IV Protease Inhibitors Induce Endoplasmic Reticulum Stress andisrupt Barrier Integrity in Intestinal Epithelial Cells
UDONG WU,* LIXIN SUN,* WEIBIN ZHA,* ELAINE STUDER,* EMILY GURLEY,* LI CHEN,* XUAN WANG,*HILLIP B. HYLEMON,*,‡ WILLIAM M. PANDAK Jr,‡ ARUN J. SANYAL,‡ LUYONG ZHANG,§ GUANGJI WANG,§
IE CHEN,� JIAN–YING WANG,� and HUIPING ZHOU*,‡,§
Department of Microbiology & Immunology and ‡Department of Internal Medicine/Gastroenterology and McGuire Veterans Affairs Medical Center, Virginiaommonwealth University, Richmond, Virginia; §China Pharmaceutical University, Nanjing, Jiangsu, China; and �Department of Surgery, University of Maryland School
f Medicine and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland
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ACKGROUND & AIMS: Human immunodeficiencyirus (HIV) protease inhibitor (PI)-induced adverse effectsave become a serious clinical problem. In addition toheir metabolic and cardiovascular complications, theserugs also frequently cause severe gastrointestinal disor-ers, including mucosal erosions, epithelial barrier dys-unction, and diarrhea. However, the exact mechanismsnderlying gastrointestinal adverse effects of HIV PIsemain unknown. This study investigated whether HIVIs disrupt intestinal epithelial barrier integrity by acti-ating endoplasmic reticulum (ER) stress. METHODS:he most commonly used HIV PIs (lopinavir, ritonavir,nd amprenavir) were used; their effects on ER stressctivation and epithelial paracellular permeability werexamined in vitro as well as in vivo using wild-type andHOP�/� mice. RESULTS: Treatment with lopinavirnd ritonavir, but not amprenavir, induced ER stress, asndicated by a decrease in secreted alkaline phosphatasectivities and an increase in the unfolded protein re-ponse. This activated ER stress partially impaired thepithelial barrier integrity by promoting intestinal epi-helial cell apoptosis. CHOP silencing by specific smallairpin RNA prevented lopinavir- and ritonavir-inducedarrier dysfunction in cultured intestinal epithelial cells,hereas CHOP�/� mice exhibited decreased mucosal in-
ury after exposure to lopinavir and ritonavir. CONCLU-IONS: HIV PIs induce ER stress and activate thenfolded protein response in intestinal epithelialells, thus resulting in disruption of the epithelialarrier integrity.
uman immunodeficiency virus (HIV) protease in-hibitors (PIs) are major components of highly ac-
ive antiretroviral therapy and have been successfullysed to suppress HIV replication. However, the benefitsf HIV PIs are compromised by various undesirable sideffects.1 HIV PI-induced gastrointestinal dysfunction rep-esents a major adverse effect of HIV PIs. Drug-inducediarrhea occurs in 16%– 62% of patients on highly active
ntiretroviral therapy.2,3
Diarrhea may be acute or chronic. In addition to ab-ormal intestinal motility and malabsorption, diarrheaan be caused either by increased active ion secretionsecretory diarrhea) or by a leaky epithelial barrier (leak-ux diarrhea). Recent studies by Bode et al showed thatIV PIs have no effect on epithelial ion secretion but
nduce apoptosis and decrease barrier function in humanntestinal epithelial cells (IECs).2– 4 However, the under-ying mechanism of HIV PI-induced disruption of barrierunction in IECs remains unclear.
The endoplasmic reticulum (ER) is a principal site forrotein synthesis and folding, calcium storage and sig-aling, and biosynthesis of corticosteroids, cholesterol,nd other lipids, and it is highly sensitive to alterations inalcium homeostasis and perturbations in its environ-ent.5,6 A number of biochemical and physiologic stim-
li can change ER homeostasis, impose stress on the ER,nd subsequently lead to accumulation of unfolded pro-ein or misfolded proteins in the ER lumen.7 The ER hasvolved highly specific signaling pathways, collectivelyermed the unfolded protein response (UPR), to ensurehat its protein-folding capacity is not overwhelmed.8
owever, persistent activation of the UPR can induce cellpoptosis. ER stress-induced apoptosis is directly impli-ated in the pathology of various human diseases, includ-ng gastrointestinal diseases.9
Our previous studies have shown that HIV PIs induceR stress, activate the UPR, and induce cell apoptosis inoth macrophages and hepatocytes.1– 4,10 Recent studiesy Djedaini et al also found that lopinavir (LOPV)-
nduced ER stress is correlated to insulin resistance in
Abbreviations used in this paper: AMPV, amprenavir; ER, endoplas-ic reticulum; ERK, extracellular signal–regulated kinase; FITC, fluo-
uman adipocytes.11 However, whether HIV PIs also in-uce ER stress in IECs has not been investigated. In theresent study, we examined the effects of different HIVIs on UPR activation and apoptosis in normal IECs.urthermore, we examined whether HIV PI-induced ERtress is responsible for HIV PI-associated cell apoptosisnd disruption of barrier function both in vitro and inivo.
Materials and MethodsCell Culture and HIV PI TreatmentThe IEC-6 cell line at passage 13 was purchased
rom American Type Culture Collection (Manassas, VA)nd used at passages 15–20 for the current experiments.he cell line was derived from normal rat intestine andas developed and characterized by Quaroni et al.12 It isontumorigenic and retains the undifferentiated charac-er of epithelial stem cells. The stable IEC-Cdx2L1 cellsere developed and characterized by Suh and Traber13
nd were kind gifts from Dr Peter G. Traber (Baylorollege of Medicine, Houston, TX). Before experiments,
ells were grown in Dulbecco’s modified Eagle mediumontaining 4 mmol/L isopropyl-�-D-thiogalactopyrano-ide for 16 days to induce cell differentiation. The differ-ntiated cells have multiple morphologic characteristicsf villus-type enterocytes with few goblet cells, and su-rase-isomaltase is highly expressed (Supplementary Fig-re 1).Amprenavir (AMPV), LOPV, and ritonavir (RITV) were
issolved in dimethyl sulfoxide and directly added toulture medium (final concentrations, 5–25 �mol/L) andncubated for 0.5–24 hours. For each result, a minimumf 3 independent experiments was performed.
Western Blot AnalysisTotal cell lysates, cytoplasmic proteins, and nu-
lear proteins were prepared, and the protein levels ofHOP, ATF-4, XBP-1, GRP78, lamin B, or �-actin wereetected and analyzed as previously described.1
Construction of IEC-6 Stable Cell LineExpressing Secreted Alkaline Phosphatase andSecreted Alkaline Phosphatase EnzymeActivity AssayActivity of secreted alkaline phosphatase (SEAP) is
apidly down-regulated by ER stress independent of tran-criptional regulation.14 IEC-6 cells were cotransfectedith pSEAP2-control and pEGFP-N1 using Lipo-
ectamine 2000 reagent (Invitrogen, Carlsbad, CA). Stablelones containing both pSEAP2 and pEGFP-N1 wereelected by G418 and purified by colonial cloning andow cytometry cell sorting. Expression of SEAP andreen fluorescent protein were confirmed by Western blotnalysis, chemiluminescent assays, and fluorescent mi-
roscopy. p
Enzyme activity of SEAP was measured by chemilumi-escent assays using the Great EscAPe SEAP Detectionit according to the manufacturer’s instructions (Pro-ega, Madison, WI). The viability of cells was detectedith CellTiter 96 AQueous One Solution Cell Prolifera-
ion Assay (Promega).
Analysis of Apoptosis by Annexin V andPropidium Iodide StainingIEC-6 cells were treated with various concentra-
ions of HIV PIs for 24 hours and stained with Annexin/fluorescein isothiocyanate (FITC) and propidium io-ide using the BD ApoAlert Annexin V Kit (Clontech,ountain View, CA) according to the protocol recom-ended by the manufacturer. Annexin V/propidium io-
ide–stained cells were visualized under an Olympus in-erted fluorescence microscope (Olympus, Center Valley,A) with a 63� oil immersion objective using FITC andhodamine filters or analyzed by flow cytometry using theeckman Coulter Elite XL-MCL single-laser flow system
Beckman Coulter, Inc, Fullerton, CA).1
RNA Isolation and Real-Time QuantitativePolymerase Chain ReactionTotal cellular RNA was isolated using the Ambion
NAqueous Kit (Austin, TX). Total RNA (5 �g) was usedor first-strand complementary DNA synthesis using theigh-Capacity cDNA Archive Kit (Applied Biosystems, Fos-
er City, CA). The messenger RNA (mRNA) levels of CHOP,RP78, and �-actin were quantified using the followingrimers: CHOP, forward:5=-GGAGCAGGAGAATGAGAG-=, reverse: 5=-GACAGACAGGAGGTGATG-3=; GRP78, for-ard: 5=-TCCTGCGTCGGTGTATTC-3=, reverse: 5=-CGT-AGT TGGTTCTTGGC-3=; �-actin, forward: 5=-TATCGG-AATGAGCGGTTCC-3=, reverse: 5=-AGCACTGTGTT GGA TAGAGG-3=. iQ SYBR Green Supermix (Bio-Rad Labo-
atories, Hercules, CA) was used as a fluorescent dye toetect the presence of double-stranded DNA. The mRNAalues for each gene were normalized to internal control-actin mRNA using the ��Ct method.15
Construction of Lentiviral Small HairpinRNA for CHOPThe small hairpin RNA (shRNA) specifically target-
ng the nucleotides of CHOP was designed through siRNAarget Finder (Ambion). The lentiviral expression vectorsontaining CHOP shRNA were constructed as previouslyescribed.15 The sequences of 3 CHOP shRNAs were asollows: shRNA1 is 5=-CTGGAAGCCTGGTATGAGGA-3=,hRNA2 is GGAAACGGAAACAGAGTGGTC-3=, andhRNA3 is 5=-GCAGGAAATCGA GCGCCTGAC-3=. Theecombinant lentiviruses were produced and titered asescribed previously.15
IEC-6 cells were incubated with lentivirus at a multi-
licity of infection of 100 for 48 hours. The CHOP RNA
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ilencing effect of the lentiviral shRNA was confirmed byeal-time reverse-transcription polymerase chain reactionRT-PCR) and Western blot analysis.
Paracellular Tracer Flux AssayFlux assays were performed with the 12-mm
ranswell (Corning Life Sciences, Corning, NY) as de-cribed by Wong and Gumbiner16 with minor modifica-ion. Briefly, cells were plated at a confluent density of 4
105 cells/cm2 on the insert and maintained under theame culture conditions for an additional 48 hours tostablish tight monolayers. The membrane-impermeableolecule, [14C]-mannitol (MW 184), served as the para-
ellular tracer. At the beginning of the flux assay, bothides of the bathing wells of Transwell filters were re-laced with fresh medium containing individual HIV PI
5–25 �mol/L) and 0.5 mmol/L unlabeled mannitol andncubated at 37°C for 24 hours. [14C]-Mannitol wasdded to a final concentration of 3.6 nmol/L to the apicalathing wells that contained 0.5 mL of medium. The basalathing well had no added tracers and contained 1.5 mL ofhe same flux assay medium as in the apical compartment.ll flux assays were performed at 37°C, and the basal me-ium was collected 2 hours after addition of [14C]-mannitolor counting in a Beckman liquid scintillation counter. Theesults were expressed as a percentage of total count valuesf each tracer.17
In Vivo StudiesC57BL/6 wild-type and CHOP�/� mice with
56BL/6 background (male, 6 – 8 weeks old; Jackson Lab-ratories, Bar Harbor, ME) were used in this study. Miceere housed and fed standard mouse chow and tap waterd libitum throughout the study following protocolspproved by the Institutional Animal Care and Use Com-ittee at Virginia Commonwealth University.Mice were randomly assigned to the following 4 groups:
ontrol group, AMPV, RITV, and LOPV. Mice were gavagedaily with individual HIV PI (50 mg/kg) or control solutionor 2 or 4 weeks. Animals were monitored daily for appear-nce of diarrhea, body weight loss, and other distress. At thend of treatment, mice were killed. Blood was collected toeasure drug concentration. The intestines and colons were
emoved, and sections were taken for formalin fixation andistologic examination.
Intestinal Permeability In VivoMice were gavaged with FITC/dextran (4.4 kilo-
altons; Sigma–Aldrich, St Louis, MO) at a dose of 600g/kg body wt 4 hours before harvest. Blood was col-
ected from the hepatic portal vein. The serum concen-ration of the FITC-dextran was determined using a flu-rescence plate reader with an excitation wavelength at
90 nm and an emission wavelength of 530 nm.18,19 w
Histologic Assessment and MicroscopicScoringSmall intestine tissues were fixed in 10% neutral
uffered formalin overnight and then washed with phos-hate-buffered saline and transferred to 70% ethanol.ormalin-fixed tissues were then embedded in paraffin,ectioned at 5 �m, and stained with H&E using standardrocedures. Images were analyzed using a Motic BA200icroscope (Motic Instruments, Inc, Baltimore, MD).
nflammation and tissue damage were assessed micro-copically and histologically based on epithelial tissueamage (0, none; 1, destruction of villous tips; 2, destruc-ion of distal half of villi; 3, complete destruction of villi)nd neutrophil infiltration (0, none; 1, rare inflammationells; 2, extravagated inflammation cells; 3, inflammationells throughout lamina propria and epithelium). Micro-copic and histologic damage were recorded and scoredor each mouse by 2 different investigators who werelind to the treatment conditions.
Terminal DeoxynucleotidylTransferase–Mediated DeoxyuridineTriphosphate Nick-End Labeling AssayTo detect the apoptosis in intestine tissue, 5-�m
ections were deparaffinized and rehydrated throughashes with graded concentrations of ethanol. Tissueas pretreated with proteinase K (20 �g/mL) for 15inutes at room temperature, followed by incubation in
% H2O2 in phosphate-buffered saline for 5 minutes atoom temperature to quench endogenous peroxidase ac-ivity. Apoptotic cells were detected using ApoAlert DNAragmentation Assay Kit following the manufacturer’srotocol (BD Biosciences, Palo Alto, CA). Control stainsere obtained by processing in parallel duplicate sec-
ions, omitting only the TnT enzyme. The apoptotic cellsere counted.20
Statistical MethodsValues are means � SD from 3–6 samples. Western
lot results were repeated at least 3 times. One-way analysisf variance was used to analyze the data. Statistics wereerformed using Prism 5 (GraphPad, San Diego, CA).
ResultsHIV PIs Disrupt Paracellular Permeabilityin IECs
Maintenance of mammalian intestinal epithelialntegrity is critical for normal gastrointestinal function.
e first examined whether HIV PIs affect intestinal epi-helial paracellular barrier function by assessing the para-ellular flux of a membrane-impermeable tracer, [14C]-annitol, across the confluent monolayer after treatment
ith individual HIV PIs in IEC-6 cells. To verify the
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ystem used for paracellular permeability assays, the ef-ect of decreased cytosolic Ca2� on the paracellular flux of14C]-mannitol was tested and used as a positive control.s expected, exposure to Ca2�-free medium for 2 hours
ignificantly increased paracellular permeability in IEC-6ells (data not shown). Both LOPV and RITV markedlyncreased paracellular permeability in IEC-6 cells, even atoncentrations as low as 5 �mol/L (Figure 1B and C). Atconcentration of 15 �mol/L, LOPV and RITV increasedaracellular permeability by 4.6- and 1.9-fold, respectively
Figure 1B and C), but AMPV had no significant effectven at a concentration of 25 �mol/L (Figure 1A).
To further determine whether individual HIV PIs have aimilar effect on paracellular permeability in differentiatedECs, we examined the effect of HIV PIs (15 �mol/L) onaracellular permeability in differentiated IEC-Cdx2L1 cells.s shown in Figure 1D, LOPV and RITV also increasedaracellular permeability in differentiated IEC-Cdx2L1 cells.aracellular permeability was increased 2.2- and 1.9-fold inEC-Cdx2L1 cells exposed to LOPV and RITV (15 �mol/L)or 24 hours, respectively. AMPV had no significant effectn paracellular permeability in differentiated IEC-Cdx2L1
igure 1. Effect of HIV PIs on paracellular permeability in IEC cells.A–C) IEC-6 cells were treated with individual HIV PIs (0–25 �mol/L) for4 hours. (D) IEC-cdx2L1 cells were cultured on the filter in Dulbecco’sodified Eagle medium containing 4 mmol/L isopropyl-�-D-thiogalac-
opyranoside for 16 days to induce differentiation and treated with in-ividual HIV PIs (25 �mol/L) for 24 hours. The paracellular permeabilityas measured as described in Materials and Methods. Values areean � SD of 3 independent experiments and analyzed using one-way
nalysis of variance. **P � .01, ***P � .001.
ells. b
HIV PIs Induce Apoptosis in IECsDysregulation of tight junction and induction of
poptosis or necrosis in IECs are responsible for epithe-ial damage. Our previous studies have shown that HIVIs induced apoptosis in macrophages and hepato-ytes.1,10 We further examined whether HIV PIs also in-uce apoptosis in IECs. IEC-6 cells were treated with an
ndividual HIV PI (15 or 25 �mol/L) for 24 hours. Ashown in Figure 2, both LOPV and RITV dose-depen-ently induced apoptosis and necrosis in IEC-6 cells, butMPV had no significant effect. At a concentration of 15mol/L, LOPV- and RITV-induced apoptotic cells were4% and 19%, respectively, which is similar to that in-uced by 10 nmol/L of thapsigargin (Figure 2C). Similaresults were obtained in IEC-6 Cdx2L1-cells (data nothown). Immunofluorescent staining also indicated thatOPV, RITV, and thapsigargin (a known ER stress in-ucer) inhibited cell differentiation, but AMPV had lessffect (Supplementary Figure 2).
HIV PIs Induce ER Stress and Activate theUPR in IECsER stress is implicated in a wide range of pathol-
gies.1,5 It has been found that HIV PIs induce apoptosisnd decrease barrier function in human HT-29/B6 cellsa human colon carcinoma cell line).2 To delineate theellular and molecular mechanisms responsible for HIVI-induced apoptosis, we first examined the HIV PI-
nduced ER stress response in IEC-6 cells using the SEAPeporter system. IEC-6 cells, which were stably trans-ected with SEAP, were treated with individual HIV PIsor 24 hours. As shown in Figure 3, both RITV and LOPVose-dependently induced ER stress, which was indicated bydecrease of SEAP activity. However, AMPV had no signif-
cant effect. Thapsigargin was used as the positive control.To further determine whether HIV PI-induced ER stress
as correlated with the activation of the UPR in IEC-6 cells,e measured the expression of the ER stress master regula-
or, GRP78/Bip, and downstream transcriptional factorsHOP, XBP-1s (spliced form of XBP-1), and ATF-4. As
hown in Supplementary Figure 3, both LOPV and RITVignificantly induced GRP78, CHOP, XBP-1s, and ATF-4xpression. Up-regulation of XBP-1s and ATF-4 could bebserved as early as 1 hour after treatment. Both LOPV- andITV-induced UPR activation were dose dependent (Figure), but AMPV had no effect on UPR activation even at aoncentration of 50 �mol/L.
To determine whether HIV PIs also activate the UPR inifferentiated IECs, we measured UPR activation in dif-erentiated IEC-Cdx2L1 cells.17 As shown in Figure 5And B, LOPV and RITV significantly increased CHOP andRP78 mRNA levels both in IEC-Cdx2L1 cells and IEC-6
ells. AMPV also increased GRP78 mRNA level in IEC-dx2L1 cells but not in IEC-6 cells. Similarly, LOPV andITV increased CHOP, XBP-1s, and ATF-4 protein levels
oth in IEC-Cdx2L1 cells and IEC-6 cells (Figure 5C and
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). Consistent with the real-time RT-PCR data, AMPVlso up-regulated the expression of the UPR genesXBP-1s and ATF-4) in IEC-Cdx2L1 cells but not inarental IEC-6 cells. These results indicate that individ-al HIV PIs have different effects on UPR activation in
ECs, which is consistent with our previous observationsn macrophages and hepatocytes.1,10
Effect of HIV PI-Induced ER Stress onParacellular Permeability in IECsAlthough it has been shown that HIV PIs induce
igure 2. HIV PIs induce apoptosis in IECs. IEC-6 cells were treated wir 25 �mol/L), or thapsigargin (TG; 100 nmol/L) for 24 hours and then saterials and Methods. (A) The representative merged images of IEC-6
re shown. (B) Representative phase-contrast images of IEC-6 cells treepresentative flow cytometry diagrams of 3 independent experiments.
poptosis and disrupt intestinal barrier function,2 the 4
xact cellular mechanisms remain to be identified. Ourrevious studies1,10 and others21 suggest that HIV PI-
nduced ER stress and activation of the UPR play impor-ant roles in HIV PI-associated adverse side effects. Toetermine whether HIV PI-induced UPR activation inEC-6 cells is responsible for HIV PI-induced disruptionf intestinal barrier integrity, we constructed 3 lentiviralhRNA to knock down the expression of CHOP in IEC-6ells. As shown in Figure 6, shRNA1 and shRNA3 signif-cantly inhibited CHOP mRNA and protein expression by
ethyl sulfoxide (DMSO), individual HIV PIs (AMPV, LOPV, and RITV; 15d with Annexin V-FITC/propidium iodide and analyzed as described intreated with individual HIV PIs or TG from 3 independent experimentsith individual HIV PI (25 �mol/L) or TG (100 nmol/L) for 24 hours. (C) The
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ad no inhibitory effect and was used as a negativeontrol in functional studies. The transfection efficiencyf traditional lipid-based reagents in IEC-6 is relatively
ow (�30%), but the infection rate of lentivirus in IEC-6ould reach as high as 90% (data not shown).
To determine whether down-regulation of CHOP ex-ression will prevent HIV PI-induced disruption of para-ellular permeability in IEC-6 cells, we infected the cellsith lentiviral-CHOP shRNA2 and shRNA3 at a multi-licity of infection of 100 for 48 hours and measured thearacellular permeability as described previously. Ashown in Figure 6C, knockdown of CHOP expressionnhibited a LOPV- and RITV-induced increase of paracel-ular permeability by 37% and 29%, respectively, in IEC-6ells. Consistently, LOPV- and RITV-induced apoptosisas also reduced after knockdown of CHOP gene expres-
ion (Figure 6D). These results indicate that HIVI-induced ER stress and subsequent activation of CHOPxpression play a critical role in HIV PI-induced disrup-ion of paracellular permeability.
HIV PIs Induce ER Stress and IncreaseIntestinal Permeability In VivoTo determine whether HIV PIs also induce ER
igure 3. HIV PIs induce ER stress in IEC-6 cells. IEC-6 cells stably traITV; 0–25 �mol/L) or thapsigargin (TG; 100 nmol/L) for 24 hours. Acxpressed as percent of control. Values are mean � SD of 5 independen
tress in intestine in vivo, we examined the effect of d
ndividual HIV PIs on UPR activation in vivo using57BL/6 wild-type male mice. As shown in Figure 7A,oth RITV and LOPV significantly increased CHOP andBP-1s expression in intestine after 4 weeks of treatment,hereas AMPV had no significant effect. We further ex-mined the effect of HIV PIs on intestinal permeability inivo using FITC-dextran as probe. As shown in Figure 6E,-week treatment of HIV PIs had no significant effect on
ntestinal permeability, but 4-week treatment with LOPVnd RITV resulted in a significant increase of the intes-inal permeability.
HIV PIs Induce Apoptosis and PathologicChanges in Intestine In VivoHistologic examination revealed that HIV PIs had
o significant effect on the colon (Supplementary Figure) and AMPV caused minor pathologic changes in intes-ine. However, both LOPV and RITV started to causeignificant pathologic changes in intestine at 2 weeks ofreatment, inducing a loss of villi, edema, neutrophilnfiltration, and necrosis. The 4-week treatment withOPV and RITV induced more severe damage in intesti-al epithelium and lamina propria (Figure 7B). In controlnd AMPV-treated animals, less tissue damage and fewerminal deoxynucleotidyl transferase–mediated deoxyuri-
ted with pSEAP plasmid were treated with HIV PIs (AMPV, LOPV, andof SEAP was measured as described in Materials and Methods anderiments. Statistical significance relative to vehicle control: ***P � .001.
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ells were detected, whereas in LOPV- and RITV-treatednimals, many more TUNEL-positive cells were observed inhe intestine (Figure 8).
Deletion of CHOP Ameliorates HIVPI-Induced Pathologic Changes in Intestineand Prevents HIV PI-Induced Increase ofIntestinal PermeabilityHistologic assessment showed that wild-type and
HOP�/� mice appeared similar under basal conditions.owever, HIV PI-induced apoptosis and pathologic
hanges in wild-type mice were diminished in CHOP�/�
ice, with significantly reduced edema and tissue dam-ge, and fewer TUNEL-positive cells were detected (Fig-res 7B–D and 8). Similarly, LOPV and RITV had lessffect on intestinal permeability in CHOP�/� mice (Fig-re 6E). Thus, deletion of the ER stress responsive gene,HOP, dramatically prevents HIV PI-induced apoptosisnd intestinal damage.
DiscussionHIV PIs have been successfully incorporated into
igure 4. Activation of the UPR by HIV PIs. (A) Representative immunf IEC-6 cells treated with individual HIV PIs (0–25 �mol/L) for 4 hoursrotein levels of CHOP, XBP-1, and ATF-4. Statistical significance relat
ighly active antiretroviral therapy to control virus rep- g
ication in HIV-infected patients. However, the benefits ofIV PIs in reducing morbidity and mortality of patientsith HIV require high compliance of the patients, which
s seriously hampered by HIV PI-associated gastrointesti-al adverse effects. The most frequently reported gastro-
ntestinal side effects of HIV PIs include abdominal pain,bnormal stools, and diarrhea.3 However, the exact cel-ular mechanisms underlying HIV PI-induced gastroin-estinal dysfunction remain largely unknown.
Recent studies by Bode et al suggest that HIV PIsnduce apoptosis and impair the barrier function in hu-
an HT-29/B6 cells, which suggests a leak-flux type ofiarrhea in patients receiving HIV PIs.2 Whether HIV PIsave similar effects on normal IECs has not been exam-
ned. In the present study, we found that the 2 mostommonly used HIV PIs, RITV and LOPV, significantlynduced apoptosis and disrupted intestinal epithelial bar-ier integrity both in vitro and in vivo, whereas AMPVad less effect.The mammalian intestinal epithelium is a rapidly self-
enewing tissue in the body, and its integrity is main-ained by a strictly regulated process of cell proliferation,
against CHOP, ATF-4, XBP-1, and lamin B from the nuclear extractsin B was used as a loading control of nuclear protein. (B–D) Relativevehicle control: *P � .05; **P � .01; ***P � .001.
oblots. Lam
rowth arrest, and apoptosis.22 Our previous studies have
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hown that HIV PI-induced ER stress response and acti-ation of the UPR are responsible for HIV PI-induced cellpoptosis both in macrophages and hepatocytes.1,10 Itlso has been reported that HIV PI-induced ER stressesponse is linked to insulin resistance and dysregulationf lipid metabolism in adipocytes.11,23 Numerous studies
igure 5. HIV PIs activate the UPR in differentiated IECs. (A and B) Reand IEC-6 cells treated with AMPV, LOPV, and RITV (25 �mol/L) for 24ontrol �-actin mRNA using the ��Ct method. DMSO, dimethyl sulfoepresentative immunoblots against CHOP, ATF-4, XBP-1, and lamin BOPV, RITV (25 �mol/L), or TG (100 nmol/L) for 4 hours. Lamin B was und ATF-4. Statistical significance relative to vehicle control: *P � .05.
ave suggested that ER stress signaling pathways are v
mplicated in various diseases, including atherosclerosis,etabolic disease, liver disease, and inflammatory bowel
isease.6,24 –27 However, whether HIV PI-induced apopto-is is also associated with the activation of the ER stressesponse in IECs has not been explored. Herein, we de-cribe our investigation of whether HIV PI-induced acti-
RT-PCR analysis of mRNA levels of CHOP and GRP78 in IEC-Cdx2L1rs. The relative mRNA levels for each gene were normalized to internalStatistical significance relative to vehicle control, *P � .05. (C and E)the nuclear extracts of IEC-Cdx2L1 and IEC-6 cells treated with AMPV,as a loading control. (D and F) Relative protein levels of CHOP, XBP-1,
l-timehou
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ion of apoptosis and disruption of normal intestinalarrier function both in in vitro cell culture models and
n in vivo mice models.Several recent studies have shown that ER stress re-
ponses contribute to the pathogenesis of chronic intes-inal inflammation.9,24,27 Disruption of ER homeostasisy adverse environmental and/or metabolic conditions
igure 6. Effect of CHOP knockdown on HIV PI-induced increase of paRNA levels in IEC-6 cells infected with individual lentiviral-CHOP-shRN-actin mRNA using the ��Ct method. Statistical significance relative tnd lamin B from the nuclear extracts of IEC-6 cells treated with the indontrol. (C) IEC-6 cells were infected with lentiviral-CHOP shRNA2 or s4 hours. The paracellular permeability was measured as described in Mtatistical significance relative to vehicle control: ***P � .001, **P � .0hase-contrast images of IEC-6 cells infected with CHOP shRNAs andermeability in vivo. Wild-type and CHOP�/� mice were treated with ineasured using FITC-dextran as described in Materials and Methods.
riggers cellular stress responses including the UPR, the d
R specific stress response. The signaling pathways of thePR are important for maintenance of normal cellularomeostasis. However, inappropriate activation of thePR can lead to cellular dysfunction and ultimately celleath. The results from our functional studies showedhat HIV PIs not only activated the UPR but also dis-upted the barrier integrity both in undifferentiated and
lular permeability in IEC-6 cells. (A) Real-time RT-PCR analysis of CHOP48 hours. The mRNA levels of CHOP were normalized to internal controlicle control: *P � .001. (B) Representative immunoblots against CHOPal lentiviral CHOP shRNA for 48 hours. Lamin B was used as a loading3 for 48 hours and then treated with individual HIV PIs (15 �mol/L) forls and Methods. Values are mean � SD of 3 independent experiments.atistical significance relative to shRNA2: #P � .05. (D) Representatived with individual HIV PIs for 24 hours. (E) Effect of HIV PIs on intestinalal HIV PIs (50 mg/kg) for 2 or 4 weeks. The intestinal permeability wastical significance relative to vehicle control: **P � .01.
racelA foro vehividuhRNAateria1. Sttreatedividu
ifferentiated IECs (Figure 1). In vivo animal studies
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igure 7. HIV PIs activate the UPR and induce damage in intestine. Mice were treated with individual HIV PIs (50 mg/kg) for 2–4 weeks. (A)epresentative immunoblots of nuclear extracts isolated from intestine tissues against CHOP, XBP-1, and lamin B are shown. (B) Representative
mages of H&E staining for each treatment group is shown. (C and D) Histologic score of HIV PI-induced epithelial tissue damage and neutrophil
January 2010 ER STRESS AND INTESTINAL BARRIER FUNCTION 207
urther showed that HIV PIs, LOPV and RITV, inducedevere damage to intestinal tissue (Figure 7). However,MPV, which had less effect on UPR activation, had littleffect on intestinal barrier function. Both in vitro and inivo studies suggest that HIV PI-induced activation of thePR is correlated to its effect on intestinal barrier function.To further demonstrate our notion that HIV PI-
nduced ER stress response is critical to HIV PI-inducedamage in intestinal epithelia, we examined the effect ofnocking down CHOP expression on HIV PI-inducedysfunction of IECs. CHOP is a UPR-induced transcrip-
igure 8. HIV PIs induce apoptosis in intestine in vivo. The representatB) CHOP�/� mice treated with individual HIV PIs as described in Materiaelative to vehicle control: *P � .05, **P � .01.
ion factor that mediates apoptosis.6 It has been reported d
hat macrophages from CHOP�/� mice are highly pro-ected from free cholesterol-induced cell death.28 Theesults from our current study also clearly showed thatown-regulation of CHOP expression significantlylocked LOPV- and RITV-induced paracellular perme-bility in IECs (Figure 6). In vivo studies with CHOP�/�
ice further demonstrated that expression of CHOPlays a critical role in HIV PI-induced intestinal tissueamage and barrier function (Figures 6 – 8). Therefore,IV PI-induced ER stress and subsequent activation of
he UPR represent an important cellular mechanism un-
NEL staining images of intestine tissue sections from (A) wild-type andMethods. (C) The apoptotic cells were counted. Statistical significance
ive TUls and
erlying HIV PI-induced dysfunction of IECs. However,
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ow HIV PIs induce ER stress in IECs remains to beurther elucidated.
It has been well established that the UPR can be acti-ated by many stress signals that cause the accumulationf unfolded or misfolded proteins in the ER lumen, suchs accumulation of free cholesterol, depletion of ER cal-ium stores, and inhibition of N-glycosylation.6 Our pre-ious studies have shown that HIV PIs induced the ac-umulation of intracellular free cholesterol and depletedhe ER calcium store in macrophages and primary hepa-ocytes.1,10 Our preliminary studies also found that LOPVnd RITV, but not AMPV, depleted the ER calcium storen IEC-6 cells (Supplementary Figure 5).
It has been reported that unresolved ER stress activateshe c-Jun-N-terminal kinase (JNK) signaling pathway andnduces inflammatory response.9,24 We also examined theffect of HIV PIs on activation of mitogen-activated pro-ein kinases both in vitro and in vivo. As shown inupplementary Figure 6A, LOPV and RITV not onlyctivated the JNK but also inhibited the extracellularignal–regulated kinase (ERK) and AKT signaling path-ays in differentiated intestinal cells. It has been shown
hat activation of ERK and AKT signaling pathways pre-ents tumor necrosis factor �–induced apoptosis inECs.29 Furthermore, RITV also activated JNK in wild-ype mice intestine but not CHOP�/� mice intestine.
Recent studies by Heazlewood et al show that activa-ion of ER stress response in goblet cells leads to spon-aneous colitis.18 It also has been shown that disruptionf Xbp1 depletes Paneth cells and reduces goblet cells inmall intestine but has no effect on epithelial barrier.24 Inhe present study, we did not observe significant changesn goblet cells. Both in vitro and in vivo studies suggesthat HIV PIs induce apoptosis of epithelial cells.
In summary, our present studies provide the first evi-ence suggesting that ER stress response is implicated inhe HIV PI-induced apoptosis and disruption of barrierntegrity both in in vitro cultured IECs and in in vivontestinal tissue. Together with our previous findings in
acrophages and hepatocytes, our studies suggest that theIV PI-induced ER stress response represents a critical cel-
ular mechanism underlying HIV PI-associated various ad-erse effects in HIV PI-treated patients. Understanding ofhe molecular mechanisms of HIV PI-induced ER stress willrovide important information for developing more effec-ive anti-HIV therapeutics that could avoid some of thedverse effects associated with current HIV PIs.
Supplementary Data
Note: To access the supplementary materialccompanying this article, visit the online version ofastroenterology at www.gastrojournal.org, and at doi:
0.1053/j.gastro.2009.08.054.
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Received January 7, 2009. Accepted August 20, 2009.
eprint requestsAddress requests for reprints to: Huiping Zhou, PhD, Department
f Microbiology & Immunology, Virginia Commonwealth University,O Box 980678, Richmond, Virginia 23298-0678. e-mail: [email protected]; fax: (804) 828-0676.
cknowledgmentsThe authors thank the AIDS Research and Reference Reagent
rogram, National Institutes of Health, and GlaxoSmithKlineamprenavir) for providing the compounds used in this research.
onflicts of interestThe authors disclose no conflicts.
undingSupported by grants from the National Institutes of Health
R21AI068432, R01AT004148, R01AI057189, P01DK38030,30CA16059, and R01DK064240), A.D. Williams fund, and Jeffress
