Translational Inhibition of Colonic Epithelial Heat Shock Proteins by IFN-γ and TNF-α in Intestinal Inflammation
2007; Elsevier BV; Volume: 133; Issue: 6 Linguagem: Inglês
10.1053/j.gastro.2007.09.026
ISSN1528-0012
AutoresShien Hu, Mae J. Ciancio, Maor Lahav, Mikihiro Fujiya, Lev Lichtenstein, Shrikant Anant, Mark W. Musch, Eugene B. Chang,
Tópico(s)thermodynamics and calorimetric analyses
ResumoBackground & Aims: Inducible heat shock proteins (iHsp), Hsp25/27 and Hsp70, play essential roles in protecting cells against stress and, in intestinal mucosal inflammation, potentially lessening the extent and severity of injury. We examined the expression and regulation of iHsp in human and experimental inflammatory bowel diseases (IBD) and in vitro. Methods: iHsp expression and regulation were assessed in normal and IBD colonic biopsy specimens, IL-10−/− mice, and young adult mouse colonic epithelial cells by immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR). Phosphorylation of double-stranded RNA-dependent protein kinase (PKR) and eukaryotic initiation factor-2α (eIF-2α) was determined by Western blot. Results: Hsp25/27 and Hsp70 levels were selectively reduced in areas of active mucosal inflammation associated with human IBD and IL-10−/− mice with colitis. Wild-type mice treated in vivo with interferon (IFN)-γ + tumor necrosis factor (TNF)-α also demonstrated reduced colonic Hsp25/27 and Hsp70. In young adult mouse colonic epithelial cells, IFN-γ+TNF-α inhibited heat induction of Hsp25/27 and Hsp70, an effect not associated with changes in iHsp messenger RNA or protein half-lives but caused by suppressed de novo iHsp synthesis. IFN-γ+TNF-α cotreatment activated PKR, resulting in phosphorylation and inactivation of eIF-2α, an essential factor in protein translation. These effects were not due to induced apoptosis and could be negated by PKR-inhibitor and short interfering RNA to PKR. Increased phosphorylation of PKR and eIF-2α were also observed in active IBD tissues. Conclusions: Mucosal inflammation is associated with iHsp down-regulation, an effect that appears mediated by translational down-regulation by proinflammatory cytokines. In the context of IBD, we propose that this mechanism contributes to the severity, extent, and persistence of inflammation-induced mucosal injury. Background & Aims: Inducible heat shock proteins (iHsp), Hsp25/27 and Hsp70, play essential roles in protecting cells against stress and, in intestinal mucosal inflammation, potentially lessening the extent and severity of injury. We examined the expression and regulation of iHsp in human and experimental inflammatory bowel diseases (IBD) and in vitro. Methods: iHsp expression and regulation were assessed in normal and IBD colonic biopsy specimens, IL-10−/− mice, and young adult mouse colonic epithelial cells by immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR). Phosphorylation of double-stranded RNA-dependent protein kinase (PKR) and eukaryotic initiation factor-2α (eIF-2α) was determined by Western blot. Results: Hsp25/27 and Hsp70 levels were selectively reduced in areas of active mucosal inflammation associated with human IBD and IL-10−/− mice with colitis. Wild-type mice treated in vivo with interferon (IFN)-γ + tumor necrosis factor (TNF)-α also demonstrated reduced colonic Hsp25/27 and Hsp70. In young adult mouse colonic epithelial cells, IFN-γ+TNF-α inhibited heat induction of Hsp25/27 and Hsp70, an effect not associated with changes in iHsp messenger RNA or protein half-lives but caused by suppressed de novo iHsp synthesis. IFN-γ+TNF-α cotreatment activated PKR, resulting in phosphorylation and inactivation of eIF-2α, an essential factor in protein translation. These effects were not due to induced apoptosis and could be negated by PKR-inhibitor and short interfering RNA to PKR. Increased phosphorylation of PKR and eIF-2α were also observed in active IBD tissues. Conclusions: Mucosal inflammation is associated with iHsp down-regulation, an effect that appears mediated by translational down-regulation by proinflammatory cytokines. In the context of IBD, we propose that this mechanism contributes to the severity, extent, and persistence of inflammation-induced mucosal injury. The extent and severity of mucosal lesions associated with IBD are dependent on the balance between aggressive, cell-destructive processes and counteracting cytoprotective and wound reparative mechanisms. In the context of IBD, investigations of the latter are few, and strategies to promote cellular protection of inflamed mucosa have been inadequately explored. In this regard, inducible heat shock proteins (iHsp) warrant further investigation because of their known cytoprotective properties, especially Hsp25 and Hsp70. In humans, Hsp27 is the homolog of Hsp25. Inducible Hsps are part of a highly conserved class of proteins that act to protect cellular structure and function.1Musch M.W. Kaplan B. Chang E.B. Role of increased basal expression of heat shock protein 72 in colonic epithelial Caco2BBE adenocarcinoma cells.Cell Growth Differ. 2001; 12: 419-426PubMed Google Scholar, 2Ropeleski M.J. Tang J. Walsh-Reitz M.M. et al.Interleukin-11-induced heat shock protein 25 confers intestinal epithelial-specific cytoprotection from oxidant stress.Gastroenterology. 2003; 124: 1358-1368Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 3Ropeleski M.J. Riehm J. Baer K.A. et al.Anti-apoptotic effects of L-glutamine-mediated transcriptional modulation of the heat shock protein 72 during heat shock.Gastroenterology. 2005; 129: 170-184Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 4Liu T.S. Musch M.W. Sugi K. et al.Protective role of HSP72 against Clostridium difficile toxin A-induced intestinal epithelial cell dysfunction.Am J Physiol Cell Physiol. 2003; 284: C1073-C1082Crossref PubMed Scopus (66) Google Scholar, 5Sussman C.R. Renfro J.L. Heat shock-induced protection and enhancement of Na+-glucose cotransport by LLC-PK1 monolayers.Am J Physiol. 1997; 273: F530-F537PubMed Google Scholar, 6Musch M.W. Sugi K. Straus D. et al.Heat-shock protein 72 protects against oxidant-induced injury of barrier function of human colonic epithelial Caco2/bbe cells.Gastroenterology. 1999; 117: 115-122Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar In normal intestinal mucosa, Hsp25/27 and Hsp70 are expressed "constitutively" by surface epithelium in the colon and stomach, areas that are continuously subjected to inducing signals from enteric flora and short-chain fatty acids.7Rakoff-Nahoum S. Paglino J. Eslami-Varzaneh F. et al.Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis.Cell. 2004; 118: 229-241Abstract Full Text Full Text PDF PubMed Scopus (3448) Google Scholar, 8Kojima K. Musch M.W. Ren H. et al.Enteric flora and lymphocyte-derived cytokines determine expression of heat shock proteins in mouse colonic epithelial cells.Gastroenterology. 2003; 124: 1395-1407Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 9Arvans D.L. Vavricka S.R. Ren H. et al.Luminal bacterial flora determines physiological expression of intestinal epithelial cytoprotective heat shock proteins 25 and 72.Am J Physiol. 2005; 288: G696-G704Crossref PubMed Scopus (100) Google Scholar Maintained expression of Hsp25/27 and Hsp70 contributes to colonic epithelial resistance to bacterial toxins and inflammation-associated stress.4Liu T.S. Musch M.W. Sugi K. et al.Protective role of HSP72 against Clostridium difficile toxin A-induced intestinal epithelial cell dysfunction.Am J Physiol Cell Physiol. 2003; 284: C1073-C1082Crossref PubMed Scopus (66) Google Scholar, 10Fujiya M. Musch M.W. Nakagawa Y. et al.The Bacillus subtilis quorum-sensing molecule CSF contributes to intestinal homeostasis via OCRN2, a host cell membrane transporter.Cell Host Microbe. 2007; 1: 299-308Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 11Ren H. Musch M.W. Kojima K. et al.Short-chain fatty acids induce intestinal epithelial heat shock protein 25 expression in rats and IEC 18 cells.Gastroenterology. 2001; 121: 631-639Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar We report here that colonic epithelial expression of Hsp25/27 and Hsp70 are significantly and selectively reduced in actively inflamed mucosa from both human IBD biopsy specimens and interleukin (IL)-10−/− mice with spontaneous colitis. A significant part of this effect is mediated by the proinflammatory cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α. We demonstrate that IFN-γ and TNF-α cotreatment down-regulates colonic epithelial Hsp25/27 and Hsp70 translation without altering messenger RNA (mRNA) levels or accelerating protein degradation. We hypothesize that IFN-γ and TNF-α cotreatment promotes tissue inflammation by stimulating the phosphorylation of the RNA-dependent protein kinase (PKR), which phosphorylates eukaryotic initiation factor-2α (eIF-2α). This results in reduced protein translation under conditions of stress, as observed with IBD and experimental colitis. However, inhibited translation of iHsp is particularly pronounced and out of proportion to that of other proteins, raising the possibility of additional translation regulatory mechanisms that are set into play in the context of mucosal inflammation. The down-regulated expression of these key cytoprotective proteins would render the intestinal mucosa more vulnerable to inflammation-associated stress and injury. Colonic pinch biopsy specimens were obtained from normal, Crohn's disease (CD) and ulcerative colitis (UC) patients undergoing colonoscopy at the University of Chicago Medical Center under a protocol approved by the Institutional Review Board. Informed consent was granted prior to obtaining tissues. After removal, biopsy specimens were placed immediately on ice and rinsed in ice-cold phosphate-buffered saline (PBS) prior to cell lysis for RNA and protein extraction as detailed below. Tissues from the same patients were also fixed in neutral buffered formalin for H&E or immunohistochemistry staining. H&E-stained sections were evaluated pathologically to distinguish actively inflamed tissue from uninvolved tissue in a blinded manner using a scoring system as previously described.12Dieleman L.A. Palmen M.J. Akol H. et al.Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines.Clin Exp Immunol. 1998; 114: 385-391Crossref PubMed Scopus (929) Google Scholar Male wild-type or IL-10−/− gene-deficient mice on the C57Bl/6 background (Jackson Laboratories, Bar Harbor, ME) were bred under conventional conditions and allowed free access to food and water. Mice were killed at 20 weeks.13Kühn R. Löhler J. Rennick D. et al.Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3761) Google Scholar For treatment with cytokines, adult C57Bl/6 male mice (20–25 g) were given 1 intraperitoneal injection of IFN-γ (2500 U; PeproTech, Rocky Hill, NJ), TNF-α (1000 ng; PeproTech), or a combination of IFN-γ and TNF-α 48 hours prior to death. For animal studies, colonic mucosa was scraped and homogenized for RNA and protein extraction as described below. Tissues from the same samples were also fixed in formalin for H&E staining. States of inflammation were assessed as above.12Dieleman L.A. Palmen M.J. Akol H. et al.Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines.Clin Exp Immunol. 1998; 114: 385-391Crossref PubMed Scopus (929) Google Scholar All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Chicago. Conditionally immortalized, young adult mouse colonic epithelial cells (YAMC) were used, which was a generous gift from Dr Robert Whitehead, Vanderbilt University, Nashville, TN. YAMCs were grown at 33°C as previously described.14Whitehead R.H. Van Eeden P.E. Noble M.D. et al.Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice.Proc Natl Acad Sci U S A. 1993; 90: 587-591Crossref PubMed Scopus (343) Google Scholar Cells were fed IFN-γ-free medium and switched to the nonpermissive conditions of 37°C for 16–24 hours. During this time, SV40 large T antigen is no longer produced, and any remaining protein misfolds because of a temperature-sensitive mutation at amino acid 58 (tsA). During this period, cells were treated with the cytokines murine IFN-γ (200 U/mL), murine TNF-α (100 ng/mL), or a combination of IFN-γ and TNF-α for 8 hours prior to heat shock and harvest. For in vitro assessment of iHsp production, cells were heated to 42°C for 23 minutes and returned to 37°C for 120 minutes, providing a maximal iHsp induction.15Musch M.W. Ciancio M.J. Sarge K. et al.Induction of heat shock protein 70 protects intestinal epithelial IEC-18 cells from oxidant and heat injury.Am J Physiol. 1996; 270: C429-C436PubMed Google Scholar To inhibit PKR and eLF-α phosphorylation, the PKR inhibitor (PKR-I; 30 nmol/L, Calbiochem, San Diego, CA) was added 30 minutes before heat induction. PKR expression was also inhibited by a Stealth silencing oligonucleotide (NM_011163; 5′-GGACCUCCACAUGACAGAAGGUUUA-3′) designed by the RNAi Designer software (Invitrogen, Carlsbad, CA). As a negative control, an oligonucleotide with the same base composition but different sequence was used. Oligonucleotides were diluted with Optimem (Invitrogen) to a final concentration of 20 nmol/L. Silentfect reagent (Bio-Rad, Hercules, CA) was diluted to 2 μL/mL separately with Optimem, mixed with diluted oligos, allowed to complex for 30 minutes at room temperature, and added to the cells for 24 hours before harvest. Decreased PKR expression was confirmed by Western blotting. In all experiments for harvest, cells were rinsed 2 times and scraped into ice-cold PBS. Cells were pelleted (14,000g × 20 seconds) and lysed for RNA and protein extraction as described below. Sections of human biopsy specimens and mouse tissues were fixed in 10% (vol/vol) neutral buffered formalin, embedded in paraffin, and 4-μm cross sections cut. Sections were stained for human Hsp27, mouse Hsp25, or Hsp70 using the Vecastain Elite ABC kit (Vector Labs, Burlingame, CA) according to the manufacturer's instructions modified to use citrate-microwave antigen recapture as previously described using antibodies described below.8Kojima K. Musch M.W. Ren H. et al.Enteric flora and lymphocyte-derived cytokines determine expression of heat shock proteins in mouse colonic epithelial cells.Gastroenterology. 2003; 124: 1395-1407Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 16Kojima K. Musch M.W. Ropeleski M.J. et al.Escherichia coli LPS induces heat shock protein 25 in intestinal epithelial cells through MAP kinase activation.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G645-G652Crossref PubMed Scopus (58) Google Scholar Human biopsy specimens, mouse mucosal samples, or YAMCs were homogenized in 10 mmol/L Tris, pH 7.4, 5 mmol/L MgCl2, complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN), 50 U/mL DNAse (Amersham, Piscataway, NJ), and 50 U/mL RNAse (Ambion Inc, Austin, TX). Ten microliters was removed for protein analysis using the bicinchoninic acid method. To the remainder, 3X Laemmli stop solution was added and heated to 65°C for 10 minutes. Twenty micrograms protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes in 25 mmol/L Tris, pH 8.8, 192 mmol/L glycine, 15% vol/vol methanol. Membranes were blocked with 5% wt/vol nonfat dry milk in Tween-Tris-buffered saline (TTBS). Primary antibodies, for Hsp25 (SPA801; Stressgen), Hsp27 (SPA800; Stressgen), Hsp70 (SPA810; Stressgen), Hsc70 (SPA815; Stressgen), β-actin (AAN01; Cytoskeleton, Denver, CO), villin (610359; BD Biosciences, San Jose, CA), eIF-2α (9722; Cell Signaling, Beverly, MA), phosphorylated eLF-α (Ser51) (9721; Cell Signaling), PKR (07-151; Upstate, Charlottesville, VA), or phosphorylated PKR (Thr446) (07-532; Upstate), were added and incubated overnight at 4°C. Membranes were washed with TTBS, incubated with horseradish peroxidase-conjugated species-appropriate secondary antibodies (Jackson Immunoresearch, West Grove, PA) for 1 hour at room temperature and developed using an enhanced chemiluminescence system (Supersignal; Pierce, Rockford, IL). Quantification of images was done by scanning densitometry using NIH Image J 1.54 software (National Institutes of Health, Bethesda, MD). To determine whether apoptosis occurred in YAMCs, analysis of the cleavage products of poly-ADP ribose polymerase (PARP) and caspase 3 were determined.17Lazebnik Y.A. Kaufmann S.H. Desnoyers S. et al.Cleavage of poly (ADP-ribose) polymerase by a proteinase with properties like ICE.Nature. 1994; 371: 346-377Crossref PubMed Scopus (2372) Google Scholar, 18Nicholson D.W. Ali A. Thornberry N.A. et al.Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature. 1995; 376: 37-43Crossref PubMed Scopus (3843) Google Scholar YAMCs were treated with IFN-γ and TNF-α for 8 hours and heat shocked as described previously. Cells treated with staurosporine (1 μmol/L) for 3 hours were analyzed as a positive control for apoptosis. The cleaved caspase 3 and cleaved PARP protein was detected by Western blotting using antibodies specific to caspase 3 (9662, Cell Signaling, Beverly, MA) and PARP (9532; Cell Signaling). Total RNA was extracted from human colonic biopsy specimens, mouse intestinal mucosal samples, or pelleted YAMCs by Trizol (Invitrogen, Grand Island, NY) according to the manufacturer's instruction. Complementary DNA was synthesized using SuperScript II (Invitrogen) and random hexonucleotide primer. The mouse Hsp25 (L07577), mouse Hsp70 (NM_010478), human Hsp27 (NM_001540), and human Hsp70 (NM_005346) sequences were downloaded from GenBank. The forward and reverse primers are as follows: for mouse Hsp25: 5′-GAAGAAAGGCAGGACGAACA-3′ and 5′-CCTCAGGGGATAGGGAAGAG-3′; for mouse Hsp70: 5′-GGCTGATCGGCCGCAAGTT-3′ and 5′-GGAAGGGCCAGTGCTTCAT-3′; for mouse GAPDH: 5′-GGCAAATTCAACGGCACAGT-3′ and 5′-AGATGGTGATGGGCTTCCC-3′; for human Hsp27: 5′-CGTGTCCCTGGATGTCAACC-3′ and 5′-TCTCCACCACGCCATCCT-3′; for human Hsp70: 5′-ACTGCCCTGATCAAGCGC-3′ and 5′-CGGGTTGGTTGTCGGAGTAG-3′. Real-time polymerase chain reaction (PCR) was performed in an iCycler (Bio-Rad) using iQSYBR Green PCR supermix (Bio-Rad). The 2-step quantification cycling protocol was used. The threshhold cycle (Ct) value is defined as the cycle number at which the fluorescence crosses a fixed threshold above the baseline. As a relative quantitation, fold changes were measured using the ΔΔCt method. For each sample, the Ct value of iHsp mRNA was measured and compared with the GAPDH endogenous control as ΔCt, (ΔCt = CtHsp − CtGAPDH). The fold change of iHsp mRNA in the unknown sample relative to control sample was determined by 2−ΔΔCt, where ΔΔCt = ΔCtUnknown − ΔCtControl.19Schmittgen T.D. Zakrajsek B.A. Mills A.G. et al.Quantitative reverse transcription polymerase chain reaction to study mRNA decay: comparison of end point and real-time methods.Anal Biochem. 2000; 285: 194-204Crossref PubMed Scopus (874) Google Scholar To measure translation, 35S-methionine or 3Ropeleski M.J. Riehm J. Baer K.A. et al.Anti-apoptotic effects of L-glutamine-mediated transcriptional modulation of the heat shock protein 72 during heat shock.Gastroenterology. 2005; 129: 170-184Abstract Full Text Full Text PDF PubMed Scopus (47) Google ScholarH-leucine labeled amino acid incorporation was used. After cytokine treatment and heat induction, YAMCs were returned to 37°C 5% CO2 incubator, incubated in either serum-free methionine-free RPMI (Biosource International, Rockville, MD) with 50 μCi/mL of 35S-methionine (Express protein labeling mix, NEG072007MC; Perkin Elmer [the mixture contains 73% L-35S-methionine and 22% L-35S-cysteine]) or serum-free leucine-free RPMI with 70 μCi/mL of 3H-leucine (L-4,5-3H-leucine, MT672; Movarek Biochemicals, Brea, CA) for 60 minutes immediately before harvest. Cells were harvested and pelleted as previously described at 60, 90, and 120 minutes after heat induction. Pelleted cells were lysed in RIPA buffer (50 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 0.1% SDS, 1% Triton X-100, 0.5% Na deoxycholate, 1 mmol/L DTT, protease inhibitors, 100 U/mL RNASIN). As a measure of total protein synthesis, 35S-methionine or 3H-leucine incorporation into trichloroacetic acid (TCA)-precipitated material was determined. Twenty microliters cell lysate was incubated in 2 mL 20% wt/vol TCA for 30 minutes. TCA precipitates were filtered onto HA filters (Millipore Corp, Bedford, MA) and washed twice with 10% TCA. Filters were solubilized, and radioactivity was quantified by liquid scintillation counting. To immunoprecipitate Hsp25, Hsp70, or β-actin, lysates were incubated overnight at 4°C with anti-Hsp25, anti-Hsp70, or anti-β-actin antibody (same as used in Western blot) coupled to Protein A-Sepharose (Sigma Chemical Co, St. Louis, MO). Immunoprecipitates were pelleted by centrifugation, washed 4 times with RIPA buffer, boiled for 5 minutes in 2X Laemmli buffer, and separated on SDS-PAGE. To measure incorporation of 35S-methionine into immunoprecipitated Hsp70 or β-actin, gels were treated for fluorography (soaking in 10% TCA for 15 minutes followed by 1 mol/L sodium salicylate for 15 minutes). Gels were dried and exposed to film at −80°C for 6–12 hours. Image quantification of Hsp70 and β-actin was done as described above.15Musch M.W. Ciancio M.J. Sarge K. et al.Induction of heat shock protein 70 protects intestinal epithelial IEC-18 cells from oxidant and heat injury.Am J Physiol. 1996; 270: C429-C436PubMed Google Scholar To measure incorporation of 3H-leucine into immunoprecipitated Hsp25, gels were sliced at approximately 25 kilodaltons and solubilized in 0.5 mL TS-2 tissue solubilizer (RPI Corp., Mt. Prospect, IL) overnight. The 3H in each sample was quantified by liquid scintillation counting. To measure the rates of Hsp70 degradation, Hsp70 was pulse labeled with 35S-methionine in methionine free medium for 60 minutes after heat induction and chased with complete medium including all amino acids nonradiolabeled. Cells were treated with cytokines for up to 8 hours and harvested at 2, 4, and 8 hours after pulse labeling. Hsp70 was immunoprecipitated and detected by fluorographs of SDS-PAGE as described above. Results are presented as the means ± SEM for the indicated number of experiments. The results of multiple experiments were analyzed by using paired Student t test or analysis of variance using a Bonferroni correction for multiple comparisons. Colonic tissues from patients with no history of IBD (control) demonstrated significant Hsp27 and Hsp70 expression in surface but not crypt epithelial cells (Figure 1A). In contrast, specimens from areas actively inflamed from CD showed significantly reduced Hsp27 and Hsp70 staining in the surface epithelial cells (Figure 1A). Specimens from active UC patients demonstrated a comparable reduction in Hsp27 and Hsp70 compared with control areas (data not shown). Western blot analyses were performed on mucosal biopsy specimens to provide a more quantitative assessment of Hsp27 and Hsp70 expression. Hsp27 and Hsp70 levels were lower in the actively inflamed specimens compared with uninvolved areas obtained from the same patients (Figure 1B). In contrast, Hsc70 and β-actin and the epithelial cell marker villin were expressed at the same level in control and colitis samples. Inducible Hsp mRNA abundance was measured by real-time PCR (Figure 1C). No significant differences in Hsp27 and Hsp70 mRNA levels were detected, suggesting that decreased iHsp expression associated with inflamed mucosa was not due to inhibited gene transcription but rather to a posttranscriptional mechanism. We next determined whether inflammation-associated iHsp down-regulation is unique to human IBD or whether similar responses could be observed in a murine model of chronic colitis. IL-10−/− mice develop colitis spontaneously. The mucosal inflammation is discontinuous, T-cell dependent, and transmural, all features that resemble human CD.13Kühn R. Löhler J. Rennick D. et al.Interleukin-10-deficient mice develop chronic enterocolitis.Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3761) Google Scholar In our animal facility, approximately 20% of IL-10−/− mice develop inflammation and injury, as demonstrated by histologic grading, weight loss, and appearance of diarrhea at 20 weeks. In mice with intestinal inflammation, colonic Hsp25 and Hsp70 were significantly and selectively down-regulated compared with wild-type controls and IL-10−/− mice without intestinal inflammation (Figure 2A). Expression of Hsc70, β-actin, and villin were unchanged in wild-type and IL-10−/− mice. No significant decreases in Hsp25 or Hsp70 mRNA levels were detected in IL-10−/− mouse colonic mucosa (Figure 2B), indicating that posttranscriptional regulation might be involved. To determine whether cytokines can contribute to the changes in Hsp25 and Hsp70 expression, C57Bl/6 mice were given an intraperitoneal injection of either IFN-γ (2500 U), TNF-α (1000 ng), or both IFN-γ and TNF-α (IFN-γ+TNF-α). Only IFN-γ+TNF-α cotreatment significantly and selectively reduced Hsp25 and Hsp70 levels in colonic mucosa without altering Hsc70, β-actin, and villin (Figure 3A). No significant differences in Hsp25 and Hsp70 mRNA levels were detected, which correlated with findings observed in human biopsy specimens (Figure 1C) and IL-10−/− mice (Figure 2B). To investigate mechanisms responsible for the inflammation-induced reduction in colonic iHsp expression, YAMCs were used to examine the direct cellular effects of specific proinflammatory cytokines on Hsp25 and Hsp70 production.11Ren H. Musch M.W. Kojima K. et al.Short-chain fatty acids induce intestinal epithelial heat shock protein 25 expression in rats and IEC 18 cells.Gastroenterology. 2001; 121: 631-639Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 16Kojima K. Musch M.W. Ropeleski M.J. et al.Escherichia coli LPS induces heat shock protein 25 in intestinal epithelial cells through MAP kinase activation.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G645-G652Crossref PubMed Scopus (58) Google Scholar YAMC cells were pretreated with either IFN-γ (200 U/mL), TNF-α (100 ng/mL), or IFN-γ+TNF-α cotreatment for 8 hours prior to inducing iHsp production by heat shock. YAMCs pretreated with IFN-γ+TNF-α demonstrated a significant reduction in Hsp25 and Hsp70 relative to cells that were only heat shocked (HS-control) (Figure 4A). IFN-γ and TNF-α alone did not affect heat-induced iHsp production. To determine whether cytokine pretreatment reduced Hsp25 and Hsp70 transcription, mRNA levels were measured by real-time PCR (Figure 4B). Heat shock rapidly induced iHsp mRNA, reaching maximal levels in 90 minutes and returning to near baseline levels within 4 hours. The transience of the increased iHsp mRNA contrasts to the prolonged expression of iHsp protein and likely results from a feedback transcriptional down-regulation as previously described.20Malago J.J. Koninkx F.J.G. van Dijk J.E. The heat shock response and cytoprotection of the intestinal epithelium.Cell Stress Chaperones. 2002; 7: 191-199Crossref PubMed Scopus (91) Google Scholar As Hsp70 protein expression increases, it binds the transcription factor heat shock factor-1, preventing transcription of heat inducible genes.20Malago J.J. Koninkx F.J.G. van Dijk J.E. The heat shock response and cytoprotection of the intestinal epithelium.Cell Stress Chaperones. 2002; 7: 191-199Crossref PubMed Scopus (91) Google Scholar Treatment with IFN-γ+TNF-α did not alter Hsp25 and Hsp70 mRNA responses induced by heat shock. Thus, the reduction in iHsp production subsequent to IFN-γ+TNF-α cotreatment arises from regulation at the translational or posttranslational level. To determine whether the effects of IFN-γ+TNF-α were due to induction of apoptosis in the YAMCs, the cleavage and activation of 2 apoptotic enzymes were determined (same samples analyzed in Figure 4A). Caspase 3 is an asparagine protease that mediates numerous apoptotic signals, including PARP cleavage. PARP is involved in cellular disassembly and serves as a marker of apoptotic cells.17Lazebnik Y.A. Kaufmann S.H. Desnoyers S. et al.Cleavage of poly (ADP-ribose) polymerase by a proteinase with properties like ICE.Nature. 1994; 371: 346-377Crossref PubMed Scopus (2372) Google Scholar, 18Nicholson D.W. Ali A. Thornberry N.A. et al.Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis.Nature. 1995; 376: 37-43Crossref PubMed Scopus (3843) Google Scholar Heat shock causes a small degree of caspase 3 and PARP activation within the time frame of the experiments (Figure 4C). However, IFN-γ+TNF-α pretreatment for 8 hours did not enhance activation of either caspase 3 or PARP. The positive control staurosporine (1 μmol/L × 3 hours, [+]), resulted in both PARP and caspase 3 cleavage. Radiolabeled amino acid incorporation into Hsp25 and Hsp70 was used to determine the influence of cytokine treatment on de novo protein synthesis.16Kojima K. Musch M.W. Ropeleski M.J. et al.Escherichia coli LPS induces heat shock protein 25 in intestinal epithelial cells through MAP kinase activation.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G645-G652Crossref PubMed Scopus (58) Google Scholar 35S-methionine or 3Ropeleski M.J. Riehm J. Baer K.A. et al.Anti-apoptotic effects of L-glutamine-mediated transcriptional modulation of the heat shock protein 72 during heat shock.Gastroenterology. 2005; 129: 170-184Abstract Full Text Full Text PDF PubMed Scopus (47) Google ScholarH-leucine was added to cells at various time points postheat shock. Cells were harvested 60 minutes later to determine 35S-methionine or 3H-leucine incorporation. Under basal conditions, IFN-γ+TNF-α cotreatment for 8 hours did not alter the rate of
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