Gpx-1 expression in mice resultsin enhanced air space enlargement and inflammation fol-lowing long-term cigarette smoke exposure [13] Expressionlevels of Atf4 Xbp1 Grp78 Grp94 Edem1 and Chop wereexamined in Gpx-1minusminus and wild type mice Long-term expo-sure to cigarette smoke did not significantly enhance ERstress marker expression in the lungs of wild type mice(Figure 5(a))We previously observed similar findings in wildtype mice [5] However Gpx-1minusminus mice exposed to cigarettesmoke had enhanced gene expression of Atf4 Xbp1 Grp78Grp94 Edem1 and Chop (Figure 5(a)) Equally loss of Gpx-1 expression resulted in elevated lung tissue protein levels ofATF4 XBP1 GRP78 GRP94 EDEM and CHOP followingsmoke exposure (Figure 5(b)) Densitometry analysis con-firmed significant increases in lung levels of ATF4 XBP1GRP78GRP94 EDEM andCHOP inGpx-1minusminusmice exposedto cigarette smoke (Figure 5(b))
Prolonged activation of CHOP by ER stress can result incellular apoptosis Increased structural and immune cellapoptosis is also observed in COPD lungs [27]Therefore weexamined whetherGpx-1minusminus mice exposed to cigarette smokehad elevated apoptosis Gpx-1minusminus mice exposed to cigarettesmoke had enhanced lung cell apoptosis observed byTUNEL caspase-3 cleavage and lactate dehydrogenase(LDH) release assays (Figure 6) Gpx-1minusminus mice exposed tocigarette smoke exhibited the highest frequency of TUNELpositive cells (Figure 6(a)) Enhanced caspase-3 cleavage wasobserved in Gpx-1minusminus mice exposed to cigarette smoke (Fig-ure 6(b)) Additionally elevated levels of LDHwere observedin the BALF of Gpx-1minusminus mice exposed to cigarette smokecompared to the other groups (Figure 6(c)) which indicatesenhanced cell membrane damage in the lung Thereforeenhanced apoptosis in the lungs could contribute to lungremodelling and failure to clear apoptotic cells could con-tribute to lung inflammation
35 Triggering the UPR Enhances GPx-1 GPx-2 and GPx-4Expression in Mouse Precision-Cut Lung Slices In Gpx-1 andGpx-2 double knockout mice apoptotic cells are increased inileal crypts [28] suggesting that GPx proteins regulate apop-tosis However the effect of ER stress on GPx proteins hasnot been directly investigated To determine the effect of ERstress on GPx-1 expression mouse precision-cut lung slices(PCLS) were exposed to the ER stress inducer tunicamycinfor 24 hoursThe concentration of tunicamycin tested did notinduce LDH release from the PCLS (Figure 7(a)) indicating
8 Mediators of Inflammation
GPx-
1 RQ
00
05
10
15
20
CSRAExposure
(a)
GPx-
1 RQ
lowastlowast
00
05
10
15
20
Cell sourceCO
PDSMNS
(b)
GPx-1
42
22
GPx
-1 D
U
lowastlowast
Cell source
COPDSMN
S
120573-Actin
00
05
10
15
20
Cell sourceCOPDSMNS
1 2 3 4 5 6
(c)
lowast
Albumin GPx-1Protein transfected
NHBE COPD cells
00
05
10
15
20
CHO
P RQ
(d)
lowastlowast
GPx-1
42
22
NHBE COPD cells
CHOP27
Albumin GPx-1Protein transfected
Albumin GPx-1Protein transfected
NHBE COPD cells
120573-Actin
Albumin GPx-1Protein transfected
1 2 3 4 5 6
0
1
2
3
4
5
GPx
-1 D
U
00
05
10
15
CHO
P D
U(e)
Figure 3 Reintroducing GPx-1 into NHBE cells isolated fromCOPD donors subdues the UPR (a) Gene expression ofGPx-1was determinedin fully differentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) and cigarette smoke (CS from 4 cigarettesevery second day (3 exposures)) using a Vitrocell VC-10 smoking robot (b) RNA and (c) protein was analyzed for GPx-1 expression fromfully differentiated NHBE cells from nonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) (d) NHBEcells isolated from COPD subjects were transfected with albumin or GPx-1 protein and CHOP expression was determined by qPCR (e)Immunoblots and corresponding densitometry analysis for CHOP Gpx-1 and 120573-actin from NHBE cells from COPD subjects followingalbumin or GPx-1 protein transfection In each immunoblot every lane represents an individual cell donor Data are shown as mean plusmn SEMwhere each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
no induction of apoptosis Tunicamycin induced all threebranches of the UPR and CHOP expression was observedin PCLS (Figures 7(b)-7(c)) Tunicamycin enhanced gene(Figure 7(b)) and protein (Figure 7(c)) expression of ATF4XBP1 ATF6 and CHOP in PCLS Interestingly tunicamycininduced GPx-1 GPx-2 and GPx-4 gene (Figure 7(d)) andprotein (Figure 7(e)) expression in PCLSTherefore an acuteER stress induces the expression of antioxidants to counterfurther oxidant and ER stress
4 Discussion
Cigarette smoking is the most relevant environmental riskfactor associated with the development of COPD Howeversmoke inhalation studies are problematic as long-term smoke
exposure is required to trigger disease formation in animalmodels and a secondary event may be required to mimic thehuman disease-state Here we observed that the loss of GPx-1 expression enhances cigarette smoke-induced ER stressGPx-1 regulates the UPR following smoke exposure and wefound that the expression of GPx-1 itself was triggered by anacute ER stress stimulus NHBE cells isolated from COPDdonors expressed significantly less GPx-1 which coincideswith elevated UPR Reintroducing GPx-1 protein into NHBEcells isolated from COPD donors reduced the UPR RSVinfection contributes to loss of lung GPx-1 expression whichis exaggerated in lungs exposed to smoke and coincides withelevated UPR Gpx-1minusminus mice exhibited greater UPR andsubsequent enhanced apoptosis following long-term cigarettesmoke exposure Interestingly triggering an acute ER stress in
Mediators of Inflammation 9
Cell source
NS SM COPD
MockRSV
Cell source
NS SM COPD
MockRSV
NHBE cells
00
05
10
15
20GP
x-1
RQ
0
2
4
6
8
10
CHO
P RQ
lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)
CHOP
42 120573-Actin
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
NHBE cells
minus + minus + minus + RSV infection
Cell sourceCOPDSMNS
(b)
Mouse lungs
RA + mockRA + RSV
Smoke + mockSmoke + RSV
RA + mockRA + RSV
Smoke + mockSmoke + RSV
00
05
10
15
GPx-
1 RQ
0
5
10
Chop
RQ
1 3 5 7 90(dpi)
1 3 5 7 90(dpi)
lowast
lowast
lowast
lowast
lowast
(c)
120573-Actin
CHOP
Room air Smoke
42
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
Mouse lungs
9dpi
(d)
Figure 4 RSV infection enhances the UPR in the lung (a) GPx-1 and CHOP gene expression were determined in NHBE cells isolated fromnonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) infected with mock or RSV and analyzed byqPCR (b) Protein expression of GPx-1 ATF6 ATF4 CHOP and 120573-actin was examined by immunoblots (c) Wild type mice were exposedto cigarette smoke or room air for six months and subsequently infected with 1 times 106 pfu of RSV Animals were euthanized at 0 1 3 5 7 and9 days after infection (dpi) and Gpx-1 and Chop expression were determined by qPCR (d) Protein expression of GPx-1 ATF6 ATF4 CHOPand 120573-actin was examined by immunoblots Data are shown as mean plusmn SEM where each measurement was performed on 3 independentdays lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line or the same infection day determined by Studentrsquos119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lungs of mice induces a potent antioxidant responseThisantioxidant response is diminished in the COPD lungs [29]which may explain the heightened UPR observed in NHBEcells isolated from COPD subjects The exact role of thisheightened UPR on the progression of COPD still remainsto be fully determined However we have established thatloss of GPx-1 in vivo leads to a marked increased in all threebranches of the UPR (see Figure 8 for proposed signalingscheme) and this coincides with enhanced apoptosis and lungtissue destruction in mice [13] Our results suggest that GPx-1 significantly regulates the UPR in COPD and enhancing
GPx-1 expression may be feasible means of offsetting theUPR and lung injury responses that drive the onset andprogression of this disease
Multiple studies utilizing the Gpx-1minusminus and transgenicmice demonstrated the protective role of GPx-1 in counteringoxidative injury and cell death mediated by ROS [13 30]GPX-1 activity also affects protein kinase phosphorylation[31] and oxidant-mediated activation of NF-120581B [32] In thiscurrent study GPx-1 was significantly reduced in NHBE cellsisolated from COPD subjects compared to nonsmokers andsmokers Others have reported that the alteration of GPx-1
Figure 5 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) Lung gene expression of Chop Atf4 Edem1 Grp78 Grp94 and Xbp1 was examined (b)Immunoblots were performed of whole lung protein for CHOP ATF4 EDEM GRP78 GRP94 and XBP1 Dot plots are represented as (a)relative quantification (RQ) compared to ACTB expression or (b) densitometry units (DU) of pixel intensity expressed as a ratio to 120573-actinEvery lane represents an individual mouse Data are shown as mean plusmn SEM where each measurement was performed on 3 independent dayson 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line determined by 2-way ANOVA withTukeyrsquos post hoc test (gt2 groups)
expression does not affect the mRNA or activity expressionof other selenoproteins [33] which suggests no compensationexpression of other selenoproteins following loss of GPx-1 expression Currently the mechanism by which cigarettesmoke regulates GPx-1 expression is not fully elucidated butconsidering our GPx-1 reintroduction data further analysis
of GPx-1 regulation is critical GPx-1 expression and activityhave been reported to be regulated by Nrf2 [34] the tran-scription factor TFAP2C [35] CpG methylation of the GPx-1promoter [35] Bcr-AblmTOR [36] selenium [37] estrogen[38] adenosine [39] Sec-insertion sequence (SECIS) factors[40] EGFR [41] and homocysteine [42] Specifically within
Mediators of Inflammation 11
Room airCigarette smoke
Wildtype
lowastlowast
00
02
04
06
08
10
TUN
EL p
ositi
ve (
)
Gpx-1minusminus
(a)
Caspase-3
42
19
Wild type
35 Pro
Cleaved
RA SM RA SM1 2 3 4 5 6 7 8
120573-Actin
Gpx-1minusminus
(b)
Wild type
lowastlowast lowast
lowastlowast
00
01
02
03
04LD
H re
leas
e (ab
s 350
nm)
0
2
4
6
8
10
Clea
ved
casp
ase D
U
Wildtype
Gpx-1minusminus Gpx-1minusminus
(c)
Figure 6 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) TUNEL analysis was performed on lung tissue from each mouse group (b) Enhancedlung tissue caspase-3 cleavage coincided with (c) elevated LDH into BALF ofGpx-1minusminus exposed to cigarette smoke Every lane in (b) representsan individual mouse and densitometry units (DU) of pixel intensity expressed as a ratio to total caspase-3 levels Dot plots are representedas mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 whencomparing both treatments connected by a line determined by 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lung during smoke exposure Singh et al show elevatedGPx-1 expression in the lungs following one-month cigarettesmoke exposure that was regulated by Nrf2 [34] HoweverNrf2 expression is lost in COPD subjects suggesting that thissecondary event could result in reduced GPx-1 expressionand heightened ER stress Loss of Nrf2 in mice resultsin enhanced susceptibility to cigarette smoke [43 44] andelastase [45] induced emphysema in mice However genomicstudies in Nrf2minusminus mouse samples suggest that Nrf2 mayregulate other GPx genes but not GPx-1 [46] Whether RSVinfection alters regulation of GPx-1 expression in a similarmanner to chronic smoke is unknown Interestingly theUPR upon RSV infection can counter viral proliferation [26]Further studies on the regulation of GPx-1 in smoke exposureand COPD and the significance of ER stress in the lungs arerequired This will be a major area for our future work
Since GPx-1 expression regulates all three branches of theUPR GPx-1 may affect a common mediator of the UPR oreach branch individually Dissociation of GRP78BiP uponER stress is required for all three branches of the UPR GPx-1expression directly regulated the gene expression of GRP78However whetherGPx-1 impacts onGRP78dissociation dur-ing ER stress is unknown Nrf2 interacts directly with PERK[47] and may play a major role in GPx-1 expression therebyregulating the UPR The ER stress inducer thapsigargininduces Nrf2 protein production in 16-HBE cells [47] whichsuggests that the UPR induces a Nrf2 response to reverseER stress We observe a similar effect on GPx-1 expressionin PCLS following tunicamycin treatment Equally XBP1regulates several antioxidants including catalase SOD1 andthioredoxinTRX1 [48]However XBP1 does not regulateGPxproteins [48] but XBP1 expression is regulated by GPx-1 The
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
Figure 3 Reintroducing GPx-1 into NHBE cells isolated fromCOPD donors subdues the UPR (a) Gene expression ofGPx-1was determinedin fully differentiated NHBE cells from nonsmoking individuals (119899 = 6) exposed to room air (RA) and cigarette smoke (CS from 4 cigarettesevery second day (3 exposures)) using a Vitrocell VC-10 smoking robot (b) RNA and (c) protein was analyzed for GPx-1 expression fromfully differentiated NHBE cells from nonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) (d) NHBEcells isolated from COPD subjects were transfected with albumin or GPx-1 protein and CHOP expression was determined by qPCR (e)Immunoblots and corresponding densitometry analysis for CHOP Gpx-1 and 120573-actin from NHBE cells from COPD subjects followingalbumin or GPx-1 protein transfection In each immunoblot every lane represents an individual cell donor Data are shown as mean plusmn SEMwhere each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing bothtreatments connected by a line determined by Studentrsquos 119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
no induction of apoptosis Tunicamycin induced all threebranches of the UPR and CHOP expression was observedin PCLS (Figures 7(b)-7(c)) Tunicamycin enhanced gene(Figure 7(b)) and protein (Figure 7(c)) expression of ATF4XBP1 ATF6 and CHOP in PCLS Interestingly tunicamycininduced GPx-1 GPx-2 and GPx-4 gene (Figure 7(d)) andprotein (Figure 7(e)) expression in PCLSTherefore an acuteER stress induces the expression of antioxidants to counterfurther oxidant and ER stress
4 Discussion
Cigarette smoking is the most relevant environmental riskfactor associated with the development of COPD Howeversmoke inhalation studies are problematic as long-term smoke
exposure is required to trigger disease formation in animalmodels and a secondary event may be required to mimic thehuman disease-state Here we observed that the loss of GPx-1 expression enhances cigarette smoke-induced ER stressGPx-1 regulates the UPR following smoke exposure and wefound that the expression of GPx-1 itself was triggered by anacute ER stress stimulus NHBE cells isolated from COPDdonors expressed significantly less GPx-1 which coincideswith elevated UPR Reintroducing GPx-1 protein into NHBEcells isolated from COPD donors reduced the UPR RSVinfection contributes to loss of lung GPx-1 expression whichis exaggerated in lungs exposed to smoke and coincides withelevated UPR Gpx-1minusminus mice exhibited greater UPR andsubsequent enhanced apoptosis following long-term cigarettesmoke exposure Interestingly triggering an acute ER stress in
Mediators of Inflammation 9
Cell source
NS SM COPD
MockRSV
Cell source
NS SM COPD
MockRSV
NHBE cells
00
05
10
15
20GP
x-1
RQ
0
2
4
6
8
10
CHO
P RQ
lowast
lowast
lowast
lowast
lowast
lowast
lowast
(a)
CHOP
42 120573-Actin
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
NHBE cells
minus + minus + minus + RSV infection
Cell sourceCOPDSMNS
(b)
Mouse lungs
RA + mockRA + RSV
Smoke + mockSmoke + RSV
RA + mockRA + RSV
Smoke + mockSmoke + RSV
00
05
10
15
GPx-
1 RQ
0
5
10
Chop
RQ
1 3 5 7 90(dpi)
1 3 5 7 90(dpi)
lowast
lowast
lowast
lowast
lowast
(c)
120573-Actin
CHOP
Room air Smoke
42
27
XBP1
49 ATF4
74
(kDa)
36 ATF6
22 GPx-1
Mouse lungs
9dpi
(d)
Figure 4 RSV infection enhances the UPR in the lung (a) GPx-1 and CHOP gene expression were determined in NHBE cells isolated fromnonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) infected with mock or RSV and analyzed byqPCR (b) Protein expression of GPx-1 ATF6 ATF4 CHOP and 120573-actin was examined by immunoblots (c) Wild type mice were exposedto cigarette smoke or room air for six months and subsequently infected with 1 times 106 pfu of RSV Animals were euthanized at 0 1 3 5 7 and9 days after infection (dpi) and Gpx-1 and Chop expression were determined by qPCR (d) Protein expression of GPx-1 ATF6 ATF4 CHOPand 120573-actin was examined by immunoblots Data are shown as mean plusmn SEM where each measurement was performed on 3 independentdays lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line or the same infection day determined by Studentrsquos119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lungs of mice induces a potent antioxidant responseThisantioxidant response is diminished in the COPD lungs [29]which may explain the heightened UPR observed in NHBEcells isolated from COPD subjects The exact role of thisheightened UPR on the progression of COPD still remainsto be fully determined However we have established thatloss of GPx-1 in vivo leads to a marked increased in all threebranches of the UPR (see Figure 8 for proposed signalingscheme) and this coincides with enhanced apoptosis and lungtissue destruction in mice [13] Our results suggest that GPx-1 significantly regulates the UPR in COPD and enhancing
GPx-1 expression may be feasible means of offsetting theUPR and lung injury responses that drive the onset andprogression of this disease
Multiple studies utilizing the Gpx-1minusminus and transgenicmice demonstrated the protective role of GPx-1 in counteringoxidative injury and cell death mediated by ROS [13 30]GPX-1 activity also affects protein kinase phosphorylation[31] and oxidant-mediated activation of NF-120581B [32] In thiscurrent study GPx-1 was significantly reduced in NHBE cellsisolated from COPD subjects compared to nonsmokers andsmokers Others have reported that the alteration of GPx-1
Figure 5 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) Lung gene expression of Chop Atf4 Edem1 Grp78 Grp94 and Xbp1 was examined (b)Immunoblots were performed of whole lung protein for CHOP ATF4 EDEM GRP78 GRP94 and XBP1 Dot plots are represented as (a)relative quantification (RQ) compared to ACTB expression or (b) densitometry units (DU) of pixel intensity expressed as a ratio to 120573-actinEvery lane represents an individual mouse Data are shown as mean plusmn SEM where each measurement was performed on 3 independent dayson 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line determined by 2-way ANOVA withTukeyrsquos post hoc test (gt2 groups)
expression does not affect the mRNA or activity expressionof other selenoproteins [33] which suggests no compensationexpression of other selenoproteins following loss of GPx-1 expression Currently the mechanism by which cigarettesmoke regulates GPx-1 expression is not fully elucidated butconsidering our GPx-1 reintroduction data further analysis
of GPx-1 regulation is critical GPx-1 expression and activityhave been reported to be regulated by Nrf2 [34] the tran-scription factor TFAP2C [35] CpG methylation of the GPx-1promoter [35] Bcr-AblmTOR [36] selenium [37] estrogen[38] adenosine [39] Sec-insertion sequence (SECIS) factors[40] EGFR [41] and homocysteine [42] Specifically within
Mediators of Inflammation 11
Room airCigarette smoke
Wildtype
lowastlowast
00
02
04
06
08
10
TUN
EL p
ositi
ve (
)
Gpx-1minusminus
(a)
Caspase-3
42
19
Wild type
35 Pro
Cleaved
RA SM RA SM1 2 3 4 5 6 7 8
120573-Actin
Gpx-1minusminus
(b)
Wild type
lowastlowast lowast
lowastlowast
00
01
02
03
04LD
H re
leas
e (ab
s 350
nm)
0
2
4
6
8
10
Clea
ved
casp
ase D
U
Wildtype
Gpx-1minusminus Gpx-1minusminus
(c)
Figure 6 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) TUNEL analysis was performed on lung tissue from each mouse group (b) Enhancedlung tissue caspase-3 cleavage coincided with (c) elevated LDH into BALF ofGpx-1minusminus exposed to cigarette smoke Every lane in (b) representsan individual mouse and densitometry units (DU) of pixel intensity expressed as a ratio to total caspase-3 levels Dot plots are representedas mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 whencomparing both treatments connected by a line determined by 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lung during smoke exposure Singh et al show elevatedGPx-1 expression in the lungs following one-month cigarettesmoke exposure that was regulated by Nrf2 [34] HoweverNrf2 expression is lost in COPD subjects suggesting that thissecondary event could result in reduced GPx-1 expressionand heightened ER stress Loss of Nrf2 in mice resultsin enhanced susceptibility to cigarette smoke [43 44] andelastase [45] induced emphysema in mice However genomicstudies in Nrf2minusminus mouse samples suggest that Nrf2 mayregulate other GPx genes but not GPx-1 [46] Whether RSVinfection alters regulation of GPx-1 expression in a similarmanner to chronic smoke is unknown Interestingly theUPR upon RSV infection can counter viral proliferation [26]Further studies on the regulation of GPx-1 in smoke exposureand COPD and the significance of ER stress in the lungs arerequired This will be a major area for our future work
Since GPx-1 expression regulates all three branches of theUPR GPx-1 may affect a common mediator of the UPR oreach branch individually Dissociation of GRP78BiP uponER stress is required for all three branches of the UPR GPx-1expression directly regulated the gene expression of GRP78However whetherGPx-1 impacts onGRP78dissociation dur-ing ER stress is unknown Nrf2 interacts directly with PERK[47] and may play a major role in GPx-1 expression therebyregulating the UPR The ER stress inducer thapsigargininduces Nrf2 protein production in 16-HBE cells [47] whichsuggests that the UPR induces a Nrf2 response to reverseER stress We observe a similar effect on GPx-1 expressionin PCLS following tunicamycin treatment Equally XBP1regulates several antioxidants including catalase SOD1 andthioredoxinTRX1 [48]However XBP1 does not regulateGPxproteins [48] but XBP1 expression is regulated by GPx-1 The
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
Figure 4 RSV infection enhances the UPR in the lung (a) GPx-1 and CHOP gene expression were determined in NHBE cells isolated fromnonsmoker (NS) smokers (SM) and COPD (COPD) individuals (119899 = 6 donors per group) infected with mock or RSV and analyzed byqPCR (b) Protein expression of GPx-1 ATF6 ATF4 CHOP and 120573-actin was examined by immunoblots (c) Wild type mice were exposedto cigarette smoke or room air for six months and subsequently infected with 1 times 106 pfu of RSV Animals were euthanized at 0 1 3 5 7 and9 days after infection (dpi) and Gpx-1 and Chop expression were determined by qPCR (d) Protein expression of GPx-1 ATF6 ATF4 CHOPand 120573-actin was examined by immunoblots Data are shown as mean plusmn SEM where each measurement was performed on 3 independentdays lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line or the same infection day determined by Studentrsquos119905-test (2 groups) or 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lungs of mice induces a potent antioxidant responseThisantioxidant response is diminished in the COPD lungs [29]which may explain the heightened UPR observed in NHBEcells isolated from COPD subjects The exact role of thisheightened UPR on the progression of COPD still remainsto be fully determined However we have established thatloss of GPx-1 in vivo leads to a marked increased in all threebranches of the UPR (see Figure 8 for proposed signalingscheme) and this coincides with enhanced apoptosis and lungtissue destruction in mice [13] Our results suggest that GPx-1 significantly regulates the UPR in COPD and enhancing
GPx-1 expression may be feasible means of offsetting theUPR and lung injury responses that drive the onset andprogression of this disease
Multiple studies utilizing the Gpx-1minusminus and transgenicmice demonstrated the protective role of GPx-1 in counteringoxidative injury and cell death mediated by ROS [13 30]GPX-1 activity also affects protein kinase phosphorylation[31] and oxidant-mediated activation of NF-120581B [32] In thiscurrent study GPx-1 was significantly reduced in NHBE cellsisolated from COPD subjects compared to nonsmokers andsmokers Others have reported that the alteration of GPx-1
Figure 5 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) Lung gene expression of Chop Atf4 Edem1 Grp78 Grp94 and Xbp1 was examined (b)Immunoblots were performed of whole lung protein for CHOP ATF4 EDEM GRP78 GRP94 and XBP1 Dot plots are represented as (a)relative quantification (RQ) compared to ACTB expression or (b) densitometry units (DU) of pixel intensity expressed as a ratio to 120573-actinEvery lane represents an individual mouse Data are shown as mean plusmn SEM where each measurement was performed on 3 independent dayson 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line determined by 2-way ANOVA withTukeyrsquos post hoc test (gt2 groups)
expression does not affect the mRNA or activity expressionof other selenoproteins [33] which suggests no compensationexpression of other selenoproteins following loss of GPx-1 expression Currently the mechanism by which cigarettesmoke regulates GPx-1 expression is not fully elucidated butconsidering our GPx-1 reintroduction data further analysis
of GPx-1 regulation is critical GPx-1 expression and activityhave been reported to be regulated by Nrf2 [34] the tran-scription factor TFAP2C [35] CpG methylation of the GPx-1promoter [35] Bcr-AblmTOR [36] selenium [37] estrogen[38] adenosine [39] Sec-insertion sequence (SECIS) factors[40] EGFR [41] and homocysteine [42] Specifically within
Mediators of Inflammation 11
Room airCigarette smoke
Wildtype
lowastlowast
00
02
04
06
08
10
TUN
EL p
ositi
ve (
)
Gpx-1minusminus
(a)
Caspase-3
42
19
Wild type
35 Pro
Cleaved
RA SM RA SM1 2 3 4 5 6 7 8
120573-Actin
Gpx-1minusminus
(b)
Wild type
lowastlowast lowast
lowastlowast
00
01
02
03
04LD
H re
leas
e (ab
s 350
nm)
0
2
4
6
8
10
Clea
ved
casp
ase D
U
Wildtype
Gpx-1minusminus Gpx-1minusminus
(c)
Figure 6 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) TUNEL analysis was performed on lung tissue from each mouse group (b) Enhancedlung tissue caspase-3 cleavage coincided with (c) elevated LDH into BALF ofGpx-1minusminus exposed to cigarette smoke Every lane in (b) representsan individual mouse and densitometry units (DU) of pixel intensity expressed as a ratio to total caspase-3 levels Dot plots are representedas mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 whencomparing both treatments connected by a line determined by 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lung during smoke exposure Singh et al show elevatedGPx-1 expression in the lungs following one-month cigarettesmoke exposure that was regulated by Nrf2 [34] HoweverNrf2 expression is lost in COPD subjects suggesting that thissecondary event could result in reduced GPx-1 expressionand heightened ER stress Loss of Nrf2 in mice resultsin enhanced susceptibility to cigarette smoke [43 44] andelastase [45] induced emphysema in mice However genomicstudies in Nrf2minusminus mouse samples suggest that Nrf2 mayregulate other GPx genes but not GPx-1 [46] Whether RSVinfection alters regulation of GPx-1 expression in a similarmanner to chronic smoke is unknown Interestingly theUPR upon RSV infection can counter viral proliferation [26]Further studies on the regulation of GPx-1 in smoke exposureand COPD and the significance of ER stress in the lungs arerequired This will be a major area for our future work
Since GPx-1 expression regulates all three branches of theUPR GPx-1 may affect a common mediator of the UPR oreach branch individually Dissociation of GRP78BiP uponER stress is required for all three branches of the UPR GPx-1expression directly regulated the gene expression of GRP78However whetherGPx-1 impacts onGRP78dissociation dur-ing ER stress is unknown Nrf2 interacts directly with PERK[47] and may play a major role in GPx-1 expression therebyregulating the UPR The ER stress inducer thapsigargininduces Nrf2 protein production in 16-HBE cells [47] whichsuggests that the UPR induces a Nrf2 response to reverseER stress We observe a similar effect on GPx-1 expressionin PCLS following tunicamycin treatment Equally XBP1regulates several antioxidants including catalase SOD1 andthioredoxinTRX1 [48]However XBP1 does not regulateGPxproteins [48] but XBP1 expression is regulated by GPx-1 The
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
Figure 5 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) Lung gene expression of Chop Atf4 Edem1 Grp78 Grp94 and Xbp1 was examined (b)Immunoblots were performed of whole lung protein for CHOP ATF4 EDEM GRP78 GRP94 and XBP1 Dot plots are represented as (a)relative quantification (RQ) compared to ACTB expression or (b) densitometry units (DU) of pixel intensity expressed as a ratio to 120573-actinEvery lane represents an individual mouse Data are shown as mean plusmn SEM where each measurement was performed on 3 independent dayson 6 donorsgroup lowast denotes a 119901 value lt 005 when comparing both treatments connected by a line determined by 2-way ANOVA withTukeyrsquos post hoc test (gt2 groups)
expression does not affect the mRNA or activity expressionof other selenoproteins [33] which suggests no compensationexpression of other selenoproteins following loss of GPx-1 expression Currently the mechanism by which cigarettesmoke regulates GPx-1 expression is not fully elucidated butconsidering our GPx-1 reintroduction data further analysis
of GPx-1 regulation is critical GPx-1 expression and activityhave been reported to be regulated by Nrf2 [34] the tran-scription factor TFAP2C [35] CpG methylation of the GPx-1promoter [35] Bcr-AblmTOR [36] selenium [37] estrogen[38] adenosine [39] Sec-insertion sequence (SECIS) factors[40] EGFR [41] and homocysteine [42] Specifically within
Mediators of Inflammation 11
Room airCigarette smoke
Wildtype
lowastlowast
00
02
04
06
08
10
TUN
EL p
ositi
ve (
)
Gpx-1minusminus
(a)
Caspase-3
42
19
Wild type
35 Pro
Cleaved
RA SM RA SM1 2 3 4 5 6 7 8
120573-Actin
Gpx-1minusminus
(b)
Wild type
lowastlowast lowast
lowastlowast
00
01
02
03
04LD
H re
leas
e (ab
s 350
nm)
0
2
4
6
8
10
Clea
ved
casp
ase D
U
Wildtype
Gpx-1minusminus Gpx-1minusminus
(c)
Figure 6 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) TUNEL analysis was performed on lung tissue from each mouse group (b) Enhancedlung tissue caspase-3 cleavage coincided with (c) elevated LDH into BALF ofGpx-1minusminus exposed to cigarette smoke Every lane in (b) representsan individual mouse and densitometry units (DU) of pixel intensity expressed as a ratio to total caspase-3 levels Dot plots are representedas mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 whencomparing both treatments connected by a line determined by 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lung during smoke exposure Singh et al show elevatedGPx-1 expression in the lungs following one-month cigarettesmoke exposure that was regulated by Nrf2 [34] HoweverNrf2 expression is lost in COPD subjects suggesting that thissecondary event could result in reduced GPx-1 expressionand heightened ER stress Loss of Nrf2 in mice resultsin enhanced susceptibility to cigarette smoke [43 44] andelastase [45] induced emphysema in mice However genomicstudies in Nrf2minusminus mouse samples suggest that Nrf2 mayregulate other GPx genes but not GPx-1 [46] Whether RSVinfection alters regulation of GPx-1 expression in a similarmanner to chronic smoke is unknown Interestingly theUPR upon RSV infection can counter viral proliferation [26]Further studies on the regulation of GPx-1 in smoke exposureand COPD and the significance of ER stress in the lungs arerequired This will be a major area for our future work
Since GPx-1 expression regulates all three branches of theUPR GPx-1 may affect a common mediator of the UPR oreach branch individually Dissociation of GRP78BiP uponER stress is required for all three branches of the UPR GPx-1expression directly regulated the gene expression of GRP78However whetherGPx-1 impacts onGRP78dissociation dur-ing ER stress is unknown Nrf2 interacts directly with PERK[47] and may play a major role in GPx-1 expression therebyregulating the UPR The ER stress inducer thapsigargininduces Nrf2 protein production in 16-HBE cells [47] whichsuggests that the UPR induces a Nrf2 response to reverseER stress We observe a similar effect on GPx-1 expressionin PCLS following tunicamycin treatment Equally XBP1regulates several antioxidants including catalase SOD1 andthioredoxinTRX1 [48]However XBP1 does not regulateGPxproteins [48] but XBP1 expression is regulated by GPx-1 The
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
Figure 6 Gpx-1 deficient mice have heightened ER stress in their lungs following exposure to cigarette smoke Gpx-1minusminus and wild type micewere exposed to cigarette smoke daily for 1 year (a) TUNEL analysis was performed on lung tissue from each mouse group (b) Enhancedlung tissue caspase-3 cleavage coincided with (c) elevated LDH into BALF ofGpx-1minusminus exposed to cigarette smoke Every lane in (b) representsan individual mouse and densitometry units (DU) of pixel intensity expressed as a ratio to total caspase-3 levels Dot plots are representedas mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotes a 119901 value lt 005 whencomparing both treatments connected by a line determined by 2-way ANOVA with Tukeyrsquos post hoc test (gt2 groups)
the lung during smoke exposure Singh et al show elevatedGPx-1 expression in the lungs following one-month cigarettesmoke exposure that was regulated by Nrf2 [34] HoweverNrf2 expression is lost in COPD subjects suggesting that thissecondary event could result in reduced GPx-1 expressionand heightened ER stress Loss of Nrf2 in mice resultsin enhanced susceptibility to cigarette smoke [43 44] andelastase [45] induced emphysema in mice However genomicstudies in Nrf2minusminus mouse samples suggest that Nrf2 mayregulate other GPx genes but not GPx-1 [46] Whether RSVinfection alters regulation of GPx-1 expression in a similarmanner to chronic smoke is unknown Interestingly theUPR upon RSV infection can counter viral proliferation [26]Further studies on the regulation of GPx-1 in smoke exposureand COPD and the significance of ER stress in the lungs arerequired This will be a major area for our future work
Since GPx-1 expression regulates all three branches of theUPR GPx-1 may affect a common mediator of the UPR oreach branch individually Dissociation of GRP78BiP uponER stress is required for all three branches of the UPR GPx-1expression directly regulated the gene expression of GRP78However whetherGPx-1 impacts onGRP78dissociation dur-ing ER stress is unknown Nrf2 interacts directly with PERK[47] and may play a major role in GPx-1 expression therebyregulating the UPR The ER stress inducer thapsigargininduces Nrf2 protein production in 16-HBE cells [47] whichsuggests that the UPR induces a Nrf2 response to reverseER stress We observe a similar effect on GPx-1 expressionin PCLS following tunicamycin treatment Equally XBP1regulates several antioxidants including catalase SOD1 andthioredoxinTRX1 [48]However XBP1 does not regulateGPxproteins [48] but XBP1 expression is regulated by GPx-1 The
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
Figure 7 Effect of ER stress-induced tunicamycin on the antioxidant expression profile in mouse precision-cut lung slices (PCLS) PCLSwere obtained from wild type mice and were exposed to tunicamycin (1 120583M) for 24 hours (a) LDH release into media and (b) Chop Atf4Atf6 and Xbp1 gene expression were examined (c) Immunoblots were conducted for Chop Atf4 Atf6 Xbp1 and 120573-actin (d) GPx-1 GPx-2GPx-3 GPx-4 and Sod1 were quantified by qPCR and (e) immunoblots analysis Every lane represents an individual mouse Dot plots arerepresented as relative quantification (RQ) compared to ACTB expression or densitometry units (DU) of pixel intensity expressed as a ratio to120573-actin Data are shown as the mean plusmn SEM where each measurement was performed on 3 independent days on 6 donorsgroup lowast denotesa 119901 value lt 005 when comparing both treatments connected by a line determined by Studentrsquos 119905-test (2 groups)
XBP1 regulated gene EDEM1 was also enhanced in Gpx-1minusminusmice exposed to smoke which further confirms that GPx-1regulation of XBP1 signaling ATF6 requires translocation tothe Golgi to undergo cleavage and subsequent translocationto the nucleus to act as a transcription factor [49] WhetherGPx-1 modulates this signaling has yet to be determinedThis crosstalk between antioxidant signaling and the UPRis partially lost in COPD and may play a critical step in thepathogenesis of this disease
The role of the UPR on apoptosis is dependent on thestimulus exposure duration and intensity of this signalingLoss of GPx-1 expression directly impacts cell death andcell death is an important factor in COPD progression [50]Smoke-induced apoptosis has been associated with severalprocesses such as ceramide signaling [51] damage-associatedmolecular pattern molecules (DAMPs) [52] and tumornecrosis factor-related apoptosis-inducing ligand (TRAIL)[53] The loss of Gpx-1 exacerbated cigarette smoke-inducedcell apoptosis in mice suggesting that Gpx-1minusminus genotypeexacerbated cell death at least partially through the induc-tion of the UPR Our group has previously demonstrated
that GPx-1 also regulates the activation of protein tyrosinephosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A)[13] Both of these phosphatases could impact smoke-inducedcell survival [54 55] Equally Nrf2 deficient cells undergoenhanced cell death following exposure to ER stress [47]whichmay be dependent on GPx-1 expressionTherefore thedata presented here suggests that enhancedCHOPexpressionin NHBE cells and mouse lungs may contribute to apoptosisOther studies also suggest that certain elements of the UPRhave several antiapoptotic and anti-inflammatory effects inother organs XBP1 reduces CSE-induced CHOP and therebyis protected fromCSE-induced apoptosis in a retinal pigmentepithelia (RPE) cell line [56] via regulation of eIF2120572 and p38phosphorylation [4] Loss of CHOP expression exacerbatedcell death through the downregulation of Nrf2 in RPE cells[4] CHOP deficiency enhances apoptosis in hippocam-pal cells and impaired memory-related behavioural perfor-mances in mice with tunicamycin treatment [57] Recentlydeficiency of CHOP exaggerated lipopolysaccharide- (LPS-)induced inflammation and kidney injury in mice [58] Theimportance of ER stress and the role of each member of
14 Mediators of Inflammation
p p p p
IRE1 PERK ATF6
ER stressGRP78BiP
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
GPx-1GPx-1
Healthy
(a)
p p p p
IRE1 PERK ATF6
elF2120572p
ATF4
ER stressGRP78BiP
XBP1u
XBP1s
ATF6
ERCy
toso
lN
ucle
usGene expression (eg CHOP EDEM GRP78and GPx-1)
COPD
Higher frequency viral infection (eg RSV)
(b)
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
Figure 8 Possible signaling mechanism for GPx-1 regulation of the UPR Evidence presented in this study indicates that following smokeexposure GPx-1 prevents ER stress (a) However in the disease-state GPx-1 expression is subdued and results in enhanced UPR (b) RSVinfection significantly contributes to reduced GPx-1 expression that coincides with enhanced UPR
the UPR in the development of lung disease still remainto be fully addressed However we have demonstrated thatenhanced UPR coincides with worsening of symptoms thatare countered with the expression of GPx-1 in mice
5 Conclusion
Here we demonstrate that GPx-1 expression is reduced inNHBE cells isolated from COPD subjects GPx-1 is a majorregulator of the UPR under smoke exposure conditions andacute ER stress induces lung GPx-1 expression Together ourdata indicate that the loss of GPX-1 expression in COPDlungs could contribute to disease progression by enhancingtheUPRThese studies suggest that enhancingGPX-1 activitymay be an effective therapeutic approach to prevent thedamage induced by UPR in the lung
Competing Interests
None of the authors have a financial relationship with acommercial entity that has an interest in the subject of thismanuscript
Acknowledgments
This work was supported by grants made available toPatrick Geraghty (Flight Attendant Medical Research Insti-tute (YCSA 113380)) Robert F Foronjy (US National Insti-tutes of Health 5R01HL098528-05 and Flight AttendantMed-ical Research Institute (CIA 130020)) Matthias A Salathe(Flight Attendant Medical Research Institute CIA 103027and 130033 the James and Esther King Biomedical ResearchProgramof the State of FL 5JK02) and JeanineMDrsquoArmiento(R01 HL086936-07)
References
[1] J Xu S LMurphy K D Kochanek and B A Basstian ldquoDeathsfinal data for 2013rdquo in National Vital Statistics Reports pp 1ndash119 National Center for Health Statistics Hyattsville Md USA2016
[2] R F Foronjy O Mirochnitchenko O Propokenko et alldquoSuperoxide dismutase expression attenuates cigarette smoke-or elastase-generated emphysema in micerdquoAmerican Journal ofRespiratory and Critical Care Medicine vol 173 no 6 pp 623ndash631 2006
Mediators of Inflammation 15
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
[3] E Bargagli C Olivieri D Bennett A Prasse J Muller-Quernheim and P Rottoli ldquoOxidative stress in the pathogenesisof diffuse lung diseases a reviewrdquo RespiratoryMedicine vol 103no 9 pp 1245ndash1256 2009
[4] C Huang J J Wang J H Ma C Jin Q Yu and S XZhang ldquoActivation of the UPR protects against cigarette smoke-induced RPE apoptosis through up-regulation of Nrf2rdquo Journalof Biological Chemistry vol 290 no 9 pp 5367ndash5380 2015
[5] P Geraghty A Wallace and J M DrsquoArmiento ldquoInduction ofthe unfolded protein response by cigarette smoke is primarilyan activating transcription factor 4-CEBP homologous proteinmediated processrdquo International Journal of Chronic ObstructivePulmonary Disease vol 6 no 1 pp 309ndash319 2011
[6] A Hengstermann and T Muller ldquoEndoplasmic reticulumstress induced by aqueous extracts of cigarette smoke in 3T3cells activates the unfolded-protein-response-dependent PERKpathway of cell survivalrdquo Free Radical Biology andMedicine vol44 no 6 pp 1097ndash1107 2008
[7] E Jorgensen A Stinson L Shan J Yang D Gietl and A PAlbino ldquoCigarette smoke induces endoplasmic reticulum stressand the unfolded protein response in normal and malignanthuman lung cellsrdquo BMC Cancer vol 8 article 229 2008
[8] S G Kelsen X Duan R Ji O Perez C Liu and S MeralildquoCigarette smoke induces an unfolded protein response inthe human lung a proteomic approachrdquo American Journal ofRespiratory Cell and Molecular Biology vol 38 no 5 pp 541ndash550 2008
[9] Y Tagawa NHiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008
[10] R J Kaufman ldquoStress signaling from the lumen of the endo-plasmic reticulum coordination of gene transcriptional andtranslational controlsrdquoGenes amp Development vol 13 no 10 pp1211ndash1233 1999
[11] P Walter and D Ron ldquoThe unfolded protein response stresspathway to homeostatic regulationrdquo Science vol 334 no 6059pp 1081ndash1086 2011
[12] A-H Lee N N Iwakoshi and L H Glimcher ldquoXBP-1 regulatesa subset of endoplasmic reticulum resident chaperone genes inthe unfolded protein responserdquoMolecular and Cellular Biologyvol 23 no 21 pp 7448ndash7459 2003
[13] P Geraghty A A Hardigan A M Wallace et al ldquoThe glu-tathione peroxidase 1-protein tyrosine phosphatase 1B-proteinphosphatase 2A axis A key determinant of airway inflamma-tion and alveolar destructionrdquo American Journal of RespiratoryCell and Molecular Biology vol 49 no 5 pp 721ndash730 2013
[14] M A Forgione N Weiss S Heydrick et al ldquoCellular glu-tathione peroxidase deficiency and endothelial dysfunctionrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 282 no 4 pp H1255ndashH1261 2002
[15] J B De Haan C Bladier M Lotfi-Miri et al ldquoFibroblastsderived from Gpx1 knockout mice display senescent-like fea-tures and are susceptible to H2O2-mediated cell deathrdquo FreeRadical Biology and Medicine vol 36 no 1 pp 53ndash64 2004
[16] P Lewis N Stefanovic J Pete et al ldquoLack of the antioxidantenzyme glutathione peroxidase-1 accelerates atherosclerosis indiabetic apolipoprotein E-deficient micerdquo Circulation vol 115no 16 pp 2178ndash2187 2007
[17] C Duong H J Seow S Bozinovski P J Crack G P Andersonand R Vlahos ldquoGlutathione peroxidase-1 protects against
cigarette smoke-induced lung inflammation inmicerdquoAmericanJournal of PhysiologymdashLung Cellular and Molecular Physiologyvol 299 no 3 pp L425ndashL433 2010
[18] M-C Nlend R J Bookman G E Conner and M SalatheldquoRegulator of G-protein signaling protein 2 modulates puriner-gic calcium and ciliary beat frequency responses in airwayepitheliardquo American Journal of Respiratory Cell and MolecularBiology vol 27 no 4 pp 436ndash445 2002
[19] A M Wallace A Hardigan P Geraghty et al ldquoProtein phos-phatase 2A regulates innate immune and proteolytic responsesto cigarette smoke exposure in the lungrdquo Toxicological Sciencesvol 126 no 2 pp 589ndash599 2012
[20] R F Foronjy M A Salathe A J Dabo et al ldquoTLR9 expressionis required for the development of cigarette smoke-inducedemphysema in micerdquo American Journal of PhysiologymdashLungCellular andMolecular Physiology vol 311 no 1 pp L154ndashL1662016
[21] A R Ressmeyer A K Larsson E Vollmer S E Dahlen SUhlig andCMartin ldquoCharacterisation of guinea pig precision-cut lung slices comparison with human tissuesrdquo EuropeanRespiratory Journal vol 28 no 3 pp 603ndash611 2006
[22] H-DHeld CMartin and SUhlig ldquoCharacterization of airwayand vascular responses in murine lungsrdquo British Journal ofPharmacology vol 126 no 5 pp 1191ndash1199 1999
[23] C Chen J JWang J Li Q Yu and S X Zhang ldquoQuinotrierixininhibits proliferation of human retinal pigment epithelial cellsrdquoMolecular Vision vol 19 pp 39ndash46 2013
[24] J A Wedzicha ldquoAirway infection accelerates decline of lungfunction in chronic obstructive pulmonary diseaserdquo AmericanJournal of Respiratory and Critical Care Medicine vol 164 no10 pp 1757ndash1758 2001
[25] P Mallia and S L Johnston ldquoMechanisms and experimentalmodels of chronic obstructive pulmonary disease exacerba-tionsrdquo Proceedings of the American Thoracic Society vol 2 no4 pp 361ndash366 2005
[26] I Hassan K S Gaines W J Hottel et al ldquoInositol-requiringenzyme 1 inhibits respiratory syncytial virus replicationrdquo Jour-nal of Biological Chemistry vol 289 no 11 pp 7537ndash7546 2014
[27] S Hodge G HodgeM Holmes and P N Reynolds ldquoIncreasedairway epithelial and T-cell apoptosis in COPD remains despitesmoking cessationrdquo European Respiratory Journal vol 25 no 3pp 447ndash454 2005
[28] F-F Chu R S Esworthy P G Chu et al ldquoBacteria-inducedintestinal cancer in mice with disrupted Gpx1 and Gpx2 genesrdquoCancer Research vol 64 no 3 pp 962ndash968 2004
[29] M Tomaki H Sugiura A Koarai et al ldquoDecreased expressionof antioxidant enzymes and increased expression of chemokinesinCOPD lungrdquoPulmonary Pharmacology andTherapeutics vol20 no 5 pp 596ndash605 2007
[30] W-H Cheng Y-S Ho B A Valentine D A Ross G FCombs Jr and X G Lei ldquoCellular glutathione peroxidase is themediator of body selenium to protect against paraquat lethalityin transgenicmicerdquo Journal of Nutrition vol 128 no 7 pp 1070ndash1076 1998
[31] M A Nasr M J Fedele K Esser and A M Diamond ldquoGPx-1 modulates AKT and P70S6K phosphorylation and Gadd45levels in MCF-7 cellsrdquo Free Radical Biology and Medicine vol37 no 2 pp 187ndash195 2004
[32] Q Li S Sanlioglu S Li T Ritchie L Oberley and J FEngelhardt ldquoGPx-1 gene delivery modulates NF120581B activationfollowing diverse environmental injuries through a specific
16 Mediators of Inflammation
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
subunit of the IKK complexrdquoAntioxidants and Redox Signalingvol 3 no 3 pp 415ndash432 2001
[33] W-H Cheng Y-S Ho D A Ross B A Valentine G F CombsJr and X G Lei ldquoCellular glutathione peroxidase knockoutmice express normal levels of selenium-dependent plasma andphospholipid hydroperoxide glutathione peroxidases in varioustissuesrdquo Journal of Nutrition vol 127 no 8 pp 1445ndash1450 1997
[34] A Singh T Rangasamy R KThimmulappa et al ldquoGlutathioneperoxidase 2 the major cigarette smoke-inducible isoform ofGPX in lungs is regulated by Nrf2rdquo American Journal ofRespiratory Cell and Molecular Biology vol 35 no 6 pp 639ndash650 2006
[35] M V Kulak A R Cyr G W Woodfield et al ldquoTranscriptionalregulation of the GPX1 gene by TFAP2C and aberrant CpGmethylation in human breast cancerrdquo Oncogene vol 32 no 34pp 4043ndash4051 2013
[36] E N Reinke D N Ekoue S Bera N Mahmud and A MDiamond ldquoTranslational regulation of GPx-1 and GPx-4 by themTOR pathwayrdquo PLoS ONE vol 9 no 4 Article ID e934722014
[37] L Flohe W A Gunzler and H H Schock ldquoGlutathioneperoxidase a selfnoenzymerdquo FEBS Letters vol 32 no 1 pp 132ndash134 1973
[38] J M Lean C J Jagger B Kirstein K Fuller and T J ChambersldquoHydrogen peroxide is essential for estrogen-deficiency boneloss and osteoclast formationrdquo Endocrinology vol 146 no 2 pp728ndash735 2005
[39] Y Zhang D E Handy and J Loscalzo ldquoAdenosine-dependentinduction of glutathione peroxidase 1 in human primaryendothelial cells and protection against oxidative stressrdquo Circu-lation Research vol 96 no 8 pp 831ndash837 2005
[40] C B Allan G M Lacourciere and T C Stadtman ldquoRespon-siveness of selenoproteins to dietary seleniumrdquo Annual Reviewof Nutrition vol 19 pp 1ndash16 1999
[41] CDuval NAugeM-F Frisach L Casteilla R Salvayre andANegre-Salvayre ldquoMitochondrial oxidative stress is modulatedby oleic acid via an epidermal growth factor receptor-dependentactivation of glutathione peroxidaserdquo Biochemical Journal vol367 no 3 pp 889ndash894 2002
[42] D E Handy Y Zhang and J Loscalzo ldquoHomocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasingtranslationrdquo Journal of Biological Chemistry vol 280 no 16 pp15518ndash15525 2005
[43] T Rangasamy C Y Cho R K Thimmulappa et al ldquoGeneticablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in micerdquo Journal of Clinical Investigationvol 114 no 9 pp 1248ndash1259 2004
[44] T Iizuka Y Ishii K Itoh et al ldquoNrf2-deficient mice are highlysusceptible to cigarette smoke-induced emphysemardquo Genes toCells vol 10 no 12 pp 1113ndash1125 2005
[45] Y Ishii K Itoh Y Morishima et al ldquoTranscription fac-tor Nrf2 plays a pivotal role in protection against elastase-induced pulmonary inflammation and emphysemardquo Journal ofImmunology vol 175 no 10 pp 6968ndash6975 2005
[46] J-M Lee M J Calkins K Chan Y W Kan and J A John-son ldquoIdentification of the NF-E2-related factor-2-dependentgenes conferring protection against oxidative stress in primarycortical astrocytes using oligonucleotide microarray analysisrdquoJournal of Biological Chemistry vol 278 no 14 pp 12029ndash120382003
[47] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrate and
effector of PERK-dependent cell survivalrdquoMolecular and Cellu-lar Biology vol 23 no 20 pp 7198ndash7209 2003
[48] Y Liu M Adachi S Zhao et al ldquoPreventing oxidative stress anew role for XBP1rdquo Cell Death and Differentiation vol 16 no 6pp 847ndash857 2009
[49] K Haze H Yoshida H Yanagi T Yura and K MorildquoMammalian transcription factor ATF6 is synthesized as atransmembrane protein and activated by proteolysis in responseto endoplasmic reticulum stressrdquoMolecular Biology of the Cellvol 10 no 11 pp 3787ndash3799 1999
[50] L Segura-Valdez A Pardo M Gaxiola B D Uhal C Becerriland M Selman ldquoUpregulation of gelatinases A and B collage-nases 1 and 2 and increased parenchymal cell death in COPDrdquoChest vol 117 no 3 pp 684ndash694 2000
[51] I Petrache V Natarajan L Zhen et al ldquoCeramide upregulationcauses pulmonary cell apoptosis and emphysema-like disease inmicerdquo Nature Medicine vol 11 no 5 pp 491ndash498 2005
[52] R F Foronjy P O Ochieng M A Salathe et al ldquoProteintyrosine phosphatase 1B negatively regulates S100A9-mediatedlung damage during respiratory syncytial virus exacerbationsrdquoMucosal Immunology vol 9 no 5 pp 1317ndash1329 2016
[53] T J Haw M R Starkey P M Nair et al ldquoA pathogenic rolefor tumor necrosis factor-related apoptosis-inducing ligand inchronic obstructive pulmonary diseaserdquo Mucosal Immunologyvol 9 no 4 pp 859ndash872 2016
[54] P Geraghty E Eden M Pillai M Campos N G McElvaneyand R F Foronjy ldquo1205721-antitrypsin activates protein phosphatase2A to counter lung inflammatory responsesrdquo American Journalof Respiratory and Critical Care Medicine vol 190 no 11 pp1229ndash1242 2014
[55] C Van Hoof and J Goris ldquoPhosphatases in apoptosis to be ornot to be PP2A is in the heart of the questionrdquo Biochimica etBiophysica Acta vol 1640 no 2-3 pp 97ndash104 2003
[56] M Cano L Wang J Wan et al ldquoOxidative stress inducesmitochondrial dysfunction and a protective unfolded proteinresponse in RPE cellsrdquo Free Radical Biology and Medicine vol69 pp 1ndash14 2014
[57] C-M Chen C-T Wu C-K Chiang B-W Liao and S-H LiuldquoCEBP homologous protein (CHOP) deficiency aggravateshippocampal cell apoptosis and impairs memory performancerdquoPLoS ONE vol 7 no 7 Article ID e40801 2012
[58] V Esposito FGrosjean J Tan et al ldquoCHOPdeficiency results inelevated lipopolysaccharide-induced inflammation and kidneyinjuryrdquo American Journal of Physiology - Renal Physiology vol304 no 4 pp F440ndashF450 2013
![Genome-Wide Association for Sensitivity to Chronic ... · Glutathione reductase (GSR), and Glutathione peroxidase (GPx) as ROS scavengers [1]. These enzymes modulate oxidative stress](https://static.documents.pub/doc/80x56/5b9226a109d3f2c05d8d2065/genome-wide-association-for-sensitivity-to-chronic-glutathione-reductase.jpg)
