Research ArticleEnhancement of the Acrolein-Induced Production of ReactiveOxygen Species and Lung Injury by GADD34
Yang Sun12 Sachiko Ito1 Naomi Nishio1 Yuriko Tanaka1
Nana Chen1 Lintao Liu1 and Ken-ichi Isobe1
1Department of Immunology Nagoya University Graduate School of Medicine Nagoya 466-8550 Japan2Department of Immunology Norman Bethune College of Medicine Jilin University Changchun 130021 China
Correspondence should be addressed to Ken-ichi Isobe kisobemednagoya-uacjp
Received 26 November 2014 Revised 28 January 2015 Accepted 28 January 2015
Academic Editor Sidhartha D Ray
Copyright copy 2015 Yang Sun et alThis is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Chronic obstructive pulmonary disease (COPD) is characterized by lung destruction and inflammation As a major compound ofcigarette smoke acrolein plays a critical role in the induction of respiratory diseases GADD34 is known as a growth arrest andDNA damage-related gene which can be overexpressed in adverse environmental conditions Here we investigated the effects ofGADD34 on acrolein-induced lung injury The intranasal exposure of acrolein induced the expression of GADD34 developingthe pulmonary damage with inflammation and increase of reactive oxygen species (ROS) Conversely the integrality of pulmonarystructure was preserved and the generation of ROSwas reduced in GADD34-knockout mice Acrolein-induced phosphorylation ofeIF2120572 in GADD34-knockout epithelial cells by shRNA protected cell death by reducing misfolded protein-caused oxidative stressThese data indicate that GADD34 participates in the development of acrolein-induced lung injury
1 Introduction
Acrolein is a reactive 120572 120573-unsaturated aldehyde which isabundant in organic combustion such as cigarette smokeautomobile exhaust and manufacturing and cooking emis-sion [1 2] Acrolein contains an active carbonyl group whichis highly reactive as biomacromolecule and induces lungdiseases such as COPD [3] chronic bronchitis emphysema[4] and lung carcinogenesis [5] Acrolein has been shownto induce G to T and G to A mutations forming mutagenica- and c-hydroxy-1 N (2)-cyclic propano-21015840-deoxyguanosineadducts which enhance oxidative DNA damage-inducedmutagenesis [6] Hochman et al showed that acrolein pro-duced reactive oxygen species (ROS) in mast cells [7]
Recently we have demonstrated that acrolein can inducepulmonary injury and macrophage infiltration Beyond thatacrolein can cause oxidative stress and generate reactiveoxygen species (ROS) [8] Although the mechanisms areincompletely understood ROS generation occurs in a widevariety of human diseases The endoplasmic reticulum (ER)is a key organelle where proteins are newly synthesized
When the newly synthesized proteins are misfolded theseproteins accumulate and cause ER stresses or unfoldedprotein response (UPR) There exist three main ER stressresponses in mammals First PERK-eIF2120572 pathway atten-uates translation to limit the accumulation of unfoldedproteins Second activation of transcription factor 6 (ATF6)induces chaperones such as glucose regulated protein-78 (Grp78)immunoglobulin binding protein (BiP) ThirdInositol-requiring enzyme 1 (IRE1) cuts the precursor XBP1mRNA twice removing an internal fragment and thus induc-ing a frame shift ER stress responses provide a conservedmechanism by reducing the folded protein load (eIF2120572phosphorylation and ERAD degradation) and increase thefolding capacity (induction of BipGRP78) [9 10] It has beenshown that acrolein induces these three UPRs [11ndash14]
Growth arrest and DNA damage-inducible protein(GADD34Ppp1r15a) was originally isolated from UV-inducible transcripts in Chinese hamster ovary (CHO) cells[15] Expression of GADD34 is upregulated by growth arrestandDNAdamage [16] It is also induced by amino acid depri-vation and several endoplasmic reticulum (ER) stresses [17
Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 170309 15 pageshttpdxdoiorg1011552015170309
2 Oxidative Medicine and Cellular Longevity
18] GADD34 dephosphorylates several kinases that functionin important signaling cascades including dephosphoryla-tion of eIF2 [17] Because acrolein is a DNA damaging agentand induces ER stresses [14] and induces myeloid infiltrationto lung [19] here we investigated whether GADD34 mightaffects pathogenesis of acrolein-induced lung injury
2 Materials and Methods
21Mice andAcroleinAdministration Eight-week-old femalewild-type C57BL6 mice were obtained from SLC Japan(Shizuoka Japan) GADD34-knockout mice were generatedas previously described [20] All mice were maintained inpathogen-free facilities in the Animal Research Center at theNagoya University Graduate School of Medicine They weremaintained at 25∘C with a 55 relative humidity and a 12 hlight-dark cycle
For the lung injury studies mice were randomly allocatedinto 3 groups (119899 = 6 per group) The mice were instilledintranasally with acrolein (5120583molkg Sigma) control micereceived equal volume of phosphate buffer saline (PBS pH =74) alone The mice were treated daily for 5 dweek for up to28 days The mice were sacrificed at 7 and 28 days then thelungs were isolated and used for histological analysis andorflow cytometry analysis
22 Cell Culture Murine bone marrow-derived macrophagecells were established previously by Ito et al [21] The cellswere cultured in RPM1640 (Sigma) with 10 fetal bovineserum (FBS Gibco Grand Island NY) and 10 GM-CSFwhich was produced by murine GMCSF-producing Chinesehamster ovary (CHO) cells (GM-CSFCM)
Lewis lung carcinoma (LLC) cells were obtained fromRiken [22] Cells were cultured in HEPES-buffered Dul-beccorsquos modified Eaglersquos medium (DMEM) containing 10FBS (Hyclone)
23 Lentivirus-Mediated shRNAKnockdown of Gene ExpressionThe translation of GADD34mRNA in LLC cells was knockeddown using the Mission TRC mouse shRNA LentivirusTransduction Particles (pLKO1-puro Sigma) The sequencesof the shRNAs used were CCGGGGCGGCTCAGATTG-TTCAAAGCTCGAGCTTTGAACAATCTGAGCCGCCTT-TTTG (shRNA TRC2 targeting exon 2) for GADD34knockdown Nontarget control shRNAs (Sigma SHC202V) were used as a control LLC cells were infectedwith viral particles and treated with 8120583gmL polybrane(Millipore TR-1003-G) and then incubated with cells for24 h Cells expressing shRNA were selected on 2 120583gmLpuromycin (Sigma P8833) for functional studies The extentof knockdown of GADD34 expression was confirmed byreal-time PCR Recombinant experiments used here wereapproved by Committee of Nagoya University GraduateSchool of Medicine Established GADD34 knockdown LLCs(shGAD34LLCs) and control LLCs (shconLLCs) wereused
24 Histology and Immunohistochemistry For histologicalanalysis the lung tissue were isolated frommice after acrolein
treatment and 5 120583m frozen lung sections were stained withhematoxylin and eosin (HampE) For immunofluorescenceanalysis cryosectionswere fixed in cold acetone and blockadewith 2 BSA-PBS for 1 h sections were incubated with rabbitanti-ProSpC (epithelial type II cellsmarker) at 4∘C overnightThe sections were stained with Alexa Flour 448 goat anti-rabbit IgG for 1 h and counterstaining with DAPI for 5min
25 Flow Cytometry The whole lung was minced in thecold PBS After centrifugation the tissue homogenate wassuspended with Tris-NH
4Cl red blood cells (RBCs) lysing
buffer (150mM sodium chloride 1 Triton 05 sodiumdeoxycholate 01 SDS 50mM Tris) to lysed RBCs Cells(1 times 105sample) were blocked in 50120583L 02 BSA-PBSstained with FITC-conjugated anti-Gr1 PE-conjugated anti-CD11b anti-CD11c andAPC-conjugated anti-F480 (BDBio-sciences) for 30min at 4∘C and analyzed by flow cytometryusing a FACS Canto flow cytometer (BD Biosciences)
26 ELISA Cells (GM-IMs) were seeded in 12-well plates (3times 105well) and treated with 10 120583M acrolein (Sigma) for 12and 24 h The level of IL-6 in culture medium was measuredby ELISA kits according to the manufacturerrsquos instructions(RampD systems)
27 ROS Measurements and Inhibition of ROS ProductionLung tissue was isolated from mouse and minced afterlysed RBCs cells (1 times 105well) were seeded in 12-wellplates stained with 2 120583M carboxy-2101584071015840-dihydrofluoresceindiacetate (H2DCFDA) (Invitrogen) for 30min at 37∘C Thecells were collected and ROS generation was determined byflow cytometry
The shconLLCs and shGADD34LLCs were plated in12-well dishes and treated with 25120583M acrolein alone for8 h or 25 120583M acrolein with 10 120583M MG132 orand 1 120583gmLcycloheximide (CHX) for 12 h Afterwashing cells were incu-bated with 2 120583M H2DCFDA for 30min at 37∘C Collectedcells were analyzed for ROS generation by flow cytometryROS inhibitor N-acetyl-L-cysteine (NAC) (Sigma-Aldrich)was dissolved at 1molL in deionized water
28 Western Blotting Lung tissue and cells RIPA lysis buffer(01M PBS pH 74 containing 1 deoxycholic acid sodium02 SDS and protease inhibitors) After measurement ofprotein concentration the sampleswere loaded and separatedby sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE) and then transferred to Immobilon transfermembranes The membranes were incubated with primaryanti-phospho-NF-120581B P65 (Ser536) antibody anti-caspase 3and anti-p-eIF2120572 (Cell Signaling) anti-GADD153 and anti-GADD34 (Santa Cruz Biotechnology) overnight at 4∘CThen the membranes were incubated with the secondaryanti-rabbit IgG for 1 h Blots were developedwithwestern blotdetection reagent (GE Healthcare)
29 Real-Time PCR Total RNA was extracted with TRIzolReagent and reverse-transcribed using a High CapacitycDNA Reverse Transcription Kit (Applied Biosystems)Quantitative real-time PCR was performed using
Oxidative Medicine and Cellular Longevity 3
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Figure 1NACprevents acrolein-induced lung injury and inflammationWild-typemicewere intranasally instilled by 5 120583molkg acroleinwithintravenous injection of 100120583L of NAC (500mgkg) or equal volume of PBS (a) Level of ROS in lung tissues wasmeasured by DCFH-DA (b)HampE staining of lung tissues (scale bar 50120583m) (c) Alveolar macrophages were stained with PE-conjugated anti-CD11c and APC-conjugatedanti-F480 and detected by FACS (d) The expression of phospho-NF-120581B p65 in the lung tissues by western blot
the MX3000P QPCR System (Agilent) according to themanufacturerrsquos protocol The sequences of the RT-PCRprimers for each pair are listed in Table 1
210 Apoptosis The shconLLCs and shGADD34LLCs wereplated in 12-well dishes and treated with 25 120583Macrolein alonefor 8 h or 25 120583M acrolein with 10 120583MMG132 orand 1 120583gmLcycloheximide (CHX) for 12 h Cells were collected and withFITC-labeled annexin V and 7-AAD (BD Bioscience) for15min Cells were analyzed by flow cytometry on a FACSCalibur Data were analyzed by FlowJo software (TreeStar)
211 Protein Synthesis Analysis ShconLLCs and shGADD34LLCs were collected after treatment To count the cell num-ber sampleswere stainedwithTrypanBlueWe centrifuge thecell in 400 rpm for 3min and the supernatant were thrownaway An equal number of live cells were extracted with RIPAlysis buffer (2 times 105 cells100 120583L) All samples were analyzedby SDS-PAGE
212 Statistics Data are expressed as means plusmn standard errorof the mean (sem) Statistical comparisons were performedby ANOVA followed by Fisherrsquos post hoc test Values of 119875 lt005 were considered statistically significant
Figure 2 Acrolein activates ER stress in lung tissue Protein expression of ER stress was analyzed Wild-type GADD34-knockout mice wereintranasally instilled by 5 120583molkg acrolein Lung tissues were collected at the indicated times for western blot analysis
31 NAC Prevents Acrolein-Induced Lung Injury and Inflam-mation ROS generation impaired oxidant defense con-tributes to the organ injury [23] We found that ROS wasgreatly produced from the lung at day 7 and was decreasedby antioxidant NAC treatment (Figure 1(a)) On the basisof analysis of lung sections stained with HampE stainingNAC prevented alveolar damage and immune cell migrationwhich were caused by acrolein in lung tissue (Figure 1(b))
F480high CD11c+ alveolar macrophages were recruited to thesite of injury (Figure 1(c)) It has been shown that smoke-associated oxidative stress may promote lung inflammationthrough NF-120581B signaling [24] We also observed the expres-sion of phospho-NF-120581B p65 by acrolein (7d) which wascompletely abolished by NAC (Figure 1(d))
32 GADD34 Mediates Acrolein-Caused Lung InjuryAcrolein has been shown to induce ER stress [14] Weexamined the expression of ER stressmarkers in lung tissue ofwild-type and GADD34-knockout mice after nasal injectionof acrolein (5 120583molkg) (Figure 2) In wild-type mice theexpression of p-eIF2120572 increased highly between 1 and 4 hafter acrolein exposure which is followed by induction ofGADD34 then p-eIF2120572 expression was decreased from 8 hAfter ER stress we observed the increase of cleaved caspase3 which might cause lung tissue destruction HoweverGADD34-knockout mice continuously express higher levelof p-eIF2120572 the cleaved caspase 3 level was lower than that inwild-type mice
Our previous studies have demonstrated that intranasalinstillation of acrolein (5120583molkg) induced lung damage andhemorrhage [8] We examined whether GADD34-knockoutmice showed same phenotypes by intranasal instillation ofacrolein Although acrolein administration caused seriousalveolar structure destruction such as airspace enlargementand hemorrhaging in the lung of wild-type mice GADD34deficiency decreased the lung injury with preserved alveolarstructure no significant hemorrhaging and sparse accu-mulation of intra-alveolar macrophages at 7 and 28 days
Oxidative Medicine and Cellular Longevity 5
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6 Oxidative Medicine and Cellular Longevity
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Figure 3 GADD34 mediates acrolein-caused lung injury The wild-type and GADD34-knockout mice were intranasally instilled by 1 and5 120583molkg acrolein The mice were treated daily for 5 dweek for up to 28 days and then were sacrificed at 7 and 28 days (a) The whole lungsof wild-type and GADD34-knockout mice were photographed at days 7 and 28 (b) HampE staining of lung tissue Scale bar 50 120583m Analysis ofalveolar length determined by mean linear intercepts (119899 = 5 to 7 mice in each group) and the number of alveolar macrophages in wild-typeand GADD34-knockout mice (119899 = 4 mice in each group) (c) Lungs stained for epithelial type II cells (ProSpC green) nuclei (DAPI blue)and the number of epithelial type II-positive cells in wild-type and GADD34-knockout mice (10 fields 119899 = 4) Scale bar 50 120583m lowast119875 lt 005lowastlowast
119875 lt 001 Data are represented as means plusmn sem (d) Levels of ROS production in the lung of wild-type and GADD34-knockout mice weremeasured by DCFH-DA after acrolein treated
(Figures 3(a) and 3(b)) The numbers of type II epithelialcells were significantly reduced in wild-type mice comparedtoGADD34-knockoutmice (Figure 3(c)) In addition higherlevel of ROS production was detected in wild-type micethan GADD34-knockout mice (Figure 3(d)) These resultscollectively indicated that GADD34 might play a crucialrole in the pathogenesis of experimental acrolein-inducedpulmonary injury
33 Low Level of Pulmonary Inflammation in GADD34-Knockout Mice Induced by Acrolein We have demon-strated that the acrolein-induced lung injury is accom-panied by inflammatory response To investigate whetherGADD34 affects the pathologies of pulmonary inflamma-tory responses the mice were instilled with acrolein for 7or 28 days to generate acute inflammation The increaseof F480highCD11c+ macrophages was lower in GADD34-knockout mice than those in wild-type mice (Figure 4(a))However the GR-1+CD11b+ neutrophils migration was notobserved at 7 or 28 days after acrolein treatment both inwild-type and in GADD34-knockout mice (Figure 4(b))Acrolein-induced lung damage may promote lung inflam-mation through NF-120581B signaling A sizable NF-120581B responsewith phosphorylation of p65 on Ser536 was observed in wild-type mice whereas this response was lowered in GADD34-knockout mice (Figure 4(c))
To assess the gene expression levels of inflammatorycytokine the real-time PCR (RT-PCR) was performedGADD34-knockout mice showed significantly lower levels ofM1 macrophages including TNF120572 IL-6 and Irf5 at 7 days
compared with wild-type mice The expression of TNF120572 andIrf5 decreased at 28 days in both wild-type and GADD34-knockout mice In addition only at late time (at 28 daysafter acrolein administration) wild-type mice expressed highlevel of M2 macrophage markers such as Arg1 and Mrc-1These expressions were lower in GADD34-knockout mice(Figure 4(d)) Then we examined the protein expression ofIL-6 in supernatant of wild-type and GADD34-knockoutmacrophage cell line By the stimulation of acrolein IL-6protein expressionwas higher inwild-typemacrophages thanthat in GADD34-knockout macrophages (Figure 4(e))
34 GADD34 Is a Mediator on ER Stress-Induced OxidativeStress In order to understand the molecular mechanismsof effects of GADD34 on acrolein-induced lung injurywe used lung cell line LLCs ShGADD34LLCs died laterthan shconLLCs by the stimulation of 25120583M acrolein(Figure 5(a)) A large amount of ROS was produced fromshconLLCs by the acrolein treatment which was blockedby NAC ROS production was attenuated by GADD34 defi-ciency (Figure 5(b)) Because ROS was produced highly inshconLLC by acrolein administration compared to that inshGADD34LLC cleaved caspase 3 was highly increasedin shconLLC at 24 h (Figure 5(c)) Then we examined theeffects of ER stresses induced by acrolein In shconLLCs theexpression of p-eIF2120572 increased early and then decreased byacrolein treatment in shconLLC Expression of CHOP wasincreased after the early increase of p-eIF2120572 in shconLLCswhich induced GADD34 then GADD34 dephosphorylated
Oxidative Medicine and Cellular Longevity 7
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8 Oxidative Medicine and Cellular Longevity
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Figure 4 Low level of pulmonary inflammation in GADD34-knockout mice induced by acrolein Lungs were collected from wild-typeand GADD34-knockout mice at days 7 and 28 after 5 120583molkg acrolein instillation (a) Alveolar macrophages as F480highCD11c+ and (b)neutrophils as Gr-1+CD11b+ were confirmed by FACS (c) The expression of phospho-NF-120581B p65 (d) The expressions of macrophage type Imarkers TNF120572 IL-6 and Irf5 and macrophage type II markers Arg-1 Mrc-2 and Retnla were analyzed by quantitative real-time PCR (e)Wild-type and GADD34-knockout mice macrophages were cultured in 12-well plastic plates and stimulated with 10 120583M acrolein for 12 and24 h Supernatants were taken and IL-6 expressionwas analyzed by ELISAData shown are themean ratiosplusmn SE of three separate experimentsData are represented as means plusmn sem lowast119875 lt 005 lowastlowast119875 lt 001
Oxidative Medicine and Cellular Longevity 9
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10 Oxidative Medicine and Cellular Longevity
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Oxidative Medicine and Cellular Longevity 11
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Figure 5 GADD34 promote recovery from a shutoff of total protein synthesis and enhance cell deathThe 25 120583Macrolein-treated shconLLCsand shGADD34LLCs were analyzed (a) Cell survival after 25 120583Macrolein treatment was measured lowast119875 lt 005 (b) Levels of ROS productionin shconLLCs and shGADD34LLCs were measured by DCFH-DA after 25120583M acrolein treated for 8 h (c) Cells were collected at theindicated times and protein expressions of ER stress signaling were detected by western blot (d)The 25 120583M acrolein-treated shconLLCs andshGADD34LLCs with 20mM NAC or without NAC for 8 h Cells were stained with Annexin V-PE7-amino-actinomycin D (7AAD) (e)Bands of proteins were analyzed by SDS-PAGE after 25120583M acrolein treatment (f) Protein concentration was measured by Bio-Rad ProteinAssay (g) The shconLLCs and shGADD34LLCs were treated by 25 120583M acrolein with 10 120583M MG132 Cells were collected and lysate wasanalyzed by SDS-PAGE (h) Amount of proteins was measured by Bio-Rad Protein Assay Data are represented as means plusmn sem lowast119875 lt 005
eIF2120572 In shGADD34LLC the expression of p-eIF2120572 con-tinued to be expressed because of the absence of GADD34
Subsequently the execution phase of apoptosis wasanalyzed to determine the effect of acrolein on shconand shGADD34 LLCs There was a significant increase inannexin V 7-AAD double positive cells in shconLLCs after8 h exposure of acrolein compared with shGADD34LLCs(Figure 5(d))
35 GADD34 Promote Recovery from a Shutoff of Total ProteinSynthesis and Enhance Cell Death In fact the extracellu-lar stimuli and changes in intracellular homeostasis causeprotein misfolding in the endoplasmic reticulum ER stresscaused unfolded protein response (UPR) is a cellular adaptiveresponse that evolved to restore protein-folding homeostasisby reducing protein synthesis Phosphorylation of eIF2120572 lim-its initiation of translation on many cellular mRNAs withinthe cells To clarify whether strongly phosphorylated eIF2120572in shGADD34LLCs can affect translation of related proteinthe protein synthesis was examined We found that proteinsin acrolein-treated WT cells (shconLLCs) do not increaseearly phase by shutoff of protein synthesis caused by p-eIF2120572Protein synthesis gradually recovered in shconLLCs after 6 hbut remained at lower levels in shGADD34LLCs (Figures5(e) and 5(f))
Ubiquitin-proteasome system controls the degradationof a large number of cellular proteins including short-lived regulatory and damaged or misfolded protein [2526] It has been assumed that accumulation of no longerneeded proteins underlies the toxicity of proteasome inhibi-tion Proteasome inhibition can induce the integrated stressresponse (ISR) [27] The ISR is an adaptive response to
many forms of stresses which converge into phosphorylationof eIF2120572 [28] We examined whether inhibition of theproteasome enhanced misfolded proteins synthesis by theaddition of MG132 to acrolein We found that the proteinexpression was enhanced in later time in both shconLLCsand shGADD34 by the addition of MG132 However thelevel of protein expression was less in shGADD34LLCs thanthat in shconLLCs by the addition of MG132 (Figures 5(g)and 5(h)) These results confirmed that protein synthesis wasdecreased by the expression of GADD34 We also discoveredthat acrolein induced higher level of ROS production andcell death in shconLLCs although these treatments hadless effects on ROS production of shGADD34LLCs (Figures6(a) and 6(b)) The addition of MG132 to acrolein treatmentenhanced ROS generation and cell death in shconLLCsProtein synthesis may play a pivotal role in ROS productionand cell death [29 30] A protein synthesis inhibitor (CHX)was added to the treatment of acrolein CHX inhibitedthe generation of ROS and cell death in both shconLLCsand shGADD34LLCs But suppressions of ROS productionand cell death were higher in shconLLCs than that inshGADD34LLCs (Figures 6(a) and 6(b))These results indi-cated that highly phosphorylated eIF2120572 in shGADD34LLCsled to a reduction of synthesized protein which decreased theROS-induced cell death
4 Discussion
COPD is characterized by chronic inflammation and destruc-tion of the lung [31] It is a major clinical challenge mostlydue to cigarette smoke exposure [32] Our findings indicated
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
18] GADD34 dephosphorylates several kinases that functionin important signaling cascades including dephosphoryla-tion of eIF2 [17] Because acrolein is a DNA damaging agentand induces ER stresses [14] and induces myeloid infiltrationto lung [19] here we investigated whether GADD34 mightaffects pathogenesis of acrolein-induced lung injury
2 Materials and Methods
21Mice andAcroleinAdministration Eight-week-old femalewild-type C57BL6 mice were obtained from SLC Japan(Shizuoka Japan) GADD34-knockout mice were generatedas previously described [20] All mice were maintained inpathogen-free facilities in the Animal Research Center at theNagoya University Graduate School of Medicine They weremaintained at 25∘C with a 55 relative humidity and a 12 hlight-dark cycle
For the lung injury studies mice were randomly allocatedinto 3 groups (119899 = 6 per group) The mice were instilledintranasally with acrolein (5120583molkg Sigma) control micereceived equal volume of phosphate buffer saline (PBS pH =74) alone The mice were treated daily for 5 dweek for up to28 days The mice were sacrificed at 7 and 28 days then thelungs were isolated and used for histological analysis andorflow cytometry analysis
22 Cell Culture Murine bone marrow-derived macrophagecells were established previously by Ito et al [21] The cellswere cultured in RPM1640 (Sigma) with 10 fetal bovineserum (FBS Gibco Grand Island NY) and 10 GM-CSFwhich was produced by murine GMCSF-producing Chinesehamster ovary (CHO) cells (GM-CSFCM)
Lewis lung carcinoma (LLC) cells were obtained fromRiken [22] Cells were cultured in HEPES-buffered Dul-beccorsquos modified Eaglersquos medium (DMEM) containing 10FBS (Hyclone)
23 Lentivirus-Mediated shRNAKnockdown of Gene ExpressionThe translation of GADD34mRNA in LLC cells was knockeddown using the Mission TRC mouse shRNA LentivirusTransduction Particles (pLKO1-puro Sigma) The sequencesof the shRNAs used were CCGGGGCGGCTCAGATTG-TTCAAAGCTCGAGCTTTGAACAATCTGAGCCGCCTT-TTTG (shRNA TRC2 targeting exon 2) for GADD34knockdown Nontarget control shRNAs (Sigma SHC202V) were used as a control LLC cells were infectedwith viral particles and treated with 8120583gmL polybrane(Millipore TR-1003-G) and then incubated with cells for24 h Cells expressing shRNA were selected on 2 120583gmLpuromycin (Sigma P8833) for functional studies The extentof knockdown of GADD34 expression was confirmed byreal-time PCR Recombinant experiments used here wereapproved by Committee of Nagoya University GraduateSchool of Medicine Established GADD34 knockdown LLCs(shGAD34LLCs) and control LLCs (shconLLCs) wereused
24 Histology and Immunohistochemistry For histologicalanalysis the lung tissue were isolated frommice after acrolein
treatment and 5 120583m frozen lung sections were stained withhematoxylin and eosin (HampE) For immunofluorescenceanalysis cryosectionswere fixed in cold acetone and blockadewith 2 BSA-PBS for 1 h sections were incubated with rabbitanti-ProSpC (epithelial type II cellsmarker) at 4∘C overnightThe sections were stained with Alexa Flour 448 goat anti-rabbit IgG for 1 h and counterstaining with DAPI for 5min
25 Flow Cytometry The whole lung was minced in thecold PBS After centrifugation the tissue homogenate wassuspended with Tris-NH
4Cl red blood cells (RBCs) lysing
buffer (150mM sodium chloride 1 Triton 05 sodiumdeoxycholate 01 SDS 50mM Tris) to lysed RBCs Cells(1 times 105sample) were blocked in 50120583L 02 BSA-PBSstained with FITC-conjugated anti-Gr1 PE-conjugated anti-CD11b anti-CD11c andAPC-conjugated anti-F480 (BDBio-sciences) for 30min at 4∘C and analyzed by flow cytometryusing a FACS Canto flow cytometer (BD Biosciences)
26 ELISA Cells (GM-IMs) were seeded in 12-well plates (3times 105well) and treated with 10 120583M acrolein (Sigma) for 12and 24 h The level of IL-6 in culture medium was measuredby ELISA kits according to the manufacturerrsquos instructions(RampD systems)
27 ROS Measurements and Inhibition of ROS ProductionLung tissue was isolated from mouse and minced afterlysed RBCs cells (1 times 105well) were seeded in 12-wellplates stained with 2 120583M carboxy-2101584071015840-dihydrofluoresceindiacetate (H2DCFDA) (Invitrogen) for 30min at 37∘C Thecells were collected and ROS generation was determined byflow cytometry
The shconLLCs and shGADD34LLCs were plated in12-well dishes and treated with 25120583M acrolein alone for8 h or 25 120583M acrolein with 10 120583M MG132 orand 1 120583gmLcycloheximide (CHX) for 12 h Afterwashing cells were incu-bated with 2 120583M H2DCFDA for 30min at 37∘C Collectedcells were analyzed for ROS generation by flow cytometryROS inhibitor N-acetyl-L-cysteine (NAC) (Sigma-Aldrich)was dissolved at 1molL in deionized water
28 Western Blotting Lung tissue and cells RIPA lysis buffer(01M PBS pH 74 containing 1 deoxycholic acid sodium02 SDS and protease inhibitors) After measurement ofprotein concentration the sampleswere loaded and separatedby sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE) and then transferred to Immobilon transfermembranes The membranes were incubated with primaryanti-phospho-NF-120581B P65 (Ser536) antibody anti-caspase 3and anti-p-eIF2120572 (Cell Signaling) anti-GADD153 and anti-GADD34 (Santa Cruz Biotechnology) overnight at 4∘CThen the membranes were incubated with the secondaryanti-rabbit IgG for 1 h Blots were developedwithwestern blotdetection reagent (GE Healthcare)
29 Real-Time PCR Total RNA was extracted with TRIzolReagent and reverse-transcribed using a High CapacitycDNA Reverse Transcription Kit (Applied Biosystems)Quantitative real-time PCR was performed using
Oxidative Medicine and Cellular Longevity 3
No dyeConCon + NAC
AcroleinAcrolein + NAC
100
80
60
40
20
0
DCF102 103 104101
(a)
Con
NAC
+
5 120583molkg acrolein
(b)
5120583molkg acrolein205 131
652 197
374 218
573 172
186 118
800 640
151 165
728 911
102 103 104 105
Con
0
102
103
104
105
0
Com
p-A
PC-A
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
Comp-PE-A Comp-PE-A
F480
Com
p-A
PC-A
F480
NAC
+
CD11c CD11c
(c)
NAC
Phospho-NF-120581B p65
NF-120581B p65
0 1 5 0 1 5 (120583molkg)
(d)
Figure 1NACprevents acrolein-induced lung injury and inflammationWild-typemicewere intranasally instilled by 5 120583molkg acroleinwithintravenous injection of 100120583L of NAC (500mgkg) or equal volume of PBS (a) Level of ROS in lung tissues wasmeasured by DCFH-DA (b)HampE staining of lung tissues (scale bar 50120583m) (c) Alveolar macrophages were stained with PE-conjugated anti-CD11c and APC-conjugatedanti-F480 and detected by FACS (d) The expression of phospho-NF-120581B p65 in the lung tissues by western blot
the MX3000P QPCR System (Agilent) according to themanufacturerrsquos protocol The sequences of the RT-PCRprimers for each pair are listed in Table 1
210 Apoptosis The shconLLCs and shGADD34LLCs wereplated in 12-well dishes and treated with 25 120583Macrolein alonefor 8 h or 25 120583M acrolein with 10 120583MMG132 orand 1 120583gmLcycloheximide (CHX) for 12 h Cells were collected and withFITC-labeled annexin V and 7-AAD (BD Bioscience) for15min Cells were analyzed by flow cytometry on a FACSCalibur Data were analyzed by FlowJo software (TreeStar)
211 Protein Synthesis Analysis ShconLLCs and shGADD34LLCs were collected after treatment To count the cell num-ber sampleswere stainedwithTrypanBlueWe centrifuge thecell in 400 rpm for 3min and the supernatant were thrownaway An equal number of live cells were extracted with RIPAlysis buffer (2 times 105 cells100 120583L) All samples were analyzedby SDS-PAGE
212 Statistics Data are expressed as means plusmn standard errorof the mean (sem) Statistical comparisons were performedby ANOVA followed by Fisherrsquos post hoc test Values of 119875 lt005 were considered statistically significant
Figure 2 Acrolein activates ER stress in lung tissue Protein expression of ER stress was analyzed Wild-type GADD34-knockout mice wereintranasally instilled by 5 120583molkg acrolein Lung tissues were collected at the indicated times for western blot analysis
31 NAC Prevents Acrolein-Induced Lung Injury and Inflam-mation ROS generation impaired oxidant defense con-tributes to the organ injury [23] We found that ROS wasgreatly produced from the lung at day 7 and was decreasedby antioxidant NAC treatment (Figure 1(a)) On the basisof analysis of lung sections stained with HampE stainingNAC prevented alveolar damage and immune cell migrationwhich were caused by acrolein in lung tissue (Figure 1(b))
F480high CD11c+ alveolar macrophages were recruited to thesite of injury (Figure 1(c)) It has been shown that smoke-associated oxidative stress may promote lung inflammationthrough NF-120581B signaling [24] We also observed the expres-sion of phospho-NF-120581B p65 by acrolein (7d) which wascompletely abolished by NAC (Figure 1(d))
32 GADD34 Mediates Acrolein-Caused Lung InjuryAcrolein has been shown to induce ER stress [14] Weexamined the expression of ER stressmarkers in lung tissue ofwild-type and GADD34-knockout mice after nasal injectionof acrolein (5 120583molkg) (Figure 2) In wild-type mice theexpression of p-eIF2120572 increased highly between 1 and 4 hafter acrolein exposure which is followed by induction ofGADD34 then p-eIF2120572 expression was decreased from 8 hAfter ER stress we observed the increase of cleaved caspase3 which might cause lung tissue destruction HoweverGADD34-knockout mice continuously express higher levelof p-eIF2120572 the cleaved caspase 3 level was lower than that inwild-type mice
Our previous studies have demonstrated that intranasalinstillation of acrolein (5120583molkg) induced lung damage andhemorrhage [8] We examined whether GADD34-knockoutmice showed same phenotypes by intranasal instillation ofacrolein Although acrolein administration caused seriousalveolar structure destruction such as airspace enlargementand hemorrhaging in the lung of wild-type mice GADD34deficiency decreased the lung injury with preserved alveolarstructure no significant hemorrhaging and sparse accu-mulation of intra-alveolar macrophages at 7 and 28 days
Oxidative Medicine and Cellular Longevity 5
WT
KO
Con 1 5Con 1 5
7d 28d
(120583molkg)
(a)
WT
010
008
006
004
002
000
WTKO
80
60
40
20
0
KO
Con
7d
28d
Con 7d 28d
WTKO
Con 7d 28d
Line
ar in
terc
ept (120583
m)
lowast
lowast
lowast
lowastlowast
Num
ber o
fal
veol
ar m
acro
phag
e (120583
m)
(b)
WT KO
Con
7d
28d
0
5
10
15
WTKO
Con 7d 28d
Num
ber o
f epi
thel
ial t
ype I
I cel
ls(p
lexl
uni
ts)
lowast
lowastlowast
lowastlowast
(c)
Figure 3 Continued
6 Oxidative Medicine and Cellular Longevity
100
80
60
40
20
0
100
80
60
40
20
0
No dyeWT conKO con
WT acroleinKO acrolein
DCF102 103 104 105101
No dyeWT conKO con
WT acroleinKO acrolein
DCF102 103 104 105101
Max
()
Max
()
7d 28d
(d)
Figure 3 GADD34 mediates acrolein-caused lung injury The wild-type and GADD34-knockout mice were intranasally instilled by 1 and5 120583molkg acrolein The mice were treated daily for 5 dweek for up to 28 days and then were sacrificed at 7 and 28 days (a) The whole lungsof wild-type and GADD34-knockout mice were photographed at days 7 and 28 (b) HampE staining of lung tissue Scale bar 50 120583m Analysis ofalveolar length determined by mean linear intercepts (119899 = 5 to 7 mice in each group) and the number of alveolar macrophages in wild-typeand GADD34-knockout mice (119899 = 4 mice in each group) (c) Lungs stained for epithelial type II cells (ProSpC green) nuclei (DAPI blue)and the number of epithelial type II-positive cells in wild-type and GADD34-knockout mice (10 fields 119899 = 4) Scale bar 50 120583m lowast119875 lt 005lowastlowast
119875 lt 001 Data are represented as means plusmn sem (d) Levels of ROS production in the lung of wild-type and GADD34-knockout mice weremeasured by DCFH-DA after acrolein treated
(Figures 3(a) and 3(b)) The numbers of type II epithelialcells were significantly reduced in wild-type mice comparedtoGADD34-knockoutmice (Figure 3(c)) In addition higherlevel of ROS production was detected in wild-type micethan GADD34-knockout mice (Figure 3(d)) These resultscollectively indicated that GADD34 might play a crucialrole in the pathogenesis of experimental acrolein-inducedpulmonary injury
33 Low Level of Pulmonary Inflammation in GADD34-Knockout Mice Induced by Acrolein We have demon-strated that the acrolein-induced lung injury is accom-panied by inflammatory response To investigate whetherGADD34 affects the pathologies of pulmonary inflamma-tory responses the mice were instilled with acrolein for 7or 28 days to generate acute inflammation The increaseof F480highCD11c+ macrophages was lower in GADD34-knockout mice than those in wild-type mice (Figure 4(a))However the GR-1+CD11b+ neutrophils migration was notobserved at 7 or 28 days after acrolein treatment both inwild-type and in GADD34-knockout mice (Figure 4(b))Acrolein-induced lung damage may promote lung inflam-mation through NF-120581B signaling A sizable NF-120581B responsewith phosphorylation of p65 on Ser536 was observed in wild-type mice whereas this response was lowered in GADD34-knockout mice (Figure 4(c))
To assess the gene expression levels of inflammatorycytokine the real-time PCR (RT-PCR) was performedGADD34-knockout mice showed significantly lower levels ofM1 macrophages including TNF120572 IL-6 and Irf5 at 7 days
compared with wild-type mice The expression of TNF120572 andIrf5 decreased at 28 days in both wild-type and GADD34-knockout mice In addition only at late time (at 28 daysafter acrolein administration) wild-type mice expressed highlevel of M2 macrophage markers such as Arg1 and Mrc-1These expressions were lower in GADD34-knockout mice(Figure 4(d)) Then we examined the protein expression ofIL-6 in supernatant of wild-type and GADD34-knockoutmacrophage cell line By the stimulation of acrolein IL-6protein expressionwas higher inwild-typemacrophages thanthat in GADD34-knockout macrophages (Figure 4(e))
34 GADD34 Is a Mediator on ER Stress-Induced OxidativeStress In order to understand the molecular mechanismsof effects of GADD34 on acrolein-induced lung injurywe used lung cell line LLCs ShGADD34LLCs died laterthan shconLLCs by the stimulation of 25120583M acrolein(Figure 5(a)) A large amount of ROS was produced fromshconLLCs by the acrolein treatment which was blockedby NAC ROS production was attenuated by GADD34 defi-ciency (Figure 5(b)) Because ROS was produced highly inshconLLC by acrolein administration compared to that inshGADD34LLC cleaved caspase 3 was highly increasedin shconLLC at 24 h (Figure 5(c)) Then we examined theeffects of ER stresses induced by acrolein In shconLLCs theexpression of p-eIF2120572 increased early and then decreased byacrolein treatment in shconLLC Expression of CHOP wasincreased after the early increase of p-eIF2120572 in shconLLCswhich induced GADD34 then GADD34 dephosphorylated
Oxidative Medicine and Cellular Longevity 7
7d 28dCon
192
621
216
560
118
543
151
411
165
514
130
516
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
WT
KO
0
5
10
15
20
25
Alv
eola
r mac
roph
age (
)
WTKO
Con 7d 28d
lowast
lowastlowast
CD11c CD11c CD11c
F480
F480
(a)
245 541
879 423
161 420
911 305
161 561
896 318
218 454
910 229
149 421
913 298
150 413
917 265
102 103 104102
103
104
102 103 104102
103
104
102 103 104102
103
104
102 103 104 102 103 104 102 103 104102
103
104
102
103
104
102
103
104
WT
KO
7d 28dCon
0
2
4
6
8
Neu
troph
il (
)
WTKO
Con 7d 28d
Gr-1 Gr-1 Gr-1
CD11
bCD
11
b
(b)
7d 28d
WT KO WT KO
0 1 5 0 1 5 0 1 5 0 1 5
Phospho-NF-120581B p65
NF-120581B p65
(120583molkg acrolein)
(c)
Figure 4 Continued
8 Oxidative Medicine and Cellular Longevity
Con 1 5 1 50
1
2
3
4Re
lativ
e qua
ntity
(dR)
7d 28d
0
1
2
3
4
5
Rela
tive q
uant
ity (d
R)
Con 1 5 1 57d 28d
0
2
4
6
Rela
tive q
uant
ity (d
R)
Con 1 5 1 57d 28d
0
1
2
3
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
0
1
2
3
4
5
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
0
1
2
3
4
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
TNF120572 IL6 Irf5
Arg-1 Retnla Mrc-1
lowastlowast lowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast lowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
lowast
lowastlowastlowast
lowastlowast
lowast
lowast
(d)20
WTKO
15
10
5
00
IL6
conc
(pg
mL)
12h 24h
(e)
Figure 4 Low level of pulmonary inflammation in GADD34-knockout mice induced by acrolein Lungs were collected from wild-typeand GADD34-knockout mice at days 7 and 28 after 5 120583molkg acrolein instillation (a) Alveolar macrophages as F480highCD11c+ and (b)neutrophils as Gr-1+CD11b+ were confirmed by FACS (c) The expression of phospho-NF-120581B p65 (d) The expressions of macrophage type Imarkers TNF120572 IL-6 and Irf5 and macrophage type II markers Arg-1 Mrc-2 and Retnla were analyzed by quantitative real-time PCR (e)Wild-type and GADD34-knockout mice macrophages were cultured in 12-well plastic plates and stimulated with 10 120583M acrolein for 12 and24 h Supernatants were taken and IL-6 expressionwas analyzed by ELISAData shown are themean ratiosplusmn SE of three separate experimentsData are represented as means plusmn sem lowast119875 lt 005 lowastlowast119875 lt 001
Oxidative Medicine and Cellular Longevity 9
00
05
10
15
Cel
l sur
viva
l (
)
Shcon
1h 4h 8h 12h 24h0
lowast
lowast
ShGADD34
(a)
0
20
40
60
80
100
Max
()
DCF102 103 104101
No dyeConNAC
AcroleinAcrolein + NAC
No dyeConNAC
AcroleinAcrolein + NAC
0
20
40
60
80
100
Max
()
DCF102 103 104101
Shcon ShGADD34
(b)
Con 1h 6h 12h 24h
P-eIF2120572
eIF2120572
GADD34
GAPDH
CHOP
30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
Caspase 3
Cleaved-caspase 3
(c)
Figure 5 Continued
10 Oxidative Medicine and Cellular Longevity
0601 232
260945
Comp-PE-A
0545 203
271947
Com
p-Pe
rCP-
Cy5-
5-A
145 181
797725
0793 287
292934
Com
p-Pe
rCP-
Cy5-
5-A
0655 209
303942
0661 415
354917
0820 632
721857
0707 266
343932
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
Annexin VComp-PE-AAnnexin V
Comp-PE-AAnnexin V
Comp-PE-AAnnexin V
7A
AD
7A
AD
NAC
+
25 120583M acrolein 25120583M acrolein
Shcon
Con Con
ShGADD34
(d)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
(e)
0
5
10
15
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
lowast
lowast
ShconShGADD34
(f)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
Acrolein + MG132
ShGADD34
(g)
Figure 5 Continued
Oxidative Medicine and Cellular Longevity 11
lowast
lowast
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
Shcon
0
5
10
15
20lowastlowast
lowastlowast
ShGADD34
(h)
Figure 5 GADD34 promote recovery from a shutoff of total protein synthesis and enhance cell deathThe 25 120583Macrolein-treated shconLLCsand shGADD34LLCs were analyzed (a) Cell survival after 25 120583Macrolein treatment was measured lowast119875 lt 005 (b) Levels of ROS productionin shconLLCs and shGADD34LLCs were measured by DCFH-DA after 25120583M acrolein treated for 8 h (c) Cells were collected at theindicated times and protein expressions of ER stress signaling were detected by western blot (d)The 25 120583M acrolein-treated shconLLCs andshGADD34LLCs with 20mM NAC or without NAC for 8 h Cells were stained with Annexin V-PE7-amino-actinomycin D (7AAD) (e)Bands of proteins were analyzed by SDS-PAGE after 25120583M acrolein treatment (f) Protein concentration was measured by Bio-Rad ProteinAssay (g) The shconLLCs and shGADD34LLCs were treated by 25 120583M acrolein with 10 120583M MG132 Cells were collected and lysate wasanalyzed by SDS-PAGE (h) Amount of proteins was measured by Bio-Rad Protein Assay Data are represented as means plusmn sem lowast119875 lt 005
eIF2120572 In shGADD34LLC the expression of p-eIF2120572 con-tinued to be expressed because of the absence of GADD34
Subsequently the execution phase of apoptosis wasanalyzed to determine the effect of acrolein on shconand shGADD34 LLCs There was a significant increase inannexin V 7-AAD double positive cells in shconLLCs after8 h exposure of acrolein compared with shGADD34LLCs(Figure 5(d))
35 GADD34 Promote Recovery from a Shutoff of Total ProteinSynthesis and Enhance Cell Death In fact the extracellu-lar stimuli and changes in intracellular homeostasis causeprotein misfolding in the endoplasmic reticulum ER stresscaused unfolded protein response (UPR) is a cellular adaptiveresponse that evolved to restore protein-folding homeostasisby reducing protein synthesis Phosphorylation of eIF2120572 lim-its initiation of translation on many cellular mRNAs withinthe cells To clarify whether strongly phosphorylated eIF2120572in shGADD34LLCs can affect translation of related proteinthe protein synthesis was examined We found that proteinsin acrolein-treated WT cells (shconLLCs) do not increaseearly phase by shutoff of protein synthesis caused by p-eIF2120572Protein synthesis gradually recovered in shconLLCs after 6 hbut remained at lower levels in shGADD34LLCs (Figures5(e) and 5(f))
Ubiquitin-proteasome system controls the degradationof a large number of cellular proteins including short-lived regulatory and damaged or misfolded protein [2526] It has been assumed that accumulation of no longerneeded proteins underlies the toxicity of proteasome inhibi-tion Proteasome inhibition can induce the integrated stressresponse (ISR) [27] The ISR is an adaptive response to
many forms of stresses which converge into phosphorylationof eIF2120572 [28] We examined whether inhibition of theproteasome enhanced misfolded proteins synthesis by theaddition of MG132 to acrolein We found that the proteinexpression was enhanced in later time in both shconLLCsand shGADD34 by the addition of MG132 However thelevel of protein expression was less in shGADD34LLCs thanthat in shconLLCs by the addition of MG132 (Figures 5(g)and 5(h)) These results confirmed that protein synthesis wasdecreased by the expression of GADD34 We also discoveredthat acrolein induced higher level of ROS production andcell death in shconLLCs although these treatments hadless effects on ROS production of shGADD34LLCs (Figures6(a) and 6(b)) The addition of MG132 to acrolein treatmentenhanced ROS generation and cell death in shconLLCsProtein synthesis may play a pivotal role in ROS productionand cell death [29 30] A protein synthesis inhibitor (CHX)was added to the treatment of acrolein CHX inhibitedthe generation of ROS and cell death in both shconLLCsand shGADD34LLCs But suppressions of ROS productionand cell death were higher in shconLLCs than that inshGADD34LLCs (Figures 6(a) and 6(b))These results indi-cated that highly phosphorylated eIF2120572 in shGADD34LLCsled to a reduction of synthesized protein which decreased theROS-induced cell death
4 Discussion
COPD is characterized by chronic inflammation and destruc-tion of the lung [31] It is a major clinical challenge mostlydue to cigarette smoke exposure [32] Our findings indicated
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
Figure 1NACprevents acrolein-induced lung injury and inflammationWild-typemicewere intranasally instilled by 5 120583molkg acroleinwithintravenous injection of 100120583L of NAC (500mgkg) or equal volume of PBS (a) Level of ROS in lung tissues wasmeasured by DCFH-DA (b)HampE staining of lung tissues (scale bar 50120583m) (c) Alveolar macrophages were stained with PE-conjugated anti-CD11c and APC-conjugatedanti-F480 and detected by FACS (d) The expression of phospho-NF-120581B p65 in the lung tissues by western blot
the MX3000P QPCR System (Agilent) according to themanufacturerrsquos protocol The sequences of the RT-PCRprimers for each pair are listed in Table 1
210 Apoptosis The shconLLCs and shGADD34LLCs wereplated in 12-well dishes and treated with 25 120583Macrolein alonefor 8 h or 25 120583M acrolein with 10 120583MMG132 orand 1 120583gmLcycloheximide (CHX) for 12 h Cells were collected and withFITC-labeled annexin V and 7-AAD (BD Bioscience) for15min Cells were analyzed by flow cytometry on a FACSCalibur Data were analyzed by FlowJo software (TreeStar)
211 Protein Synthesis Analysis ShconLLCs and shGADD34LLCs were collected after treatment To count the cell num-ber sampleswere stainedwithTrypanBlueWe centrifuge thecell in 400 rpm for 3min and the supernatant were thrownaway An equal number of live cells were extracted with RIPAlysis buffer (2 times 105 cells100 120583L) All samples were analyzedby SDS-PAGE
212 Statistics Data are expressed as means plusmn standard errorof the mean (sem) Statistical comparisons were performedby ANOVA followed by Fisherrsquos post hoc test Values of 119875 lt005 were considered statistically significant
Figure 2 Acrolein activates ER stress in lung tissue Protein expression of ER stress was analyzed Wild-type GADD34-knockout mice wereintranasally instilled by 5 120583molkg acrolein Lung tissues were collected at the indicated times for western blot analysis
31 NAC Prevents Acrolein-Induced Lung Injury and Inflam-mation ROS generation impaired oxidant defense con-tributes to the organ injury [23] We found that ROS wasgreatly produced from the lung at day 7 and was decreasedby antioxidant NAC treatment (Figure 1(a)) On the basisof analysis of lung sections stained with HampE stainingNAC prevented alveolar damage and immune cell migrationwhich were caused by acrolein in lung tissue (Figure 1(b))
F480high CD11c+ alveolar macrophages were recruited to thesite of injury (Figure 1(c)) It has been shown that smoke-associated oxidative stress may promote lung inflammationthrough NF-120581B signaling [24] We also observed the expres-sion of phospho-NF-120581B p65 by acrolein (7d) which wascompletely abolished by NAC (Figure 1(d))
32 GADD34 Mediates Acrolein-Caused Lung InjuryAcrolein has been shown to induce ER stress [14] Weexamined the expression of ER stressmarkers in lung tissue ofwild-type and GADD34-knockout mice after nasal injectionof acrolein (5 120583molkg) (Figure 2) In wild-type mice theexpression of p-eIF2120572 increased highly between 1 and 4 hafter acrolein exposure which is followed by induction ofGADD34 then p-eIF2120572 expression was decreased from 8 hAfter ER stress we observed the increase of cleaved caspase3 which might cause lung tissue destruction HoweverGADD34-knockout mice continuously express higher levelof p-eIF2120572 the cleaved caspase 3 level was lower than that inwild-type mice
Our previous studies have demonstrated that intranasalinstillation of acrolein (5120583molkg) induced lung damage andhemorrhage [8] We examined whether GADD34-knockoutmice showed same phenotypes by intranasal instillation ofacrolein Although acrolein administration caused seriousalveolar structure destruction such as airspace enlargementand hemorrhaging in the lung of wild-type mice GADD34deficiency decreased the lung injury with preserved alveolarstructure no significant hemorrhaging and sparse accu-mulation of intra-alveolar macrophages at 7 and 28 days
Oxidative Medicine and Cellular Longevity 5
WT
KO
Con 1 5Con 1 5
7d 28d
(120583molkg)
(a)
WT
010
008
006
004
002
000
WTKO
80
60
40
20
0
KO
Con
7d
28d
Con 7d 28d
WTKO
Con 7d 28d
Line
ar in
terc
ept (120583
m)
lowast
lowast
lowast
lowastlowast
Num
ber o
fal
veol
ar m
acro
phag
e (120583
m)
(b)
WT KO
Con
7d
28d
0
5
10
15
WTKO
Con 7d 28d
Num
ber o
f epi
thel
ial t
ype I
I cel
ls(p
lexl
uni
ts)
lowast
lowastlowast
lowastlowast
(c)
Figure 3 Continued
6 Oxidative Medicine and Cellular Longevity
100
80
60
40
20
0
100
80
60
40
20
0
No dyeWT conKO con
WT acroleinKO acrolein
DCF102 103 104 105101
No dyeWT conKO con
WT acroleinKO acrolein
DCF102 103 104 105101
Max
()
Max
()
7d 28d
(d)
Figure 3 GADD34 mediates acrolein-caused lung injury The wild-type and GADD34-knockout mice were intranasally instilled by 1 and5 120583molkg acrolein The mice were treated daily for 5 dweek for up to 28 days and then were sacrificed at 7 and 28 days (a) The whole lungsof wild-type and GADD34-knockout mice were photographed at days 7 and 28 (b) HampE staining of lung tissue Scale bar 50 120583m Analysis ofalveolar length determined by mean linear intercepts (119899 = 5 to 7 mice in each group) and the number of alveolar macrophages in wild-typeand GADD34-knockout mice (119899 = 4 mice in each group) (c) Lungs stained for epithelial type II cells (ProSpC green) nuclei (DAPI blue)and the number of epithelial type II-positive cells in wild-type and GADD34-knockout mice (10 fields 119899 = 4) Scale bar 50 120583m lowast119875 lt 005lowastlowast
119875 lt 001 Data are represented as means plusmn sem (d) Levels of ROS production in the lung of wild-type and GADD34-knockout mice weremeasured by DCFH-DA after acrolein treated
(Figures 3(a) and 3(b)) The numbers of type II epithelialcells were significantly reduced in wild-type mice comparedtoGADD34-knockoutmice (Figure 3(c)) In addition higherlevel of ROS production was detected in wild-type micethan GADD34-knockout mice (Figure 3(d)) These resultscollectively indicated that GADD34 might play a crucialrole in the pathogenesis of experimental acrolein-inducedpulmonary injury
33 Low Level of Pulmonary Inflammation in GADD34-Knockout Mice Induced by Acrolein We have demon-strated that the acrolein-induced lung injury is accom-panied by inflammatory response To investigate whetherGADD34 affects the pathologies of pulmonary inflamma-tory responses the mice were instilled with acrolein for 7or 28 days to generate acute inflammation The increaseof F480highCD11c+ macrophages was lower in GADD34-knockout mice than those in wild-type mice (Figure 4(a))However the GR-1+CD11b+ neutrophils migration was notobserved at 7 or 28 days after acrolein treatment both inwild-type and in GADD34-knockout mice (Figure 4(b))Acrolein-induced lung damage may promote lung inflam-mation through NF-120581B signaling A sizable NF-120581B responsewith phosphorylation of p65 on Ser536 was observed in wild-type mice whereas this response was lowered in GADD34-knockout mice (Figure 4(c))
To assess the gene expression levels of inflammatorycytokine the real-time PCR (RT-PCR) was performedGADD34-knockout mice showed significantly lower levels ofM1 macrophages including TNF120572 IL-6 and Irf5 at 7 days
compared with wild-type mice The expression of TNF120572 andIrf5 decreased at 28 days in both wild-type and GADD34-knockout mice In addition only at late time (at 28 daysafter acrolein administration) wild-type mice expressed highlevel of M2 macrophage markers such as Arg1 and Mrc-1These expressions were lower in GADD34-knockout mice(Figure 4(d)) Then we examined the protein expression ofIL-6 in supernatant of wild-type and GADD34-knockoutmacrophage cell line By the stimulation of acrolein IL-6protein expressionwas higher inwild-typemacrophages thanthat in GADD34-knockout macrophages (Figure 4(e))
34 GADD34 Is a Mediator on ER Stress-Induced OxidativeStress In order to understand the molecular mechanismsof effects of GADD34 on acrolein-induced lung injurywe used lung cell line LLCs ShGADD34LLCs died laterthan shconLLCs by the stimulation of 25120583M acrolein(Figure 5(a)) A large amount of ROS was produced fromshconLLCs by the acrolein treatment which was blockedby NAC ROS production was attenuated by GADD34 defi-ciency (Figure 5(b)) Because ROS was produced highly inshconLLC by acrolein administration compared to that inshGADD34LLC cleaved caspase 3 was highly increasedin shconLLC at 24 h (Figure 5(c)) Then we examined theeffects of ER stresses induced by acrolein In shconLLCs theexpression of p-eIF2120572 increased early and then decreased byacrolein treatment in shconLLC Expression of CHOP wasincreased after the early increase of p-eIF2120572 in shconLLCswhich induced GADD34 then GADD34 dephosphorylated
Oxidative Medicine and Cellular Longevity 7
7d 28dCon
192
621
216
560
118
543
151
411
165
514
130
516
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
WT
KO
0
5
10
15
20
25
Alv
eola
r mac
roph
age (
)
WTKO
Con 7d 28d
lowast
lowastlowast
CD11c CD11c CD11c
F480
F480
(a)
245 541
879 423
161 420
911 305
161 561
896 318
218 454
910 229
149 421
913 298
150 413
917 265
102 103 104102
103
104
102 103 104102
103
104
102 103 104102
103
104
102 103 104 102 103 104 102 103 104102
103
104
102
103
104
102
103
104
WT
KO
7d 28dCon
0
2
4
6
8
Neu
troph
il (
)
WTKO
Con 7d 28d
Gr-1 Gr-1 Gr-1
CD11
bCD
11
b
(b)
7d 28d
WT KO WT KO
0 1 5 0 1 5 0 1 5 0 1 5
Phospho-NF-120581B p65
NF-120581B p65
(120583molkg acrolein)
(c)
Figure 4 Continued
8 Oxidative Medicine and Cellular Longevity
Con 1 5 1 50
1
2
3
4Re
lativ
e qua
ntity
(dR)
7d 28d
0
1
2
3
4
5
Rela
tive q
uant
ity (d
R)
Con 1 5 1 57d 28d
0
2
4
6
Rela
tive q
uant
ity (d
R)
Con 1 5 1 57d 28d
0
1
2
3
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
0
1
2
3
4
5
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
0
1
2
3
4
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
TNF120572 IL6 Irf5
Arg-1 Retnla Mrc-1
lowastlowast lowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast lowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
lowast
lowastlowastlowast
lowastlowast
lowast
lowast
(d)20
WTKO
15
10
5
00
IL6
conc
(pg
mL)
12h 24h
(e)
Figure 4 Low level of pulmonary inflammation in GADD34-knockout mice induced by acrolein Lungs were collected from wild-typeand GADD34-knockout mice at days 7 and 28 after 5 120583molkg acrolein instillation (a) Alveolar macrophages as F480highCD11c+ and (b)neutrophils as Gr-1+CD11b+ were confirmed by FACS (c) The expression of phospho-NF-120581B p65 (d) The expressions of macrophage type Imarkers TNF120572 IL-6 and Irf5 and macrophage type II markers Arg-1 Mrc-2 and Retnla were analyzed by quantitative real-time PCR (e)Wild-type and GADD34-knockout mice macrophages were cultured in 12-well plastic plates and stimulated with 10 120583M acrolein for 12 and24 h Supernatants were taken and IL-6 expressionwas analyzed by ELISAData shown are themean ratiosplusmn SE of three separate experimentsData are represented as means plusmn sem lowast119875 lt 005 lowastlowast119875 lt 001
Oxidative Medicine and Cellular Longevity 9
00
05
10
15
Cel
l sur
viva
l (
)
Shcon
1h 4h 8h 12h 24h0
lowast
lowast
ShGADD34
(a)
0
20
40
60
80
100
Max
()
DCF102 103 104101
No dyeConNAC
AcroleinAcrolein + NAC
No dyeConNAC
AcroleinAcrolein + NAC
0
20
40
60
80
100
Max
()
DCF102 103 104101
Shcon ShGADD34
(b)
Con 1h 6h 12h 24h
P-eIF2120572
eIF2120572
GADD34
GAPDH
CHOP
30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
Caspase 3
Cleaved-caspase 3
(c)
Figure 5 Continued
10 Oxidative Medicine and Cellular Longevity
0601 232
260945
Comp-PE-A
0545 203
271947
Com
p-Pe
rCP-
Cy5-
5-A
145 181
797725
0793 287
292934
Com
p-Pe
rCP-
Cy5-
5-A
0655 209
303942
0661 415
354917
0820 632
721857
0707 266
343932
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
Annexin VComp-PE-AAnnexin V
Comp-PE-AAnnexin V
Comp-PE-AAnnexin V
7A
AD
7A
AD
NAC
+
25 120583M acrolein 25120583M acrolein
Shcon
Con Con
ShGADD34
(d)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
(e)
0
5
10
15
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
lowast
lowast
ShconShGADD34
(f)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
Acrolein + MG132
ShGADD34
(g)
Figure 5 Continued
Oxidative Medicine and Cellular Longevity 11
lowast
lowast
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
Shcon
0
5
10
15
20lowastlowast
lowastlowast
ShGADD34
(h)
Figure 5 GADD34 promote recovery from a shutoff of total protein synthesis and enhance cell deathThe 25 120583Macrolein-treated shconLLCsand shGADD34LLCs were analyzed (a) Cell survival after 25 120583Macrolein treatment was measured lowast119875 lt 005 (b) Levels of ROS productionin shconLLCs and shGADD34LLCs were measured by DCFH-DA after 25120583M acrolein treated for 8 h (c) Cells were collected at theindicated times and protein expressions of ER stress signaling were detected by western blot (d)The 25 120583M acrolein-treated shconLLCs andshGADD34LLCs with 20mM NAC or without NAC for 8 h Cells were stained with Annexin V-PE7-amino-actinomycin D (7AAD) (e)Bands of proteins were analyzed by SDS-PAGE after 25120583M acrolein treatment (f) Protein concentration was measured by Bio-Rad ProteinAssay (g) The shconLLCs and shGADD34LLCs were treated by 25 120583M acrolein with 10 120583M MG132 Cells were collected and lysate wasanalyzed by SDS-PAGE (h) Amount of proteins was measured by Bio-Rad Protein Assay Data are represented as means plusmn sem lowast119875 lt 005
eIF2120572 In shGADD34LLC the expression of p-eIF2120572 con-tinued to be expressed because of the absence of GADD34
Subsequently the execution phase of apoptosis wasanalyzed to determine the effect of acrolein on shconand shGADD34 LLCs There was a significant increase inannexin V 7-AAD double positive cells in shconLLCs after8 h exposure of acrolein compared with shGADD34LLCs(Figure 5(d))
35 GADD34 Promote Recovery from a Shutoff of Total ProteinSynthesis and Enhance Cell Death In fact the extracellu-lar stimuli and changes in intracellular homeostasis causeprotein misfolding in the endoplasmic reticulum ER stresscaused unfolded protein response (UPR) is a cellular adaptiveresponse that evolved to restore protein-folding homeostasisby reducing protein synthesis Phosphorylation of eIF2120572 lim-its initiation of translation on many cellular mRNAs withinthe cells To clarify whether strongly phosphorylated eIF2120572in shGADD34LLCs can affect translation of related proteinthe protein synthesis was examined We found that proteinsin acrolein-treated WT cells (shconLLCs) do not increaseearly phase by shutoff of protein synthesis caused by p-eIF2120572Protein synthesis gradually recovered in shconLLCs after 6 hbut remained at lower levels in shGADD34LLCs (Figures5(e) and 5(f))
Ubiquitin-proteasome system controls the degradationof a large number of cellular proteins including short-lived regulatory and damaged or misfolded protein [2526] It has been assumed that accumulation of no longerneeded proteins underlies the toxicity of proteasome inhibi-tion Proteasome inhibition can induce the integrated stressresponse (ISR) [27] The ISR is an adaptive response to
many forms of stresses which converge into phosphorylationof eIF2120572 [28] We examined whether inhibition of theproteasome enhanced misfolded proteins synthesis by theaddition of MG132 to acrolein We found that the proteinexpression was enhanced in later time in both shconLLCsand shGADD34 by the addition of MG132 However thelevel of protein expression was less in shGADD34LLCs thanthat in shconLLCs by the addition of MG132 (Figures 5(g)and 5(h)) These results confirmed that protein synthesis wasdecreased by the expression of GADD34 We also discoveredthat acrolein induced higher level of ROS production andcell death in shconLLCs although these treatments hadless effects on ROS production of shGADD34LLCs (Figures6(a) and 6(b)) The addition of MG132 to acrolein treatmentenhanced ROS generation and cell death in shconLLCsProtein synthesis may play a pivotal role in ROS productionand cell death [29 30] A protein synthesis inhibitor (CHX)was added to the treatment of acrolein CHX inhibitedthe generation of ROS and cell death in both shconLLCsand shGADD34LLCs But suppressions of ROS productionand cell death were higher in shconLLCs than that inshGADD34LLCs (Figures 6(a) and 6(b))These results indi-cated that highly phosphorylated eIF2120572 in shGADD34LLCsled to a reduction of synthesized protein which decreased theROS-induced cell death
4 Discussion
COPD is characterized by chronic inflammation and destruc-tion of the lung [31] It is a major clinical challenge mostlydue to cigarette smoke exposure [32] Our findings indicated
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
Figure 2 Acrolein activates ER stress in lung tissue Protein expression of ER stress was analyzed Wild-type GADD34-knockout mice wereintranasally instilled by 5 120583molkg acrolein Lung tissues were collected at the indicated times for western blot analysis
31 NAC Prevents Acrolein-Induced Lung Injury and Inflam-mation ROS generation impaired oxidant defense con-tributes to the organ injury [23] We found that ROS wasgreatly produced from the lung at day 7 and was decreasedby antioxidant NAC treatment (Figure 1(a)) On the basisof analysis of lung sections stained with HampE stainingNAC prevented alveolar damage and immune cell migrationwhich were caused by acrolein in lung tissue (Figure 1(b))
F480high CD11c+ alveolar macrophages were recruited to thesite of injury (Figure 1(c)) It has been shown that smoke-associated oxidative stress may promote lung inflammationthrough NF-120581B signaling [24] We also observed the expres-sion of phospho-NF-120581B p65 by acrolein (7d) which wascompletely abolished by NAC (Figure 1(d))
32 GADD34 Mediates Acrolein-Caused Lung InjuryAcrolein has been shown to induce ER stress [14] Weexamined the expression of ER stressmarkers in lung tissue ofwild-type and GADD34-knockout mice after nasal injectionof acrolein (5 120583molkg) (Figure 2) In wild-type mice theexpression of p-eIF2120572 increased highly between 1 and 4 hafter acrolein exposure which is followed by induction ofGADD34 then p-eIF2120572 expression was decreased from 8 hAfter ER stress we observed the increase of cleaved caspase3 which might cause lung tissue destruction HoweverGADD34-knockout mice continuously express higher levelof p-eIF2120572 the cleaved caspase 3 level was lower than that inwild-type mice
Our previous studies have demonstrated that intranasalinstillation of acrolein (5120583molkg) induced lung damage andhemorrhage [8] We examined whether GADD34-knockoutmice showed same phenotypes by intranasal instillation ofacrolein Although acrolein administration caused seriousalveolar structure destruction such as airspace enlargementand hemorrhaging in the lung of wild-type mice GADD34deficiency decreased the lung injury with preserved alveolarstructure no significant hemorrhaging and sparse accu-mulation of intra-alveolar macrophages at 7 and 28 days
Oxidative Medicine and Cellular Longevity 5
WT
KO
Con 1 5Con 1 5
7d 28d
(120583molkg)
(a)
WT
010
008
006
004
002
000
WTKO
80
60
40
20
0
KO
Con
7d
28d
Con 7d 28d
WTKO
Con 7d 28d
Line
ar in
terc
ept (120583
m)
lowast
lowast
lowast
lowastlowast
Num
ber o
fal
veol
ar m
acro
phag
e (120583
m)
(b)
WT KO
Con
7d
28d
0
5
10
15
WTKO
Con 7d 28d
Num
ber o
f epi
thel
ial t
ype I
I cel
ls(p
lexl
uni
ts)
lowast
lowastlowast
lowastlowast
(c)
Figure 3 Continued
6 Oxidative Medicine and Cellular Longevity
100
80
60
40
20
0
100
80
60
40
20
0
No dyeWT conKO con
WT acroleinKO acrolein
DCF102 103 104 105101
No dyeWT conKO con
WT acroleinKO acrolein
DCF102 103 104 105101
Max
()
Max
()
7d 28d
(d)
Figure 3 GADD34 mediates acrolein-caused lung injury The wild-type and GADD34-knockout mice were intranasally instilled by 1 and5 120583molkg acrolein The mice were treated daily for 5 dweek for up to 28 days and then were sacrificed at 7 and 28 days (a) The whole lungsof wild-type and GADD34-knockout mice were photographed at days 7 and 28 (b) HampE staining of lung tissue Scale bar 50 120583m Analysis ofalveolar length determined by mean linear intercepts (119899 = 5 to 7 mice in each group) and the number of alveolar macrophages in wild-typeand GADD34-knockout mice (119899 = 4 mice in each group) (c) Lungs stained for epithelial type II cells (ProSpC green) nuclei (DAPI blue)and the number of epithelial type II-positive cells in wild-type and GADD34-knockout mice (10 fields 119899 = 4) Scale bar 50 120583m lowast119875 lt 005lowastlowast
119875 lt 001 Data are represented as means plusmn sem (d) Levels of ROS production in the lung of wild-type and GADD34-knockout mice weremeasured by DCFH-DA after acrolein treated
(Figures 3(a) and 3(b)) The numbers of type II epithelialcells were significantly reduced in wild-type mice comparedtoGADD34-knockoutmice (Figure 3(c)) In addition higherlevel of ROS production was detected in wild-type micethan GADD34-knockout mice (Figure 3(d)) These resultscollectively indicated that GADD34 might play a crucialrole in the pathogenesis of experimental acrolein-inducedpulmonary injury
33 Low Level of Pulmonary Inflammation in GADD34-Knockout Mice Induced by Acrolein We have demon-strated that the acrolein-induced lung injury is accom-panied by inflammatory response To investigate whetherGADD34 affects the pathologies of pulmonary inflamma-tory responses the mice were instilled with acrolein for 7or 28 days to generate acute inflammation The increaseof F480highCD11c+ macrophages was lower in GADD34-knockout mice than those in wild-type mice (Figure 4(a))However the GR-1+CD11b+ neutrophils migration was notobserved at 7 or 28 days after acrolein treatment both inwild-type and in GADD34-knockout mice (Figure 4(b))Acrolein-induced lung damage may promote lung inflam-mation through NF-120581B signaling A sizable NF-120581B responsewith phosphorylation of p65 on Ser536 was observed in wild-type mice whereas this response was lowered in GADD34-knockout mice (Figure 4(c))
To assess the gene expression levels of inflammatorycytokine the real-time PCR (RT-PCR) was performedGADD34-knockout mice showed significantly lower levels ofM1 macrophages including TNF120572 IL-6 and Irf5 at 7 days
compared with wild-type mice The expression of TNF120572 andIrf5 decreased at 28 days in both wild-type and GADD34-knockout mice In addition only at late time (at 28 daysafter acrolein administration) wild-type mice expressed highlevel of M2 macrophage markers such as Arg1 and Mrc-1These expressions were lower in GADD34-knockout mice(Figure 4(d)) Then we examined the protein expression ofIL-6 in supernatant of wild-type and GADD34-knockoutmacrophage cell line By the stimulation of acrolein IL-6protein expressionwas higher inwild-typemacrophages thanthat in GADD34-knockout macrophages (Figure 4(e))
34 GADD34 Is a Mediator on ER Stress-Induced OxidativeStress In order to understand the molecular mechanismsof effects of GADD34 on acrolein-induced lung injurywe used lung cell line LLCs ShGADD34LLCs died laterthan shconLLCs by the stimulation of 25120583M acrolein(Figure 5(a)) A large amount of ROS was produced fromshconLLCs by the acrolein treatment which was blockedby NAC ROS production was attenuated by GADD34 defi-ciency (Figure 5(b)) Because ROS was produced highly inshconLLC by acrolein administration compared to that inshGADD34LLC cleaved caspase 3 was highly increasedin shconLLC at 24 h (Figure 5(c)) Then we examined theeffects of ER stresses induced by acrolein In shconLLCs theexpression of p-eIF2120572 increased early and then decreased byacrolein treatment in shconLLC Expression of CHOP wasincreased after the early increase of p-eIF2120572 in shconLLCswhich induced GADD34 then GADD34 dephosphorylated
Oxidative Medicine and Cellular Longevity 7
7d 28dCon
192
621
216
560
118
543
151
411
165
514
130
516
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
WT
KO
0
5
10
15
20
25
Alv
eola
r mac
roph
age (
)
WTKO
Con 7d 28d
lowast
lowastlowast
CD11c CD11c CD11c
F480
F480
(a)
245 541
879 423
161 420
911 305
161 561
896 318
218 454
910 229
149 421
913 298
150 413
917 265
102 103 104102
103
104
102 103 104102
103
104
102 103 104102
103
104
102 103 104 102 103 104 102 103 104102
103
104
102
103
104
102
103
104
WT
KO
7d 28dCon
0
2
4
6
8
Neu
troph
il (
)
WTKO
Con 7d 28d
Gr-1 Gr-1 Gr-1
CD11
bCD
11
b
(b)
7d 28d
WT KO WT KO
0 1 5 0 1 5 0 1 5 0 1 5
Phospho-NF-120581B p65
NF-120581B p65
(120583molkg acrolein)
(c)
Figure 4 Continued
8 Oxidative Medicine and Cellular Longevity
Con 1 5 1 50
1
2
3
4Re
lativ
e qua
ntity
(dR)
7d 28d
0
1
2
3
4
5
Rela
tive q
uant
ity (d
R)
Con 1 5 1 57d 28d
0
2
4
6
Rela
tive q
uant
ity (d
R)
Con 1 5 1 57d 28d
0
1
2
3
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
0
1
2
3
4
5
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
0
1
2
3
4
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
TNF120572 IL6 Irf5
Arg-1 Retnla Mrc-1
lowastlowast lowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast lowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
lowast
lowastlowastlowast
lowastlowast
lowast
lowast
(d)20
WTKO
15
10
5
00
IL6
conc
(pg
mL)
12h 24h
(e)
Figure 4 Low level of pulmonary inflammation in GADD34-knockout mice induced by acrolein Lungs were collected from wild-typeand GADD34-knockout mice at days 7 and 28 after 5 120583molkg acrolein instillation (a) Alveolar macrophages as F480highCD11c+ and (b)neutrophils as Gr-1+CD11b+ were confirmed by FACS (c) The expression of phospho-NF-120581B p65 (d) The expressions of macrophage type Imarkers TNF120572 IL-6 and Irf5 and macrophage type II markers Arg-1 Mrc-2 and Retnla were analyzed by quantitative real-time PCR (e)Wild-type and GADD34-knockout mice macrophages were cultured in 12-well plastic plates and stimulated with 10 120583M acrolein for 12 and24 h Supernatants were taken and IL-6 expressionwas analyzed by ELISAData shown are themean ratiosplusmn SE of three separate experimentsData are represented as means plusmn sem lowast119875 lt 005 lowastlowast119875 lt 001
Oxidative Medicine and Cellular Longevity 9
00
05
10
15
Cel
l sur
viva
l (
)
Shcon
1h 4h 8h 12h 24h0
lowast
lowast
ShGADD34
(a)
0
20
40
60
80
100
Max
()
DCF102 103 104101
No dyeConNAC
AcroleinAcrolein + NAC
No dyeConNAC
AcroleinAcrolein + NAC
0
20
40
60
80
100
Max
()
DCF102 103 104101
Shcon ShGADD34
(b)
Con 1h 6h 12h 24h
P-eIF2120572
eIF2120572
GADD34
GAPDH
CHOP
30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
Caspase 3
Cleaved-caspase 3
(c)
Figure 5 Continued
10 Oxidative Medicine and Cellular Longevity
0601 232
260945
Comp-PE-A
0545 203
271947
Com
p-Pe
rCP-
Cy5-
5-A
145 181
797725
0793 287
292934
Com
p-Pe
rCP-
Cy5-
5-A
0655 209
303942
0661 415
354917
0820 632
721857
0707 266
343932
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
Annexin VComp-PE-AAnnexin V
Comp-PE-AAnnexin V
Comp-PE-AAnnexin V
7A
AD
7A
AD
NAC
+
25 120583M acrolein 25120583M acrolein
Shcon
Con Con
ShGADD34
(d)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
(e)
0
5
10
15
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
lowast
lowast
ShconShGADD34
(f)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
Acrolein + MG132
ShGADD34
(g)
Figure 5 Continued
Oxidative Medicine and Cellular Longevity 11
lowast
lowast
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
Shcon
0
5
10
15
20lowastlowast
lowastlowast
ShGADD34
(h)
Figure 5 GADD34 promote recovery from a shutoff of total protein synthesis and enhance cell deathThe 25 120583Macrolein-treated shconLLCsand shGADD34LLCs were analyzed (a) Cell survival after 25 120583Macrolein treatment was measured lowast119875 lt 005 (b) Levels of ROS productionin shconLLCs and shGADD34LLCs were measured by DCFH-DA after 25120583M acrolein treated for 8 h (c) Cells were collected at theindicated times and protein expressions of ER stress signaling were detected by western blot (d)The 25 120583M acrolein-treated shconLLCs andshGADD34LLCs with 20mM NAC or without NAC for 8 h Cells were stained with Annexin V-PE7-amino-actinomycin D (7AAD) (e)Bands of proteins were analyzed by SDS-PAGE after 25120583M acrolein treatment (f) Protein concentration was measured by Bio-Rad ProteinAssay (g) The shconLLCs and shGADD34LLCs were treated by 25 120583M acrolein with 10 120583M MG132 Cells were collected and lysate wasanalyzed by SDS-PAGE (h) Amount of proteins was measured by Bio-Rad Protein Assay Data are represented as means plusmn sem lowast119875 lt 005
eIF2120572 In shGADD34LLC the expression of p-eIF2120572 con-tinued to be expressed because of the absence of GADD34
Subsequently the execution phase of apoptosis wasanalyzed to determine the effect of acrolein on shconand shGADD34 LLCs There was a significant increase inannexin V 7-AAD double positive cells in shconLLCs after8 h exposure of acrolein compared with shGADD34LLCs(Figure 5(d))
35 GADD34 Promote Recovery from a Shutoff of Total ProteinSynthesis and Enhance Cell Death In fact the extracellu-lar stimuli and changes in intracellular homeostasis causeprotein misfolding in the endoplasmic reticulum ER stresscaused unfolded protein response (UPR) is a cellular adaptiveresponse that evolved to restore protein-folding homeostasisby reducing protein synthesis Phosphorylation of eIF2120572 lim-its initiation of translation on many cellular mRNAs withinthe cells To clarify whether strongly phosphorylated eIF2120572in shGADD34LLCs can affect translation of related proteinthe protein synthesis was examined We found that proteinsin acrolein-treated WT cells (shconLLCs) do not increaseearly phase by shutoff of protein synthesis caused by p-eIF2120572Protein synthesis gradually recovered in shconLLCs after 6 hbut remained at lower levels in shGADD34LLCs (Figures5(e) and 5(f))
Ubiquitin-proteasome system controls the degradationof a large number of cellular proteins including short-lived regulatory and damaged or misfolded protein [2526] It has been assumed that accumulation of no longerneeded proteins underlies the toxicity of proteasome inhibi-tion Proteasome inhibition can induce the integrated stressresponse (ISR) [27] The ISR is an adaptive response to
many forms of stresses which converge into phosphorylationof eIF2120572 [28] We examined whether inhibition of theproteasome enhanced misfolded proteins synthesis by theaddition of MG132 to acrolein We found that the proteinexpression was enhanced in later time in both shconLLCsand shGADD34 by the addition of MG132 However thelevel of protein expression was less in shGADD34LLCs thanthat in shconLLCs by the addition of MG132 (Figures 5(g)and 5(h)) These results confirmed that protein synthesis wasdecreased by the expression of GADD34 We also discoveredthat acrolein induced higher level of ROS production andcell death in shconLLCs although these treatments hadless effects on ROS production of shGADD34LLCs (Figures6(a) and 6(b)) The addition of MG132 to acrolein treatmentenhanced ROS generation and cell death in shconLLCsProtein synthesis may play a pivotal role in ROS productionand cell death [29 30] A protein synthesis inhibitor (CHX)was added to the treatment of acrolein CHX inhibitedthe generation of ROS and cell death in both shconLLCsand shGADD34LLCs But suppressions of ROS productionand cell death were higher in shconLLCs than that inshGADD34LLCs (Figures 6(a) and 6(b))These results indi-cated that highly phosphorylated eIF2120572 in shGADD34LLCsled to a reduction of synthesized protein which decreased theROS-induced cell death
4 Discussion
COPD is characterized by chronic inflammation and destruc-tion of the lung [31] It is a major clinical challenge mostlydue to cigarette smoke exposure [32] Our findings indicated
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
Figure 3 GADD34 mediates acrolein-caused lung injury The wild-type and GADD34-knockout mice were intranasally instilled by 1 and5 120583molkg acrolein The mice were treated daily for 5 dweek for up to 28 days and then were sacrificed at 7 and 28 days (a) The whole lungsof wild-type and GADD34-knockout mice were photographed at days 7 and 28 (b) HampE staining of lung tissue Scale bar 50 120583m Analysis ofalveolar length determined by mean linear intercepts (119899 = 5 to 7 mice in each group) and the number of alveolar macrophages in wild-typeand GADD34-knockout mice (119899 = 4 mice in each group) (c) Lungs stained for epithelial type II cells (ProSpC green) nuclei (DAPI blue)and the number of epithelial type II-positive cells in wild-type and GADD34-knockout mice (10 fields 119899 = 4) Scale bar 50 120583m lowast119875 lt 005lowastlowast
119875 lt 001 Data are represented as means plusmn sem (d) Levels of ROS production in the lung of wild-type and GADD34-knockout mice weremeasured by DCFH-DA after acrolein treated
(Figures 3(a) and 3(b)) The numbers of type II epithelialcells were significantly reduced in wild-type mice comparedtoGADD34-knockoutmice (Figure 3(c)) In addition higherlevel of ROS production was detected in wild-type micethan GADD34-knockout mice (Figure 3(d)) These resultscollectively indicated that GADD34 might play a crucialrole in the pathogenesis of experimental acrolein-inducedpulmonary injury
33 Low Level of Pulmonary Inflammation in GADD34-Knockout Mice Induced by Acrolein We have demon-strated that the acrolein-induced lung injury is accom-panied by inflammatory response To investigate whetherGADD34 affects the pathologies of pulmonary inflamma-tory responses the mice were instilled with acrolein for 7or 28 days to generate acute inflammation The increaseof F480highCD11c+ macrophages was lower in GADD34-knockout mice than those in wild-type mice (Figure 4(a))However the GR-1+CD11b+ neutrophils migration was notobserved at 7 or 28 days after acrolein treatment both inwild-type and in GADD34-knockout mice (Figure 4(b))Acrolein-induced lung damage may promote lung inflam-mation through NF-120581B signaling A sizable NF-120581B responsewith phosphorylation of p65 on Ser536 was observed in wild-type mice whereas this response was lowered in GADD34-knockout mice (Figure 4(c))
To assess the gene expression levels of inflammatorycytokine the real-time PCR (RT-PCR) was performedGADD34-knockout mice showed significantly lower levels ofM1 macrophages including TNF120572 IL-6 and Irf5 at 7 days
compared with wild-type mice The expression of TNF120572 andIrf5 decreased at 28 days in both wild-type and GADD34-knockout mice In addition only at late time (at 28 daysafter acrolein administration) wild-type mice expressed highlevel of M2 macrophage markers such as Arg1 and Mrc-1These expressions were lower in GADD34-knockout mice(Figure 4(d)) Then we examined the protein expression ofIL-6 in supernatant of wild-type and GADD34-knockoutmacrophage cell line By the stimulation of acrolein IL-6protein expressionwas higher inwild-typemacrophages thanthat in GADD34-knockout macrophages (Figure 4(e))
34 GADD34 Is a Mediator on ER Stress-Induced OxidativeStress In order to understand the molecular mechanismsof effects of GADD34 on acrolein-induced lung injurywe used lung cell line LLCs ShGADD34LLCs died laterthan shconLLCs by the stimulation of 25120583M acrolein(Figure 5(a)) A large amount of ROS was produced fromshconLLCs by the acrolein treatment which was blockedby NAC ROS production was attenuated by GADD34 defi-ciency (Figure 5(b)) Because ROS was produced highly inshconLLC by acrolein administration compared to that inshGADD34LLC cleaved caspase 3 was highly increasedin shconLLC at 24 h (Figure 5(c)) Then we examined theeffects of ER stresses induced by acrolein In shconLLCs theexpression of p-eIF2120572 increased early and then decreased byacrolein treatment in shconLLC Expression of CHOP wasincreased after the early increase of p-eIF2120572 in shconLLCswhich induced GADD34 then GADD34 dephosphorylated
Oxidative Medicine and Cellular Longevity 7
7d 28dCon
192
621
216
560
118
543
151
411
165
514
130
516
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
WT
KO
0
5
10
15
20
25
Alv
eola
r mac
roph
age (
)
WTKO
Con 7d 28d
lowast
lowastlowast
CD11c CD11c CD11c
F480
F480
(a)
245 541
879 423
161 420
911 305
161 561
896 318
218 454
910 229
149 421
913 298
150 413
917 265
102 103 104102
103
104
102 103 104102
103
104
102 103 104102
103
104
102 103 104 102 103 104 102 103 104102
103
104
102
103
104
102
103
104
WT
KO
7d 28dCon
0
2
4
6
8
Neu
troph
il (
)
WTKO
Con 7d 28d
Gr-1 Gr-1 Gr-1
CD11
bCD
11
b
(b)
7d 28d
WT KO WT KO
0 1 5 0 1 5 0 1 5 0 1 5
Phospho-NF-120581B p65
NF-120581B p65
(120583molkg acrolein)
(c)
Figure 4 Continued
8 Oxidative Medicine and Cellular Longevity
Con 1 5 1 50
1
2
3
4Re
lativ
e qua
ntity
(dR)
7d 28d
0
1
2
3
4
5
Rela
tive q
uant
ity (d
R)
Con 1 5 1 57d 28d
0
2
4
6
Rela
tive q
uant
ity (d
R)
Con 1 5 1 57d 28d
0
1
2
3
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
0
1
2
3
4
5
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
0
1
2
3
4
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
TNF120572 IL6 Irf5
Arg-1 Retnla Mrc-1
lowastlowast lowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast lowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
lowast
lowastlowastlowast
lowastlowast
lowast
lowast
(d)20
WTKO
15
10
5
00
IL6
conc
(pg
mL)
12h 24h
(e)
Figure 4 Low level of pulmonary inflammation in GADD34-knockout mice induced by acrolein Lungs were collected from wild-typeand GADD34-knockout mice at days 7 and 28 after 5 120583molkg acrolein instillation (a) Alveolar macrophages as F480highCD11c+ and (b)neutrophils as Gr-1+CD11b+ were confirmed by FACS (c) The expression of phospho-NF-120581B p65 (d) The expressions of macrophage type Imarkers TNF120572 IL-6 and Irf5 and macrophage type II markers Arg-1 Mrc-2 and Retnla were analyzed by quantitative real-time PCR (e)Wild-type and GADD34-knockout mice macrophages were cultured in 12-well plastic plates and stimulated with 10 120583M acrolein for 12 and24 h Supernatants were taken and IL-6 expressionwas analyzed by ELISAData shown are themean ratiosplusmn SE of three separate experimentsData are represented as means plusmn sem lowast119875 lt 005 lowastlowast119875 lt 001
Oxidative Medicine and Cellular Longevity 9
00
05
10
15
Cel
l sur
viva
l (
)
Shcon
1h 4h 8h 12h 24h0
lowast
lowast
ShGADD34
(a)
0
20
40
60
80
100
Max
()
DCF102 103 104101
No dyeConNAC
AcroleinAcrolein + NAC
No dyeConNAC
AcroleinAcrolein + NAC
0
20
40
60
80
100
Max
()
DCF102 103 104101
Shcon ShGADD34
(b)
Con 1h 6h 12h 24h
P-eIF2120572
eIF2120572
GADD34
GAPDH
CHOP
30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
Caspase 3
Cleaved-caspase 3
(c)
Figure 5 Continued
10 Oxidative Medicine and Cellular Longevity
0601 232
260945
Comp-PE-A
0545 203
271947
Com
p-Pe
rCP-
Cy5-
5-A
145 181
797725
0793 287
292934
Com
p-Pe
rCP-
Cy5-
5-A
0655 209
303942
0661 415
354917
0820 632
721857
0707 266
343932
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
Annexin VComp-PE-AAnnexin V
Comp-PE-AAnnexin V
Comp-PE-AAnnexin V
7A
AD
7A
AD
NAC
+
25 120583M acrolein 25120583M acrolein
Shcon
Con Con
ShGADD34
(d)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
(e)
0
5
10
15
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
lowast
lowast
ShconShGADD34
(f)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
Acrolein + MG132
ShGADD34
(g)
Figure 5 Continued
Oxidative Medicine and Cellular Longevity 11
lowast
lowast
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
Shcon
0
5
10
15
20lowastlowast
lowastlowast
ShGADD34
(h)
Figure 5 GADD34 promote recovery from a shutoff of total protein synthesis and enhance cell deathThe 25 120583Macrolein-treated shconLLCsand shGADD34LLCs were analyzed (a) Cell survival after 25 120583Macrolein treatment was measured lowast119875 lt 005 (b) Levels of ROS productionin shconLLCs and shGADD34LLCs were measured by DCFH-DA after 25120583M acrolein treated for 8 h (c) Cells were collected at theindicated times and protein expressions of ER stress signaling were detected by western blot (d)The 25 120583M acrolein-treated shconLLCs andshGADD34LLCs with 20mM NAC or without NAC for 8 h Cells were stained with Annexin V-PE7-amino-actinomycin D (7AAD) (e)Bands of proteins were analyzed by SDS-PAGE after 25120583M acrolein treatment (f) Protein concentration was measured by Bio-Rad ProteinAssay (g) The shconLLCs and shGADD34LLCs were treated by 25 120583M acrolein with 10 120583M MG132 Cells were collected and lysate wasanalyzed by SDS-PAGE (h) Amount of proteins was measured by Bio-Rad Protein Assay Data are represented as means plusmn sem lowast119875 lt 005
eIF2120572 In shGADD34LLC the expression of p-eIF2120572 con-tinued to be expressed because of the absence of GADD34
Subsequently the execution phase of apoptosis wasanalyzed to determine the effect of acrolein on shconand shGADD34 LLCs There was a significant increase inannexin V 7-AAD double positive cells in shconLLCs after8 h exposure of acrolein compared with shGADD34LLCs(Figure 5(d))
35 GADD34 Promote Recovery from a Shutoff of Total ProteinSynthesis and Enhance Cell Death In fact the extracellu-lar stimuli and changes in intracellular homeostasis causeprotein misfolding in the endoplasmic reticulum ER stresscaused unfolded protein response (UPR) is a cellular adaptiveresponse that evolved to restore protein-folding homeostasisby reducing protein synthesis Phosphorylation of eIF2120572 lim-its initiation of translation on many cellular mRNAs withinthe cells To clarify whether strongly phosphorylated eIF2120572in shGADD34LLCs can affect translation of related proteinthe protein synthesis was examined We found that proteinsin acrolein-treated WT cells (shconLLCs) do not increaseearly phase by shutoff of protein synthesis caused by p-eIF2120572Protein synthesis gradually recovered in shconLLCs after 6 hbut remained at lower levels in shGADD34LLCs (Figures5(e) and 5(f))
Ubiquitin-proteasome system controls the degradationof a large number of cellular proteins including short-lived regulatory and damaged or misfolded protein [2526] It has been assumed that accumulation of no longerneeded proteins underlies the toxicity of proteasome inhibi-tion Proteasome inhibition can induce the integrated stressresponse (ISR) [27] The ISR is an adaptive response to
many forms of stresses which converge into phosphorylationof eIF2120572 [28] We examined whether inhibition of theproteasome enhanced misfolded proteins synthesis by theaddition of MG132 to acrolein We found that the proteinexpression was enhanced in later time in both shconLLCsand shGADD34 by the addition of MG132 However thelevel of protein expression was less in shGADD34LLCs thanthat in shconLLCs by the addition of MG132 (Figures 5(g)and 5(h)) These results confirmed that protein synthesis wasdecreased by the expression of GADD34 We also discoveredthat acrolein induced higher level of ROS production andcell death in shconLLCs although these treatments hadless effects on ROS production of shGADD34LLCs (Figures6(a) and 6(b)) The addition of MG132 to acrolein treatmentenhanced ROS generation and cell death in shconLLCsProtein synthesis may play a pivotal role in ROS productionand cell death [29 30] A protein synthesis inhibitor (CHX)was added to the treatment of acrolein CHX inhibitedthe generation of ROS and cell death in both shconLLCsand shGADD34LLCs But suppressions of ROS productionand cell death were higher in shconLLCs than that inshGADD34LLCs (Figures 6(a) and 6(b))These results indi-cated that highly phosphorylated eIF2120572 in shGADD34LLCsled to a reduction of synthesized protein which decreased theROS-induced cell death
4 Discussion
COPD is characterized by chronic inflammation and destruc-tion of the lung [31] It is a major clinical challenge mostlydue to cigarette smoke exposure [32] Our findings indicated
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
Figure 3 GADD34 mediates acrolein-caused lung injury The wild-type and GADD34-knockout mice were intranasally instilled by 1 and5 120583molkg acrolein The mice were treated daily for 5 dweek for up to 28 days and then were sacrificed at 7 and 28 days (a) The whole lungsof wild-type and GADD34-knockout mice were photographed at days 7 and 28 (b) HampE staining of lung tissue Scale bar 50 120583m Analysis ofalveolar length determined by mean linear intercepts (119899 = 5 to 7 mice in each group) and the number of alveolar macrophages in wild-typeand GADD34-knockout mice (119899 = 4 mice in each group) (c) Lungs stained for epithelial type II cells (ProSpC green) nuclei (DAPI blue)and the number of epithelial type II-positive cells in wild-type and GADD34-knockout mice (10 fields 119899 = 4) Scale bar 50 120583m lowast119875 lt 005lowastlowast
119875 lt 001 Data are represented as means plusmn sem (d) Levels of ROS production in the lung of wild-type and GADD34-knockout mice weremeasured by DCFH-DA after acrolein treated
(Figures 3(a) and 3(b)) The numbers of type II epithelialcells were significantly reduced in wild-type mice comparedtoGADD34-knockoutmice (Figure 3(c)) In addition higherlevel of ROS production was detected in wild-type micethan GADD34-knockout mice (Figure 3(d)) These resultscollectively indicated that GADD34 might play a crucialrole in the pathogenesis of experimental acrolein-inducedpulmonary injury
33 Low Level of Pulmonary Inflammation in GADD34-Knockout Mice Induced by Acrolein We have demon-strated that the acrolein-induced lung injury is accom-panied by inflammatory response To investigate whetherGADD34 affects the pathologies of pulmonary inflamma-tory responses the mice were instilled with acrolein for 7or 28 days to generate acute inflammation The increaseof F480highCD11c+ macrophages was lower in GADD34-knockout mice than those in wild-type mice (Figure 4(a))However the GR-1+CD11b+ neutrophils migration was notobserved at 7 or 28 days after acrolein treatment both inwild-type and in GADD34-knockout mice (Figure 4(b))Acrolein-induced lung damage may promote lung inflam-mation through NF-120581B signaling A sizable NF-120581B responsewith phosphorylation of p65 on Ser536 was observed in wild-type mice whereas this response was lowered in GADD34-knockout mice (Figure 4(c))
To assess the gene expression levels of inflammatorycytokine the real-time PCR (RT-PCR) was performedGADD34-knockout mice showed significantly lower levels ofM1 macrophages including TNF120572 IL-6 and Irf5 at 7 days
compared with wild-type mice The expression of TNF120572 andIrf5 decreased at 28 days in both wild-type and GADD34-knockout mice In addition only at late time (at 28 daysafter acrolein administration) wild-type mice expressed highlevel of M2 macrophage markers such as Arg1 and Mrc-1These expressions were lower in GADD34-knockout mice(Figure 4(d)) Then we examined the protein expression ofIL-6 in supernatant of wild-type and GADD34-knockoutmacrophage cell line By the stimulation of acrolein IL-6protein expressionwas higher inwild-typemacrophages thanthat in GADD34-knockout macrophages (Figure 4(e))
34 GADD34 Is a Mediator on ER Stress-Induced OxidativeStress In order to understand the molecular mechanismsof effects of GADD34 on acrolein-induced lung injurywe used lung cell line LLCs ShGADD34LLCs died laterthan shconLLCs by the stimulation of 25120583M acrolein(Figure 5(a)) A large amount of ROS was produced fromshconLLCs by the acrolein treatment which was blockedby NAC ROS production was attenuated by GADD34 defi-ciency (Figure 5(b)) Because ROS was produced highly inshconLLC by acrolein administration compared to that inshGADD34LLC cleaved caspase 3 was highly increasedin shconLLC at 24 h (Figure 5(c)) Then we examined theeffects of ER stresses induced by acrolein In shconLLCs theexpression of p-eIF2120572 increased early and then decreased byacrolein treatment in shconLLC Expression of CHOP wasincreased after the early increase of p-eIF2120572 in shconLLCswhich induced GADD34 then GADD34 dephosphorylated
Oxidative Medicine and Cellular Longevity 7
7d 28dCon
192
621
216
560
118
543
151
411
165
514
130
516
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
102 103 104 1050
102
103
104
105
0
WT
KO
0
5
10
15
20
25
Alv
eola
r mac
roph
age (
)
WTKO
Con 7d 28d
lowast
lowastlowast
CD11c CD11c CD11c
F480
F480
(a)
245 541
879 423
161 420
911 305
161 561
896 318
218 454
910 229
149 421
913 298
150 413
917 265
102 103 104102
103
104
102 103 104102
103
104
102 103 104102
103
104
102 103 104 102 103 104 102 103 104102
103
104
102
103
104
102
103
104
WT
KO
7d 28dCon
0
2
4
6
8
Neu
troph
il (
)
WTKO
Con 7d 28d
Gr-1 Gr-1 Gr-1
CD11
bCD
11
b
(b)
7d 28d
WT KO WT KO
0 1 5 0 1 5 0 1 5 0 1 5
Phospho-NF-120581B p65
NF-120581B p65
(120583molkg acrolein)
(c)
Figure 4 Continued
8 Oxidative Medicine and Cellular Longevity
Con 1 5 1 50
1
2
3
4Re
lativ
e qua
ntity
(dR)
7d 28d
0
1
2
3
4
5
Rela
tive q
uant
ity (d
R)
Con 1 5 1 57d 28d
0
2
4
6
Rela
tive q
uant
ity (d
R)
Con 1 5 1 57d 28d
0
1
2
3
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
0
1
2
3
4
5
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
0
1
2
3
4
Rela
tive q
uant
ity (d
R)
Con 1 5 1 5
WTKO
7d 28d
TNF120572 IL6 Irf5
Arg-1 Retnla Mrc-1
lowastlowast lowastlowastlowastlowast
lowastlowast
lowastlowast
lowastlowast
lowastlowastlowast
lowast lowast
lowastlowast
lowastlowastlowastlowast
lowastlowastlowastlowast
lowast
lowastlowastlowast
lowastlowast
lowast
lowast
(d)20
WTKO
15
10
5
00
IL6
conc
(pg
mL)
12h 24h
(e)
Figure 4 Low level of pulmonary inflammation in GADD34-knockout mice induced by acrolein Lungs were collected from wild-typeand GADD34-knockout mice at days 7 and 28 after 5 120583molkg acrolein instillation (a) Alveolar macrophages as F480highCD11c+ and (b)neutrophils as Gr-1+CD11b+ were confirmed by FACS (c) The expression of phospho-NF-120581B p65 (d) The expressions of macrophage type Imarkers TNF120572 IL-6 and Irf5 and macrophage type II markers Arg-1 Mrc-2 and Retnla were analyzed by quantitative real-time PCR (e)Wild-type and GADD34-knockout mice macrophages were cultured in 12-well plastic plates and stimulated with 10 120583M acrolein for 12 and24 h Supernatants were taken and IL-6 expressionwas analyzed by ELISAData shown are themean ratiosplusmn SE of three separate experimentsData are represented as means plusmn sem lowast119875 lt 005 lowastlowast119875 lt 001
Oxidative Medicine and Cellular Longevity 9
00
05
10
15
Cel
l sur
viva
l (
)
Shcon
1h 4h 8h 12h 24h0
lowast
lowast
ShGADD34
(a)
0
20
40
60
80
100
Max
()
DCF102 103 104101
No dyeConNAC
AcroleinAcrolein + NAC
No dyeConNAC
AcroleinAcrolein + NAC
0
20
40
60
80
100
Max
()
DCF102 103 104101
Shcon ShGADD34
(b)
Con 1h 6h 12h 24h
P-eIF2120572
eIF2120572
GADD34
GAPDH
CHOP
30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
Caspase 3
Cleaved-caspase 3
(c)
Figure 5 Continued
10 Oxidative Medicine and Cellular Longevity
0601 232
260945
Comp-PE-A
0545 203
271947
Com
p-Pe
rCP-
Cy5-
5-A
145 181
797725
0793 287
292934
Com
p-Pe
rCP-
Cy5-
5-A
0655 209
303942
0661 415
354917
0820 632
721857
0707 266
343932
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
Annexin VComp-PE-AAnnexin V
Comp-PE-AAnnexin V
Comp-PE-AAnnexin V
7A
AD
7A
AD
NAC
+
25 120583M acrolein 25120583M acrolein
Shcon
Con Con
ShGADD34
(d)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
(e)
0
5
10
15
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
lowast
lowast
ShconShGADD34
(f)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
Acrolein + MG132
ShGADD34
(g)
Figure 5 Continued
Oxidative Medicine and Cellular Longevity 11
lowast
lowast
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
Shcon
0
5
10
15
20lowastlowast
lowastlowast
ShGADD34
(h)
Figure 5 GADD34 promote recovery from a shutoff of total protein synthesis and enhance cell deathThe 25 120583Macrolein-treated shconLLCsand shGADD34LLCs were analyzed (a) Cell survival after 25 120583Macrolein treatment was measured lowast119875 lt 005 (b) Levels of ROS productionin shconLLCs and shGADD34LLCs were measured by DCFH-DA after 25120583M acrolein treated for 8 h (c) Cells were collected at theindicated times and protein expressions of ER stress signaling were detected by western blot (d)The 25 120583M acrolein-treated shconLLCs andshGADD34LLCs with 20mM NAC or without NAC for 8 h Cells were stained with Annexin V-PE7-amino-actinomycin D (7AAD) (e)Bands of proteins were analyzed by SDS-PAGE after 25120583M acrolein treatment (f) Protein concentration was measured by Bio-Rad ProteinAssay (g) The shconLLCs and shGADD34LLCs were treated by 25 120583M acrolein with 10 120583M MG132 Cells were collected and lysate wasanalyzed by SDS-PAGE (h) Amount of proteins was measured by Bio-Rad Protein Assay Data are represented as means plusmn sem lowast119875 lt 005
eIF2120572 In shGADD34LLC the expression of p-eIF2120572 con-tinued to be expressed because of the absence of GADD34
Subsequently the execution phase of apoptosis wasanalyzed to determine the effect of acrolein on shconand shGADD34 LLCs There was a significant increase inannexin V 7-AAD double positive cells in shconLLCs after8 h exposure of acrolein compared with shGADD34LLCs(Figure 5(d))
35 GADD34 Promote Recovery from a Shutoff of Total ProteinSynthesis and Enhance Cell Death In fact the extracellu-lar stimuli and changes in intracellular homeostasis causeprotein misfolding in the endoplasmic reticulum ER stresscaused unfolded protein response (UPR) is a cellular adaptiveresponse that evolved to restore protein-folding homeostasisby reducing protein synthesis Phosphorylation of eIF2120572 lim-its initiation of translation on many cellular mRNAs withinthe cells To clarify whether strongly phosphorylated eIF2120572in shGADD34LLCs can affect translation of related proteinthe protein synthesis was examined We found that proteinsin acrolein-treated WT cells (shconLLCs) do not increaseearly phase by shutoff of protein synthesis caused by p-eIF2120572Protein synthesis gradually recovered in shconLLCs after 6 hbut remained at lower levels in shGADD34LLCs (Figures5(e) and 5(f))
Ubiquitin-proteasome system controls the degradationof a large number of cellular proteins including short-lived regulatory and damaged or misfolded protein [2526] It has been assumed that accumulation of no longerneeded proteins underlies the toxicity of proteasome inhibi-tion Proteasome inhibition can induce the integrated stressresponse (ISR) [27] The ISR is an adaptive response to
many forms of stresses which converge into phosphorylationof eIF2120572 [28] We examined whether inhibition of theproteasome enhanced misfolded proteins synthesis by theaddition of MG132 to acrolein We found that the proteinexpression was enhanced in later time in both shconLLCsand shGADD34 by the addition of MG132 However thelevel of protein expression was less in shGADD34LLCs thanthat in shconLLCs by the addition of MG132 (Figures 5(g)and 5(h)) These results confirmed that protein synthesis wasdecreased by the expression of GADD34 We also discoveredthat acrolein induced higher level of ROS production andcell death in shconLLCs although these treatments hadless effects on ROS production of shGADD34LLCs (Figures6(a) and 6(b)) The addition of MG132 to acrolein treatmentenhanced ROS generation and cell death in shconLLCsProtein synthesis may play a pivotal role in ROS productionand cell death [29 30] A protein synthesis inhibitor (CHX)was added to the treatment of acrolein CHX inhibitedthe generation of ROS and cell death in both shconLLCsand shGADD34LLCs But suppressions of ROS productionand cell death were higher in shconLLCs than that inshGADD34LLCs (Figures 6(a) and 6(b))These results indi-cated that highly phosphorylated eIF2120572 in shGADD34LLCsled to a reduction of synthesized protein which decreased theROS-induced cell death
4 Discussion
COPD is characterized by chronic inflammation and destruc-tion of the lung [31] It is a major clinical challenge mostlydue to cigarette smoke exposure [32] Our findings indicated
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
Figure 4 Low level of pulmonary inflammation in GADD34-knockout mice induced by acrolein Lungs were collected from wild-typeand GADD34-knockout mice at days 7 and 28 after 5 120583molkg acrolein instillation (a) Alveolar macrophages as F480highCD11c+ and (b)neutrophils as Gr-1+CD11b+ were confirmed by FACS (c) The expression of phospho-NF-120581B p65 (d) The expressions of macrophage type Imarkers TNF120572 IL-6 and Irf5 and macrophage type II markers Arg-1 Mrc-2 and Retnla were analyzed by quantitative real-time PCR (e)Wild-type and GADD34-knockout mice macrophages were cultured in 12-well plastic plates and stimulated with 10 120583M acrolein for 12 and24 h Supernatants were taken and IL-6 expressionwas analyzed by ELISAData shown are themean ratiosplusmn SE of three separate experimentsData are represented as means plusmn sem lowast119875 lt 005 lowastlowast119875 lt 001
Oxidative Medicine and Cellular Longevity 9
00
05
10
15
Cel
l sur
viva
l (
)
Shcon
1h 4h 8h 12h 24h0
lowast
lowast
ShGADD34
(a)
0
20
40
60
80
100
Max
()
DCF102 103 104101
No dyeConNAC
AcroleinAcrolein + NAC
No dyeConNAC
AcroleinAcrolein + NAC
0
20
40
60
80
100
Max
()
DCF102 103 104101
Shcon ShGADD34
(b)
Con 1h 6h 12h 24h
P-eIF2120572
eIF2120572
GADD34
GAPDH
CHOP
30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
Caspase 3
Cleaved-caspase 3
(c)
Figure 5 Continued
10 Oxidative Medicine and Cellular Longevity
0601 232
260945
Comp-PE-A
0545 203
271947
Com
p-Pe
rCP-
Cy5-
5-A
145 181
797725
0793 287
292934
Com
p-Pe
rCP-
Cy5-
5-A
0655 209
303942
0661 415
354917
0820 632
721857
0707 266
343932
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
Annexin VComp-PE-AAnnexin V
Comp-PE-AAnnexin V
Comp-PE-AAnnexin V
7A
AD
7A
AD
NAC
+
25 120583M acrolein 25120583M acrolein
Shcon
Con Con
ShGADD34
(d)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
(e)
0
5
10
15
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
lowast
lowast
ShconShGADD34
(f)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
Acrolein + MG132
ShGADD34
(g)
Figure 5 Continued
Oxidative Medicine and Cellular Longevity 11
lowast
lowast
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
Shcon
0
5
10
15
20lowastlowast
lowastlowast
ShGADD34
(h)
Figure 5 GADD34 promote recovery from a shutoff of total protein synthesis and enhance cell deathThe 25 120583Macrolein-treated shconLLCsand shGADD34LLCs were analyzed (a) Cell survival after 25 120583Macrolein treatment was measured lowast119875 lt 005 (b) Levels of ROS productionin shconLLCs and shGADD34LLCs were measured by DCFH-DA after 25120583M acrolein treated for 8 h (c) Cells were collected at theindicated times and protein expressions of ER stress signaling were detected by western blot (d)The 25 120583M acrolein-treated shconLLCs andshGADD34LLCs with 20mM NAC or without NAC for 8 h Cells were stained with Annexin V-PE7-amino-actinomycin D (7AAD) (e)Bands of proteins were analyzed by SDS-PAGE after 25120583M acrolein treatment (f) Protein concentration was measured by Bio-Rad ProteinAssay (g) The shconLLCs and shGADD34LLCs were treated by 25 120583M acrolein with 10 120583M MG132 Cells were collected and lysate wasanalyzed by SDS-PAGE (h) Amount of proteins was measured by Bio-Rad Protein Assay Data are represented as means plusmn sem lowast119875 lt 005
eIF2120572 In shGADD34LLC the expression of p-eIF2120572 con-tinued to be expressed because of the absence of GADD34
Subsequently the execution phase of apoptosis wasanalyzed to determine the effect of acrolein on shconand shGADD34 LLCs There was a significant increase inannexin V 7-AAD double positive cells in shconLLCs after8 h exposure of acrolein compared with shGADD34LLCs(Figure 5(d))
35 GADD34 Promote Recovery from a Shutoff of Total ProteinSynthesis and Enhance Cell Death In fact the extracellu-lar stimuli and changes in intracellular homeostasis causeprotein misfolding in the endoplasmic reticulum ER stresscaused unfolded protein response (UPR) is a cellular adaptiveresponse that evolved to restore protein-folding homeostasisby reducing protein synthesis Phosphorylation of eIF2120572 lim-its initiation of translation on many cellular mRNAs withinthe cells To clarify whether strongly phosphorylated eIF2120572in shGADD34LLCs can affect translation of related proteinthe protein synthesis was examined We found that proteinsin acrolein-treated WT cells (shconLLCs) do not increaseearly phase by shutoff of protein synthesis caused by p-eIF2120572Protein synthesis gradually recovered in shconLLCs after 6 hbut remained at lower levels in shGADD34LLCs (Figures5(e) and 5(f))
Ubiquitin-proteasome system controls the degradationof a large number of cellular proteins including short-lived regulatory and damaged or misfolded protein [2526] It has been assumed that accumulation of no longerneeded proteins underlies the toxicity of proteasome inhibi-tion Proteasome inhibition can induce the integrated stressresponse (ISR) [27] The ISR is an adaptive response to
many forms of stresses which converge into phosphorylationof eIF2120572 [28] We examined whether inhibition of theproteasome enhanced misfolded proteins synthesis by theaddition of MG132 to acrolein We found that the proteinexpression was enhanced in later time in both shconLLCsand shGADD34 by the addition of MG132 However thelevel of protein expression was less in shGADD34LLCs thanthat in shconLLCs by the addition of MG132 (Figures 5(g)and 5(h)) These results confirmed that protein synthesis wasdecreased by the expression of GADD34 We also discoveredthat acrolein induced higher level of ROS production andcell death in shconLLCs although these treatments hadless effects on ROS production of shGADD34LLCs (Figures6(a) and 6(b)) The addition of MG132 to acrolein treatmentenhanced ROS generation and cell death in shconLLCsProtein synthesis may play a pivotal role in ROS productionand cell death [29 30] A protein synthesis inhibitor (CHX)was added to the treatment of acrolein CHX inhibitedthe generation of ROS and cell death in both shconLLCsand shGADD34LLCs But suppressions of ROS productionand cell death were higher in shconLLCs than that inshGADD34LLCs (Figures 6(a) and 6(b))These results indi-cated that highly phosphorylated eIF2120572 in shGADD34LLCsled to a reduction of synthesized protein which decreased theROS-induced cell death
4 Discussion
COPD is characterized by chronic inflammation and destruc-tion of the lung [31] It is a major clinical challenge mostlydue to cigarette smoke exposure [32] Our findings indicated
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
Figure 4 Low level of pulmonary inflammation in GADD34-knockout mice induced by acrolein Lungs were collected from wild-typeand GADD34-knockout mice at days 7 and 28 after 5 120583molkg acrolein instillation (a) Alveolar macrophages as F480highCD11c+ and (b)neutrophils as Gr-1+CD11b+ were confirmed by FACS (c) The expression of phospho-NF-120581B p65 (d) The expressions of macrophage type Imarkers TNF120572 IL-6 and Irf5 and macrophage type II markers Arg-1 Mrc-2 and Retnla were analyzed by quantitative real-time PCR (e)Wild-type and GADD34-knockout mice macrophages were cultured in 12-well plastic plates and stimulated with 10 120583M acrolein for 12 and24 h Supernatants were taken and IL-6 expressionwas analyzed by ELISAData shown are themean ratiosplusmn SE of three separate experimentsData are represented as means plusmn sem lowast119875 lt 005 lowastlowast119875 lt 001
Oxidative Medicine and Cellular Longevity 9
00
05
10
15
Cel
l sur
viva
l (
)
Shcon
1h 4h 8h 12h 24h0
lowast
lowast
ShGADD34
(a)
0
20
40
60
80
100
Max
()
DCF102 103 104101
No dyeConNAC
AcroleinAcrolein + NAC
No dyeConNAC
AcroleinAcrolein + NAC
0
20
40
60
80
100
Max
()
DCF102 103 104101
Shcon ShGADD34
(b)
Con 1h 6h 12h 24h
P-eIF2120572
eIF2120572
GADD34
GAPDH
CHOP
30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
Caspase 3
Cleaved-caspase 3
(c)
Figure 5 Continued
10 Oxidative Medicine and Cellular Longevity
0601 232
260945
Comp-PE-A
0545 203
271947
Com
p-Pe
rCP-
Cy5-
5-A
145 181
797725
0793 287
292934
Com
p-Pe
rCP-
Cy5-
5-A
0655 209
303942
0661 415
354917
0820 632
721857
0707 266
343932
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
102 103 104
102
103
104
105
Annexin VComp-PE-AAnnexin V
Comp-PE-AAnnexin V
Comp-PE-AAnnexin V
7A
AD
7A
AD
NAC
+
25 120583M acrolein 25120583M acrolein
Shcon
Con Con
ShGADD34
(d)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
(e)
0
5
10
15
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
lowast
lowast
ShconShGADD34
(f)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
Acrolein + MG132
ShGADD34
(g)
Figure 5 Continued
Oxidative Medicine and Cellular Longevity 11
lowast
lowast
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
Shcon
0
5
10
15
20lowastlowast
lowastlowast
ShGADD34
(h)
Figure 5 GADD34 promote recovery from a shutoff of total protein synthesis and enhance cell deathThe 25 120583Macrolein-treated shconLLCsand shGADD34LLCs were analyzed (a) Cell survival after 25 120583Macrolein treatment was measured lowast119875 lt 005 (b) Levels of ROS productionin shconLLCs and shGADD34LLCs were measured by DCFH-DA after 25120583M acrolein treated for 8 h (c) Cells were collected at theindicated times and protein expressions of ER stress signaling were detected by western blot (d)The 25 120583M acrolein-treated shconLLCs andshGADD34LLCs with 20mM NAC or without NAC for 8 h Cells were stained with Annexin V-PE7-amino-actinomycin D (7AAD) (e)Bands of proteins were analyzed by SDS-PAGE after 25120583M acrolein treatment (f) Protein concentration was measured by Bio-Rad ProteinAssay (g) The shconLLCs and shGADD34LLCs were treated by 25 120583M acrolein with 10 120583M MG132 Cells were collected and lysate wasanalyzed by SDS-PAGE (h) Amount of proteins was measured by Bio-Rad Protein Assay Data are represented as means plusmn sem lowast119875 lt 005
eIF2120572 In shGADD34LLC the expression of p-eIF2120572 con-tinued to be expressed because of the absence of GADD34
Subsequently the execution phase of apoptosis wasanalyzed to determine the effect of acrolein on shconand shGADD34 LLCs There was a significant increase inannexin V 7-AAD double positive cells in shconLLCs after8 h exposure of acrolein compared with shGADD34LLCs(Figure 5(d))
35 GADD34 Promote Recovery from a Shutoff of Total ProteinSynthesis and Enhance Cell Death In fact the extracellu-lar stimuli and changes in intracellular homeostasis causeprotein misfolding in the endoplasmic reticulum ER stresscaused unfolded protein response (UPR) is a cellular adaptiveresponse that evolved to restore protein-folding homeostasisby reducing protein synthesis Phosphorylation of eIF2120572 lim-its initiation of translation on many cellular mRNAs withinthe cells To clarify whether strongly phosphorylated eIF2120572in shGADD34LLCs can affect translation of related proteinthe protein synthesis was examined We found that proteinsin acrolein-treated WT cells (shconLLCs) do not increaseearly phase by shutoff of protein synthesis caused by p-eIF2120572Protein synthesis gradually recovered in shconLLCs after 6 hbut remained at lower levels in shGADD34LLCs (Figures5(e) and 5(f))
Ubiquitin-proteasome system controls the degradationof a large number of cellular proteins including short-lived regulatory and damaged or misfolded protein [2526] It has been assumed that accumulation of no longerneeded proteins underlies the toxicity of proteasome inhibi-tion Proteasome inhibition can induce the integrated stressresponse (ISR) [27] The ISR is an adaptive response to
many forms of stresses which converge into phosphorylationof eIF2120572 [28] We examined whether inhibition of theproteasome enhanced misfolded proteins synthesis by theaddition of MG132 to acrolein We found that the proteinexpression was enhanced in later time in both shconLLCsand shGADD34 by the addition of MG132 However thelevel of protein expression was less in shGADD34LLCs thanthat in shconLLCs by the addition of MG132 (Figures 5(g)and 5(h)) These results confirmed that protein synthesis wasdecreased by the expression of GADD34 We also discoveredthat acrolein induced higher level of ROS production andcell death in shconLLCs although these treatments hadless effects on ROS production of shGADD34LLCs (Figures6(a) and 6(b)) The addition of MG132 to acrolein treatmentenhanced ROS generation and cell death in shconLLCsProtein synthesis may play a pivotal role in ROS productionand cell death [29 30] A protein synthesis inhibitor (CHX)was added to the treatment of acrolein CHX inhibitedthe generation of ROS and cell death in both shconLLCsand shGADD34LLCs But suppressions of ROS productionand cell death were higher in shconLLCs than that inshGADD34LLCs (Figures 6(a) and 6(b))These results indi-cated that highly phosphorylated eIF2120572 in shGADD34LLCsled to a reduction of synthesized protein which decreased theROS-induced cell death
4 Discussion
COPD is characterized by chronic inflammation and destruc-tion of the lung [31] It is a major clinical challenge mostlydue to cigarette smoke exposure [32] Our findings indicated
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
(e)
0
5
10
15
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
lowast
lowast
ShconShGADD34
(f)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
Acrolein + MG132
ShGADD34
(g)
Figure 5 Continued
Oxidative Medicine and Cellular Longevity 11
lowast
lowast
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
Shcon
0
5
10
15
20lowastlowast
lowastlowast
ShGADD34
(h)
Figure 5 GADD34 promote recovery from a shutoff of total protein synthesis and enhance cell deathThe 25 120583Macrolein-treated shconLLCsand shGADD34LLCs were analyzed (a) Cell survival after 25 120583Macrolein treatment was measured lowast119875 lt 005 (b) Levels of ROS productionin shconLLCs and shGADD34LLCs were measured by DCFH-DA after 25120583M acrolein treated for 8 h (c) Cells were collected at theindicated times and protein expressions of ER stress signaling were detected by western blot (d)The 25 120583M acrolein-treated shconLLCs andshGADD34LLCs with 20mM NAC or without NAC for 8 h Cells were stained with Annexin V-PE7-amino-actinomycin D (7AAD) (e)Bands of proteins were analyzed by SDS-PAGE after 25120583M acrolein treatment (f) Protein concentration was measured by Bio-Rad ProteinAssay (g) The shconLLCs and shGADD34LLCs were treated by 25 120583M acrolein with 10 120583M MG132 Cells were collected and lysate wasanalyzed by SDS-PAGE (h) Amount of proteins was measured by Bio-Rad Protein Assay Data are represented as means plusmn sem lowast119875 lt 005
eIF2120572 In shGADD34LLC the expression of p-eIF2120572 con-tinued to be expressed because of the absence of GADD34
Subsequently the execution phase of apoptosis wasanalyzed to determine the effect of acrolein on shconand shGADD34 LLCs There was a significant increase inannexin V 7-AAD double positive cells in shconLLCs after8 h exposure of acrolein compared with shGADD34LLCs(Figure 5(d))
35 GADD34 Promote Recovery from a Shutoff of Total ProteinSynthesis and Enhance Cell Death In fact the extracellu-lar stimuli and changes in intracellular homeostasis causeprotein misfolding in the endoplasmic reticulum ER stresscaused unfolded protein response (UPR) is a cellular adaptiveresponse that evolved to restore protein-folding homeostasisby reducing protein synthesis Phosphorylation of eIF2120572 lim-its initiation of translation on many cellular mRNAs withinthe cells To clarify whether strongly phosphorylated eIF2120572in shGADD34LLCs can affect translation of related proteinthe protein synthesis was examined We found that proteinsin acrolein-treated WT cells (shconLLCs) do not increaseearly phase by shutoff of protein synthesis caused by p-eIF2120572Protein synthesis gradually recovered in shconLLCs after 6 hbut remained at lower levels in shGADD34LLCs (Figures5(e) and 5(f))
Ubiquitin-proteasome system controls the degradationof a large number of cellular proteins including short-lived regulatory and damaged or misfolded protein [2526] It has been assumed that accumulation of no longerneeded proteins underlies the toxicity of proteasome inhibi-tion Proteasome inhibition can induce the integrated stressresponse (ISR) [27] The ISR is an adaptive response to
many forms of stresses which converge into phosphorylationof eIF2120572 [28] We examined whether inhibition of theproteasome enhanced misfolded proteins synthesis by theaddition of MG132 to acrolein We found that the proteinexpression was enhanced in later time in both shconLLCsand shGADD34 by the addition of MG132 However thelevel of protein expression was less in shGADD34LLCs thanthat in shconLLCs by the addition of MG132 (Figures 5(g)and 5(h)) These results confirmed that protein synthesis wasdecreased by the expression of GADD34 We also discoveredthat acrolein induced higher level of ROS production andcell death in shconLLCs although these treatments hadless effects on ROS production of shGADD34LLCs (Figures6(a) and 6(b)) The addition of MG132 to acrolein treatmentenhanced ROS generation and cell death in shconLLCsProtein synthesis may play a pivotal role in ROS productionand cell death [29 30] A protein synthesis inhibitor (CHX)was added to the treatment of acrolein CHX inhibitedthe generation of ROS and cell death in both shconLLCsand shGADD34LLCs But suppressions of ROS productionand cell death were higher in shconLLCs than that inshGADD34LLCs (Figures 6(a) and 6(b))These results indi-cated that highly phosphorylated eIF2120572 in shGADD34LLCsled to a reduction of synthesized protein which decreased theROS-induced cell death
4 Discussion
COPD is characterized by chronic inflammation and destruc-tion of the lung [31] It is a major clinical challenge mostlydue to cigarette smoke exposure [32] Our findings indicated
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
AcroleinShGADD34
(e)
0
5
10
15
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
lowast
lowast
ShconShGADD34
(f)
Con 1h 6h 12h 24h30min Con 1h 6h 12h 24h30minShcon
Acrolein + MG132
ShGADD34
(g)
Figure 5 Continued
Oxidative Medicine and Cellular Longevity 11
lowast
lowast
Prot
ein
(mg
mL)
Con 1h 6h 12h 24h30min
Shcon
0
5
10
15
20lowastlowast
lowastlowast
ShGADD34
(h)
Figure 5 GADD34 promote recovery from a shutoff of total protein synthesis and enhance cell deathThe 25 120583Macrolein-treated shconLLCsand shGADD34LLCs were analyzed (a) Cell survival after 25 120583Macrolein treatment was measured lowast119875 lt 005 (b) Levels of ROS productionin shconLLCs and shGADD34LLCs were measured by DCFH-DA after 25120583M acrolein treated for 8 h (c) Cells were collected at theindicated times and protein expressions of ER stress signaling were detected by western blot (d)The 25 120583M acrolein-treated shconLLCs andshGADD34LLCs with 20mM NAC or without NAC for 8 h Cells were stained with Annexin V-PE7-amino-actinomycin D (7AAD) (e)Bands of proteins were analyzed by SDS-PAGE after 25120583M acrolein treatment (f) Protein concentration was measured by Bio-Rad ProteinAssay (g) The shconLLCs and shGADD34LLCs were treated by 25 120583M acrolein with 10 120583M MG132 Cells were collected and lysate wasanalyzed by SDS-PAGE (h) Amount of proteins was measured by Bio-Rad Protein Assay Data are represented as means plusmn sem lowast119875 lt 005
eIF2120572 In shGADD34LLC the expression of p-eIF2120572 con-tinued to be expressed because of the absence of GADD34
Subsequently the execution phase of apoptosis wasanalyzed to determine the effect of acrolein on shconand shGADD34 LLCs There was a significant increase inannexin V 7-AAD double positive cells in shconLLCs after8 h exposure of acrolein compared with shGADD34LLCs(Figure 5(d))
35 GADD34 Promote Recovery from a Shutoff of Total ProteinSynthesis and Enhance Cell Death In fact the extracellu-lar stimuli and changes in intracellular homeostasis causeprotein misfolding in the endoplasmic reticulum ER stresscaused unfolded protein response (UPR) is a cellular adaptiveresponse that evolved to restore protein-folding homeostasisby reducing protein synthesis Phosphorylation of eIF2120572 lim-its initiation of translation on many cellular mRNAs withinthe cells To clarify whether strongly phosphorylated eIF2120572in shGADD34LLCs can affect translation of related proteinthe protein synthesis was examined We found that proteinsin acrolein-treated WT cells (shconLLCs) do not increaseearly phase by shutoff of protein synthesis caused by p-eIF2120572Protein synthesis gradually recovered in shconLLCs after 6 hbut remained at lower levels in shGADD34LLCs (Figures5(e) and 5(f))
Ubiquitin-proteasome system controls the degradationof a large number of cellular proteins including short-lived regulatory and damaged or misfolded protein [2526] It has been assumed that accumulation of no longerneeded proteins underlies the toxicity of proteasome inhibi-tion Proteasome inhibition can induce the integrated stressresponse (ISR) [27] The ISR is an adaptive response to
many forms of stresses which converge into phosphorylationof eIF2120572 [28] We examined whether inhibition of theproteasome enhanced misfolded proteins synthesis by theaddition of MG132 to acrolein We found that the proteinexpression was enhanced in later time in both shconLLCsand shGADD34 by the addition of MG132 However thelevel of protein expression was less in shGADD34LLCs thanthat in shconLLCs by the addition of MG132 (Figures 5(g)and 5(h)) These results confirmed that protein synthesis wasdecreased by the expression of GADD34 We also discoveredthat acrolein induced higher level of ROS production andcell death in shconLLCs although these treatments hadless effects on ROS production of shGADD34LLCs (Figures6(a) and 6(b)) The addition of MG132 to acrolein treatmentenhanced ROS generation and cell death in shconLLCsProtein synthesis may play a pivotal role in ROS productionand cell death [29 30] A protein synthesis inhibitor (CHX)was added to the treatment of acrolein CHX inhibitedthe generation of ROS and cell death in both shconLLCsand shGADD34LLCs But suppressions of ROS productionand cell death were higher in shconLLCs than that inshGADD34LLCs (Figures 6(a) and 6(b))These results indi-cated that highly phosphorylated eIF2120572 in shGADD34LLCsled to a reduction of synthesized protein which decreased theROS-induced cell death
4 Discussion
COPD is characterized by chronic inflammation and destruc-tion of the lung [31] It is a major clinical challenge mostlydue to cigarette smoke exposure [32] Our findings indicated
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
Figure 5 GADD34 promote recovery from a shutoff of total protein synthesis and enhance cell deathThe 25 120583Macrolein-treated shconLLCsand shGADD34LLCs were analyzed (a) Cell survival after 25 120583Macrolein treatment was measured lowast119875 lt 005 (b) Levels of ROS productionin shconLLCs and shGADD34LLCs were measured by DCFH-DA after 25120583M acrolein treated for 8 h (c) Cells were collected at theindicated times and protein expressions of ER stress signaling were detected by western blot (d)The 25 120583M acrolein-treated shconLLCs andshGADD34LLCs with 20mM NAC or without NAC for 8 h Cells were stained with Annexin V-PE7-amino-actinomycin D (7AAD) (e)Bands of proteins were analyzed by SDS-PAGE after 25120583M acrolein treatment (f) Protein concentration was measured by Bio-Rad ProteinAssay (g) The shconLLCs and shGADD34LLCs were treated by 25 120583M acrolein with 10 120583M MG132 Cells were collected and lysate wasanalyzed by SDS-PAGE (h) Amount of proteins was measured by Bio-Rad Protein Assay Data are represented as means plusmn sem lowast119875 lt 005
eIF2120572 In shGADD34LLC the expression of p-eIF2120572 con-tinued to be expressed because of the absence of GADD34
Subsequently the execution phase of apoptosis wasanalyzed to determine the effect of acrolein on shconand shGADD34 LLCs There was a significant increase inannexin V 7-AAD double positive cells in shconLLCs after8 h exposure of acrolein compared with shGADD34LLCs(Figure 5(d))
35 GADD34 Promote Recovery from a Shutoff of Total ProteinSynthesis and Enhance Cell Death In fact the extracellu-lar stimuli and changes in intracellular homeostasis causeprotein misfolding in the endoplasmic reticulum ER stresscaused unfolded protein response (UPR) is a cellular adaptiveresponse that evolved to restore protein-folding homeostasisby reducing protein synthesis Phosphorylation of eIF2120572 lim-its initiation of translation on many cellular mRNAs withinthe cells To clarify whether strongly phosphorylated eIF2120572in shGADD34LLCs can affect translation of related proteinthe protein synthesis was examined We found that proteinsin acrolein-treated WT cells (shconLLCs) do not increaseearly phase by shutoff of protein synthesis caused by p-eIF2120572Protein synthesis gradually recovered in shconLLCs after 6 hbut remained at lower levels in shGADD34LLCs (Figures5(e) and 5(f))
Ubiquitin-proteasome system controls the degradationof a large number of cellular proteins including short-lived regulatory and damaged or misfolded protein [2526] It has been assumed that accumulation of no longerneeded proteins underlies the toxicity of proteasome inhibi-tion Proteasome inhibition can induce the integrated stressresponse (ISR) [27] The ISR is an adaptive response to
many forms of stresses which converge into phosphorylationof eIF2120572 [28] We examined whether inhibition of theproteasome enhanced misfolded proteins synthesis by theaddition of MG132 to acrolein We found that the proteinexpression was enhanced in later time in both shconLLCsand shGADD34 by the addition of MG132 However thelevel of protein expression was less in shGADD34LLCs thanthat in shconLLCs by the addition of MG132 (Figures 5(g)and 5(h)) These results confirmed that protein synthesis wasdecreased by the expression of GADD34 We also discoveredthat acrolein induced higher level of ROS production andcell death in shconLLCs although these treatments hadless effects on ROS production of shGADD34LLCs (Figures6(a) and 6(b)) The addition of MG132 to acrolein treatmentenhanced ROS generation and cell death in shconLLCsProtein synthesis may play a pivotal role in ROS productionand cell death [29 30] A protein synthesis inhibitor (CHX)was added to the treatment of acrolein CHX inhibitedthe generation of ROS and cell death in both shconLLCsand shGADD34LLCs But suppressions of ROS productionand cell death were higher in shconLLCs than that inshGADD34LLCs (Figures 6(a) and 6(b))These results indi-cated that highly phosphorylated eIF2120572 in shGADD34LLCsled to a reduction of synthesized protein which decreased theROS-induced cell death
4 Discussion
COPD is characterized by chronic inflammation and destruc-tion of the lung [31] It is a major clinical challenge mostlydue to cigarette smoke exposure [32] Our findings indicated
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
Figure 6 Protein synthesis promotes ROS production and cell deathThe shconLLCs and shGADD34LLCs were treated by 25120583Macroleinwith 10120583MMG132 andor 1 120583gmL CHX or without these agents for 12 h (a) ROS levels were measured by DCFH-DA fluorescence by flowcytometry Right mean fluorescence after subtracting autofluorescence lowast119875 lt 005 lowastlowast119875 lt 001 (b) Cells were stained with Annexin V-PE7-AAD and then analyzed by flow cytometry lowast119875 lt 005 lowastlowast119875 lt 001
14 Oxidative Medicine and Cellular Longevity
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
that acrolein not only caused pulmonary structure damagebut also promoted pulmonary inflammation through NF-120581Bsignaling in vivo Alveolar injury caused by acrolein exposuremight be relevant to cigarette smoke-induced chronic lungdestruction
It has been shown that ER stresses induce cell deathby CHOP following PERK-mediated eIF2120572 phosphorylation[24 33] We examined whether acrolein induces eIF2120572phosphorylation and CHOP expression in vitro We showedthat CHOP was strongly expressed by the treatment ofacrolein in shconLLC In shGADD34LLC expression of p-eIF2120572 was also increased by acrolein treatment In contrastto shconLLC expression of p-eIF2120572 in shGADD34LLCcontinued long time because of the lack of GADD34 expres-sion Continued expression of p-eIF2120572 in shGADD34LLCinduced the later expression of CHOP in these cells Fromthese results cell death in shconLLCs is not caused by CHOPexpression
In LLCshcon the acrolein-induced protein synthesis wasupregulated which induced oxidative stress Generated ROScaused cell death by upregulating caspase 3 In contrastthe continuous expressions of p-eIF2120572 in shGADD34LLCcells shut off the synthesis of protein which caused theremission of oxidative stress in shGADD34LLCs We usedproteasome and protein synthesis inhibitors to supportour results MG132 is a proteasome inhibitor which accu-mulates proteins Accumulated proteins induce oxidativestress which induce cell death Because p-eIF2120572 inducedby acrolein in shGADD34LLCs was higher than that inshconLLCs protein accumulation by MG132 was higherin shGADD34LLCs than that in shconLLCs Further weshowed that proteins in shconLLC were greatly decreasedby the stimulation of acrolein which reduced the formationof ROS as well as cell death Oxidative stress is recog-nized as a major predisposing factor in the pathogenesisof COPD [34] Alveolar macrophages from patients withCOPD are more activated and release increased amount ofROS [35] The endogenous oxidative stress is generated bymitochondria when the lung gets injury and then the ROSinduces strong inflammatory responses and severe damagein lung Previous studies have confirmed that the generationof ROS contributes to bactericidal activity of macrophages[36] Because we observed higher caspase 3 expression inshconLLCs ROS might produce mitochondrial damages bydirect acrolein exposure It has been shown that exposure ofacrolein leads to mitochondrial dysfunction which inducesaccumulation of ROS [37] We found that GADD34 washighly expressed under the stimulation of acrolein indicatingGADD34 might be involved in the pathogenesis of alveolarinjury by producing ROS
In conclusion our results demonstrate that GADD34 isupregulated in vivo and in vitro by the exposure of acroleinGADD34 reverts the phosphorylation of eIF2120572 induced byacrolein Dephosphorylation of eIF2120572 accumulatesmisfoldedproteins which induces oxidative stress Generated ROSfrom direct mitochondrial dysfunction by acrolein or ROSproduced by ER stress induce cell death and macrophagesinfiltration GADD34 is one of the key proteins in acrolein-induced lung inflammation and tissue injury
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] R L Stedman ldquoThe chemical composition of tobacco andtobacco smokerdquo Chemical Reviews vol 68 no 2 pp 153ndash2071968
[2] C Aranyi W J OrsquoShea J A Graham and F J Miller ldquoTheeffects of inhalation of organic chemical air contaminants onmurine lung host defensesrdquo Fundamental and Applied Toxicol-ogy vol 6 no 4 pp 713ndash720 1986
[3] J C Hogg ldquoChronic obstructive pulmonary disease anoverview of pathology and pathogenesisrdquo Novartis FoundationSymposium vol 234 pp 4ndash19 26 2001
[4] M Saetta ldquoAirway inflammation in chronic obstructive pul-monary diseaserdquo The American Journal of Respiratory andCritical Care Medicine vol 160 no 5 part 2 pp S17ndashS20 1999
[5] L Lacroix G Feng and R Lotan ldquoIdentification of genesexpressed differentially in an in vitro human lung carcinogen-esis modelrdquo Cancer Biology and Therapy vol 5 no 6 pp 665ndash673 2006
[6] H TWang YHuD Tong et al ldquoEffect of carcinogenic acroleinon DNA repair and mutagenic susceptibilityrdquo The Journal ofBiological Chemistry vol 287 no 15 pp 12379ndash12386 2012
[7] D J Hochman C R Collaco and E G Brooks ldquoAcroleininduction of oxidative stress and degranulation in mast cellsrdquoEnvironmental Toxicology vol 29 no 8 pp 908ndash915 2014
[8] Y Sun S Ito N Nishio Y Tanaka N Chen and K IsobeldquoAcrolein induced both pulmonary inflammation and the deathof lung epithelial cellsrdquo Toxicology Letters vol 229 no 2 pp384ndash392 2014
[9] P Walter and D Ron ldquoThe unfolded protein response fromstress pathway to homeostatic regulationrdquo Science vol 334 no6059 pp 1081ndash1086 2011
[10] SWang and R J Kaufman ldquoThe impact of the unfolded proteinresponse on human diseaserdquo Journal of Cell Biology vol 197 no7 pp 857ndash867 2012
[11] A Tanel P Pallepati A Bettaieb P Morin and D A Averill-Bates ldquoAcrolein activates cell survival and apoptotic deathresponses involving the endoplasmic reticulum in A549 lungcellsrdquo Biochimica et Biophysica Acta Molecular Cell Researchvol 1843 no 5 pp 827ndash835 2014
[12] M K Mohammad D Avila J Zhang et al ldquoAcrolein cyto-toxicity in hepatocytes involves endoplasmic reticulum stressmitochondrial dysfunction and oxidative stressrdquoToxicology andApplied Pharmacology vol 265 no 1 pp 73ndash82 2012
[13] Y Kitaguchi L Taraseviciene-Stewart M Hanaoka R Natara-jan D Kraskauskas and N F Voelkel ldquoAcrolein inducesendoplasmic reticulum stress and causes airspace enlargementrdquoPLoS ONE vol 7 no 5 Article ID e38038 2012
[14] PHaberzettl E Vladykovskaya S Srivastava andA BhatnagarldquoRole of endoplasmic reticulum stress in acrolein-inducedendothelial activationrdquo Toxicology and Applied Pharmacologyvol 234 no 1 pp 14ndash24 2009
[15] A J Fornace Jr I Alamo Jr and M C Hollander ldquoDNAdamage-inducible transcripts in mammalian cellsrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 85 no 23 pp 8800ndash8804 1988
Oxidative Medicine and Cellular Longevity 15
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
[16] Q ZhanKA Lord I Alamo Jr et al ldquoThegadd andMyDgenesdefine a novel set of mammalian genes encoding acidic proteinsthat synergistically suppress cell growthrdquoMolecular andCellularBiology vol 14 no 4 pp 2361ndash2371 1994
[17] I Novoa H Zeng H P Harding and D Ron ldquoFeedbackinhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2120572rdquo The Journal of CellBiology vol 153 no 5 pp 1011ndash1022 2001
[18] T Mengesdorf S Althausen I Oberndorfer and W PaschenldquoResponse of neurons to an irreversible inhibition of endoplas-mic reticulum Ca2+-atpase relationship between global proteinsynthesis and expression and translation of individual genesrdquoBiochemical Journal vol 356 no 3 pp 805ndash812 2001
[19] M T Borchers S C Wesselkamper N L Harris et al ldquoCD8+T cells contribute to macrophage accumulation and airspaceenlargement following repeated irritant exposurerdquo Experimen-tal and Molecular Pathology vol 83 no 3 pp 301ndash310 2007
[20] E Kojima A Takeuchi M Haneda et al ldquoThe function ofGADD34 is a recovery from a shutoff of protein synthesisinduced by ER stress elucidation by GADD34-deficient micerdquoThe FASEB Journal vol 17 no 11 pp 1573ndash1575 2003
[21] S Ito Y Tanaka N Nishio S Thanasegaran and K-I IsobeldquoEstablishment of self-renewable GM-CSF-dependent imma-ture macrophages in vitro from murine bone marrowrdquo PLoSONE vol 8 no 10 Article ID e76943 2013
[22] J S Bertram and P Janik ldquoEstablishment of a cloned lineof lewis lung carcinoma cells adapted to cell culturerdquo CancerLetters vol 11 no 1 pp 63ndash73 1980
[23] W Zhao D I Diz and M E Robbins ldquoOxidative damagepathways in relation to normal tissue injuryrdquo British Journal ofRadiology vol 80 no 1 pp S23ndashS31 2007
[24] D Scheuner B B Song EMcEwen et al ldquoTranslational controlis required for the unfolded protein response and in vivo glucosehomeostasisrdquoMolecular Cell vol 7 no 6 pp 1165ndash1176 2001
[26] A L Schwartz and A Ciechanover ldquoTargeting proteins fordestruction by the ubiquitin system implications for humanpathobiologyrdquo Annual Review of Pharmacology and Toxicologyvol 49 pp 73ndash96 2009
[27] H-Y Jiang and R C Wek ldquoPhosphorylation of the 120572-subunitof the eukaryotic initiation factor-2 (eIF2120572) reduces proteinsynthesis and enhances apoptosis in response to proteasomeinhibitionrdquoThe Journal of Biological Chemistry vol 280 no 14pp 14189ndash14202 2005
[28] A Suraweera C Munch A Hanssum and A BertolottildquoFailure of amino acid homeostasis causes cell death followingproteasome inhibitionrdquo Molecular Cell vol 48 no 2 pp 242ndash253 2012
[29] H J Clarke J E Chambers E Liniker and S J MarciniakldquoEndoplasmic reticulum stress in malignancyrdquo Cancer Cell vol25 no 5 pp 563ndash573 2014
[30] J Han S H Back J Hur et al ldquoER-stress-induced transcrip-tional regulation increases protein synthesis leading to celldeathrdquo Nature Cell Biology vol 15 no 5 pp 481ndash490 2013
[31] J C Hogg F Chu S Utokaparch et al ldquoThe nature of small-airway obstruction in chronic obstructive pulmonary diseaserdquoTheNew England Journal ofMedicine vol 350 no 26 pp 2645ndash2653 2004
[32] M Pichavant G Remy S Bekaert et al ldquoOxidative stress-mediated iNKT-cell activation is involved in COPD pathogen-esisrdquoMucosal Immunology vol 7 no 3 pp 568ndash578 2014
[33] H P Harding I Novoa Y Zhang et al ldquoRegulated translationinitiation controls stress-induced gene expression in mam-malian cellsrdquoMolecular Cell vol 6 no 5 pp 1099ndash1108 2000
[34] P A Kirkham and P J Barnes ldquoOxidative stress in COPDrdquoChest vol 144 no 1 pp 266ndash273 2013
[35] T Schaberg U Klein M Rau J Eller and H Lode ldquoSubpop-ulations of alveolar macrophages in smokers and nonsmokersrelation to the expression of CD11CD18 molecules and super-oxide anion productionrdquo The American Journal of Respiratoryand Critical Care Medicine vol 151 no 5 pp 1551ndash1558 1995
[36] A P West I E Brodsky C Rahner et al ldquoTLR signalling aug-ments macrophage bactericidal activity through mitochondrialROSrdquo Nature vol 472 no 7344 pp 476ndash480 2011
[37] J Luo J P Robinson and R Shi ldquoAcrolein-induced cell deathin PC12 cells role of mitochondria-mediated oxidative stressrdquoNeurochemistry International vol 47 no 7 pp 449ndash457 2005
