A Role for HSP70 in Protecting against Indomethacin-induced Gastric Lesions
2009; Elsevier BV; Volume: 284; Issue: 29 Linguagem: Inglês
10.1074/jbc.m109.006817
ISSN1083-351X
AutoresShintaro Suemasu, Ken‐ichiro Tanaka, Takushi Namba, Tomoaki Ishihara, Takashi Katsu, Mitsuaki Fujimoto, Hiroaki Adachi, Gen Sobue, Koji Takeuchi, Akira Nakai, Tohru Mizushima,
Tópico(s)Vitamin C and Antioxidants Research
ResumoA major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, is gastrointestinal complications. Both NSAID-dependent cyclooxygenase inhibition and gastric mucosal apoptosis are involved in NSAID-produced gastric lesions, and this apoptosis is mediated by the endoplasmic reticulum stress response and resulting activation of Bax. Heat shock proteins (HSPs) have been suggested to protect gastric mucosa from NSAID-induced lesions; here we have tested this idea genetically. The severity of gastric lesions produced by indomethacin was worse in mice lacking heat shock factor 1 (HSF1), a transcription factor for hsp genes, than in control mice. Indomethacin administration up-regulated the expression of gastric mucosal HSP70. Indomethacin-induced gastric lesions were ameliorated in transgenic mice expressing HSP70. After indomethacin administration, fewer apoptotic cells were observed in the gastric mucosa of transgenic mice expressing HSP70 than in wild-type mice, whereas the gastric levels of prostaglandin E2 for the two were indistinguishable. This suggests that expression of HSP70 ameliorates indomethacin-induced gastric lesions by affecting mucosal apoptosis. Suppression of HSP70 expression in vitro stimulated indomethacin-induced apoptosis and activation of Bax but not the endoplasmic reticulum stress response. Geranylgeranylacetone induced HSP70 at gastric mucosa in an HSF1-dependent manner and suppressed the formation of indomethacin-induced gastric lesions in wild-type mice but not in HSF1-null mice. The results of this study provide direct genetic evidence that expression of HSP70 confers gastric protection against indomethacin-induced lesions by inhibiting the activation of Bax. The HSP inducing activity of geranylgeranylacetone seems to contribute to its gastroprotective activity against indomethacin. A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, is gastrointestinal complications. Both NSAID-dependent cyclooxygenase inhibition and gastric mucosal apoptosis are involved in NSAID-produced gastric lesions, and this apoptosis is mediated by the endoplasmic reticulum stress response and resulting activation of Bax. Heat shock proteins (HSPs) have been suggested to protect gastric mucosa from NSAID-induced lesions; here we have tested this idea genetically. The severity of gastric lesions produced by indomethacin was worse in mice lacking heat shock factor 1 (HSF1), a transcription factor for hsp genes, than in control mice. Indomethacin administration up-regulated the expression of gastric mucosal HSP70. Indomethacin-induced gastric lesions were ameliorated in transgenic mice expressing HSP70. After indomethacin administration, fewer apoptotic cells were observed in the gastric mucosa of transgenic mice expressing HSP70 than in wild-type mice, whereas the gastric levels of prostaglandin E2 for the two were indistinguishable. This suggests that expression of HSP70 ameliorates indomethacin-induced gastric lesions by affecting mucosal apoptosis. Suppression of HSP70 expression in vitro stimulated indomethacin-induced apoptosis and activation of Bax but not the endoplasmic reticulum stress response. Geranylgeranylacetone induced HSP70 at gastric mucosa in an HSF1-dependent manner and suppressed the formation of indomethacin-induced gastric lesions in wild-type mice but not in HSF1-null mice. The results of this study provide direct genetic evidence that expression of HSP70 confers gastric protection against indomethacin-induced lesions by inhibiting the activation of Bax. The HSP inducing activity of geranylgeranylacetone seems to contribute to its gastroprotective activity against indomethacin. Nonsteroidal anti-inflammatory drugs (NSAIDs), 2The abbreviations used are: NSAIDnonsteroidal anti-inflammatory drugCHOPCCAAT enhancer-binding protein homologous transcription factorCOXcyclooxygenaseDAPI4,6-diamidino-2-phenylindole dihydrochlorideERendoplasmic reticulumFACSfluorescence activated cell sortingGGAgeranylgeranylacetoneHSFheat shock factorHSPheat shock proteinILinterleukinPGprostaglandinPUMAp53 up-regulated modulator of apoptosisRTreverse transcriptionTUNELterminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labelingTdTterminal deoxynucleotidyl transferasesiRNAsmall interfering RNAPIpropidium iodide.2The abbreviations used are: NSAIDnonsteroidal anti-inflammatory drugCHOPCCAAT enhancer-binding protein homologous transcription factorCOXcyclooxygenaseDAPI4,6-diamidino-2-phenylindole dihydrochlorideERendoplasmic reticulumFACSfluorescence activated cell sortingGGAgeranylgeranylacetoneHSFheat shock factorHSPheat shock proteinILinterleukinPGprostaglandinPUMAp53 up-regulated modulator of apoptosisRTreverse transcriptionTUNELterminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labelingTdTterminal deoxynucleotidyl transferasesiRNAsmall interfering RNAPIpropidium iodide. such as indomethacin, are a useful family of therapeutics (1.Smalley W.E. Ray W.A. Daugherty J.R. Griffin M.R. Am. J. Epidemiol. 1995; 141: 539-545Crossref PubMed Scopus (270) Google Scholar). An inhibitory effect of NSAIDs on cyclooxygenase (COX) activity is responsible for their anti-inflammatory actions because COX is an enzyme essential for the synthesis of prostaglandins (PGs), such as PGE2, which have a strong capacity to induce inflammation. On the other hand, NSAID use is associated with gastrointestinal complications (2.Hawkey C.J. Gastroenterology. 2000; 119: 521-535Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 3.Barrier C.H. Hirschowitz B.I. Arthritis Rheum. 1989; 32: 926-932PubMed Google Scholar, 4.Fries J.F. Miller S.R. Spitz P.W. Williams C.A. Hubert H.B. Bloch D.A. 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Although PGE2 has a strong protective effect on gastrointestinal mucosa, the inhibition of COX by NSAIDs is not the sole explanation for the gastrointestinal side effects of NSAIDs (5.Lichtenberger L.M. Biochem. Pharmacol. 2001; 61: 631-637Crossref PubMed Scopus (126) Google Scholar). We have recently demonstrated that NSAIDs induce apoptosis in cultured gastric cells and at gastric mucosa in a manner independent of COX inhibition (6.Tanaka K. Tomisato W. Hoshino T. Ishihara T. Namba T. Aburaya M. Katsu T. Suzuki K. Tsutsumi S. Mizushima T. J. Biol. Chem. 2005; 280: 31059-31067Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 7.Tsutsumi S. Gotoh T. Tomisato W. Mima S. Hoshino T. Hwang H.J. Takenaka H. Tsuchiya T. Mori M. Mizushima T. Cell Death Differ. 2004; 11: 1009-1016Crossref PubMed Scopus (214) Google Scholar, 8.Tomisato W. Tanaka K. Katsu T. Kakuta H. Sasaki K. Tsutsumi S. Hoshino T. Aburaya M. Li D. Tsuchiya T. Suzuki K. Yokomizo K. Mizushima T. Biochem. Biophys. Res. Commun. 2004; 323: 1032-1039Crossref PubMed Scopus (84) Google Scholar, 9.Tomisato W. Tsutsumi S. Rokutan K. Tsuchiya T. Mizushima T. Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 281: G1092-1100Crossref PubMed Google Scholar, 10.Aburaya M. Tanaka K. Hoshino T. Tsutsumi S. Suzuki K. Makise M. Akagi R. Mizushima T. J. Biol. Chem. 2006; 281: 33422-33432Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). As for the molecular mechanism governing this apoptosis, we have proposed the following pathway. Permeabilization of cytoplasmic membranes by NSAIDs stimulates Ca2+ influx and increases intracellular Ca2+ levels, which in turn induces the endoplasmic reticulum (ER) stress response (6.Tanaka K. Tomisato W. Hoshino T. Ishihara T. Namba T. Aburaya M. Katsu T. Suzuki K. Tsutsumi S. Mizushima T. J. Biol. Chem. 2005; 280: 31059-31067Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 11.Tsutsumi S. Namba T. Tanaka K.I. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (114) Google Scholar, 12.Namba T. Hoshino T. Tanaka K. Tsutsumi S. Ishihara T. Mima S. Suzuki K. Ogawa S. Mizushima T. Mol. Pharmacol. 2007; 71: 860-870Crossref PubMed Scopus (53) Google Scholar). In the ER stress response, an apoptosis-inducing transcription factor, CCAAT enhancer-binding protein homologous transcription factor (CHOP), is induced, as we have previously shown, CHOP is essential for NSAID-induced apoptosis (7.Tsutsumi S. Gotoh T. Tomisato W. Mima S. Hoshino T. Hwang H.J. Takenaka H. Tsuchiya T. Mori M. Mizushima T. Cell Death Differ. 2004; 11: 1009-1016Crossref PubMed Scopus (214) Google Scholar). CHOP induces expression of p53 up-regulated modulator of apoptosis (PUMA) and the resulting translocation and activation of Bax. We have already shown that both PUMA and Bax play an important role in NSAID-induced mitochondrial dysfunction, activation of caspases and apoptosis (13.Ishihara T. Hoshino T. Namba T. Tanaka K. Mizushima T. Biochem. Biophys. Res. Commun. 2007; 356: 711-717Crossref PubMed Scopus (34) Google Scholar). Furthermore, we have suggested that both COX inhibition (measured as a decrease in the gastric PGE2 level) and gastric mucosal apoptosis are required for the formation of NSAID-induced gastric lesions in vivo (10.Aburaya M. Tanaka K. Hoshino T. Tsutsumi S. Suzuki K. Makise M. Akagi R. Mizushima T. J. Biol. Chem. 2006; 281: 33422-33432Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 14.Tomisato W. Tsutsumi S. Hoshino T. Hwang H.J. Mio M. Tsuchiya T. Mizushima T. Biochem. Pharmacol. 2004; 67: 575-585Crossref PubMed Scopus (103) Google Scholar). Therefore, maintenance of gastric PGE2 levels or protection against gastric mucosal apoptosis is important for protection of gastric mucosa from NSAID-induced lesions. Heat shock proteins (HSPs) are induced by various stressors, including NSAIDs, and induction of HSPs, especially HSP70, provides cellular resistance to NSAIDs (15.Mathew A. Morimoto R.I. Ann. N.Y. Acad. Sci. 1998; 851: 99-111Crossref PubMed Scopus (131) Google Scholar, 16.Gehrmann M. Brunner M. Pfister K. Reichle A. Kremmer E. Multhoff G. Clin. Cancer Res. 2004; 10: 3354-3364Crossref PubMed Scopus (54) Google Scholar). Interestingly, geranylgeranylacetone (GGA), a leading anti-ulcer drug on the Japanese market, has been reported to be a nontoxic HSP inducer (17.Hirakawa T. Rokutan K. Nikawa T. Kishi K. Gastroenterology. 1996; 111: 345-357Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). We have previously reported that treatment of cultured gastric mucosal cells with GGA protects cells from NSAID-induced cell death and is accompanied by induction of HSP70 (18.Tomisato W. Takahashi N. Komoto C. Rokutan K. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2000; 45: 1674-1679Crossref PubMed Scopus (42) Google Scholar). These previous results suggest that HSP70 protects gastric mucosa from NSAID-induced gastric lesions; however, no direct evidence exists. The results also suggest that the protective effect of GGA against NSAID-produced gastric lesions is due to its HSP inducing activity. However, because GGA mediates various other gastroprotective mechanisms, such as an increase in gastric mucosal blood flow, stimulation of surface mucus production, and direct protection of gastric mucosal cell membranes (19.Terano A. Hiraishi H. Ota S. Sugimoto T. Digestion. 1986; 33: 206-210Crossref PubMed Scopus (80) Google Scholar, 20.Kunisaki C. Sugiyama M. Digestion. 1992; 53: 45-53Crossref PubMed Scopus (21) Google Scholar, 21.Ushijima H. Tanaka K. Takeda M. Katsu T. Mima S. Mizushima T. Mol. Pharmacol. 2005; 68: 1156-1161Crossref PubMed Scopus (28) Google Scholar), it remains unclear whether up-regulation of HSPs represents major mode of gastroprotective activity of GGA against NSAIDs. Up-regulation of HSP expression by stressors is achieved at the level of transcription through a consensus cis-element (heat shock element) and a transcription factor (heat shock factor 1 (HSF1)), which specifically binds to a heat shock element located upstream of the hsp genes (22.Morimoto R.I. Genes Dev. 1998; 12: 3788-3796Crossref PubMed Scopus (1533) Google Scholar). Disruption to the activity of HSF1 leads to the loss of stressor-induced HSP up-regulation (22.Morimoto R.I. Genes Dev. 1998; 12: 3788-3796Crossref PubMed Scopus (1533) Google Scholar, 23.McMillan D.R. Xiao X. Shao L. Graves K. Benjamin I.J. J. Biol. Chem. 1998; 273: 7523-7528Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). We recently used HSF1-null mice to obtain genetic evidence for a protective role for HSPs against production of gastric lesions (24.Tanaka K. Tsutsumi S. Arai Y. Hoshino T. Suzuki K. Takaki E. Ito T. Takeuchi K. Nakai A. Mizushima T. Mol. Pharmacol. 2007; 71: 985-993Crossref PubMed Scopus (51) Google Scholar). However, NSAID-produced gastric lesions, which involve not only irritant-induced mucosal damage but also a decrease in the PGE2 level, were not tested. Furthermore, although transgenic mice expressing HSP27 display a phenotype of resistance to irritant-induced gastric lesions (25.Ebert M.P. Schäfer C. Chen J. Hoffmann J. Gu P. Kubisch C. Carl-McGrath S. Treiber G. Malfertheiner P. Röcken C. J. Pathol. 2005; 207: 177-184Crossref PubMed Scopus (31) Google Scholar), the effect of artificial up-regulation of HSP70 on the production of gastric lesions has not been tested. In this study, we used HSF1-null mice and transgenic mice expressing HSP70 to examine the role of HSP70 in the pathogenesis of NSAID-produced gastric lesions. The results suggest that HSP70 is protective against the production of indomethacin-induced gastric lesions. Furthermore, the results suggest that HSP70 achieves this protective effect through inhibiting mucosal apoptosis rather than affecting the gastric level of PGE2. RPMI 1640 was obtained from Nissui Pharmaceutical Co. (Osaka, Japan). Paraformaldehyde, fetal bovine serum, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide were obtained from Sigma. Indomethacin was obtained from Wako Co. (Osaka, Japan). A PGE2 enzyme-linked immunosorbent assay kit was purchased from Cayman Chemical (Ann Arbor, MI). Antibodies against HSP70 were purchased from StressGen (San Diego, CA), and actin and the N-terminal region of Bax (Bax N20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Terminal deoxynucleotidyl transferase (TdT) was obtained from TOYOBO (Osaka, Japan). Biotin 14-ATP, Alexa Fluor 488 goat anti-rabbit (or anti-mice) immunoglobulin G, and Alexa Fluor 488 conjugated with streptavidin were purchased from Invitrogen. Mounting medium for immunohistochemical analysis (VECTASHIELD) was from Vector Laboratories (Burlingame, CA). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was from Dojindo (Kumamoto, Japan). The RNeasy kit and HiPerFect were obtained from Qiagen, the first strand cDNA synthesis kit was from Takara (Kyoto, Japan), and SYBR GREEN PCR Master Mix was from ABI (Foster City, CA). HSF1-null mice and their wild-type counterparts (ICR) (10–12 weeks of age and 30–35 g) or transgenic mice expressing HSP70 (gifts from Drs. Angelidis and Pagoulatos (University of Ioannina, Greece)) and their wild-type counterparts (C57/BL6) (6–8 weeks of age and 20–25 g) were prepared as described previously (27.Tanaka K. Namba T. Arai Y. Fujimoto M. Adachi H. Sobue G. Takeuchi K. Nakai A. Mizushima T. J. Biol. Chem. 2007; 282: 23240-23252Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Homozygotic transgenic mice were used in experiments. The experiments and procedures described here were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health (Bethesda, MD) and were approved by the Animal Care Committee of Kumamoto University. The gastric ulcerogenic response was examined as described previously (14.Tomisato W. Tsutsumi S. Hoshino T. Hwang H.J. Mio M. Tsuchiya T. Mizushima T. Biochem. Pharmacol. 2004; 67: 575-585Crossref PubMed Scopus (103) Google Scholar), with some modifications. Mice fasted for 18 h were orally administered with indomethacin. Eight hours later, the animals were sacrificed, after which their stomachs were removed, and the areas of gastric mucosal lesions were measured by an observer unaware of the treatment they had received. Calculation of the scores involved measuring the area of all the lesions in square millimeters and summing the values to give an overall gastric lesion index. The gastric PGE2 level was determined by enzyme-linked immunosorbent assay according to the manufacturer's instructions. Human gastric adenocarcinoma (AGS) cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 95% air with 5% CO2 at 37 °C. The cells were exposed to indomethacin by changing the medium. The cells were cultured for 24 h and then used in experiments. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide method as previously described (28.Hoshino T. Tsutsumi S. Tomisato W. Hwang H.J. Tsuchiya T. Mizushima T. J. Biol. Chem. 2003; 278: 12752-12758Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The measurement of caspase-3-like activity was as previously described (9.Tomisato W. Tsutsumi S. Rokutan K. Tsuchiya T. Mizushima T. Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 281: G1092-1100Crossref PubMed Google Scholar, 11.Tsutsumi S. Namba T. Tanaka K.I. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (114) Google Scholar). K+ efflux from cells was monitored as previously described (21.Ushijima H. Tanaka K. Takeda M. Katsu T. Mima S. Mizushima T. Mol. Pharmacol. 2005; 68: 1156-1161Crossref PubMed Scopus (28) Google Scholar). The transfection with pcDNA3.1 containing the hsp70 gene (29.Fujimoto M. Takaki E. Hayashi T. Kitaura Y. Tanaka Y. Inouye S. Nakai A. J. Biol. Chem. 2005; 280: 34908-34916Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) was carried out using Lipofectamine (TM2000) according to the manufacturer's instructions. The cells were used for experiments after a 20-h recovery period. We used siRNA, with the sequences 5′-ggagcugcaggugugudTdT-3′ and 5′-acacaccugcuccagcuccdTdT-3′, as annealed oligonucleotides for repressing HSP70 expression. AGS cells were transfected with siRNA using HiPerFect transfection reagent according to the manufacturer's instructions. Nonsilencing siRNA (5′-uucuccgaacgugucacgudTdT-3′ and 5′-acgugacacguucggagaadTdT-3′) were used as a negative control. Apoptosis was monitored using PI analysis by FACS as described previously (11.Tsutsumi S. Namba T. Tanaka K.I. Arai Y. Ishihara T. Aburaya M. Mima S. Hoshino T. Mizushima T. Oncogene. 2006; 25: 1018-1029Crossref PubMed Scopus (114) Google Scholar). The cells were collected by centrifugation, and the pellets were fixed with 70% ethanol and recentrifuged. The pellets were resuspended in phosphate-citrate buffer (0.2 m Na2HPO4 and 4 mm citric acid) and incubated for 20 min at room temperature. After centrifugation, the pellets were resuspended in DNA staining solution (50 mg/ml PI and 10 μg/ml RNase A) and incubated for 20 min at room temperature. Samples were scanned with a FACSCalibur (Becton Dickinson) cell sorter. For measuring only PI-DNA mediated fluorescence specifically, we scanned cells without PI staining and only PI solution (without cells) to determine the signal threshold. For excluding debris and cell fragments, thresholds were set for eliminating the low channels (from 1 to 15), in other words, cells with low PI fluorescence (the G1 peak was set as channel 400 of total 1024 channels) or for eliminating small cells by adjusting FSC and SSC light scatter parameters (according to the manufacturer's protocols). Apoptotic cells appeared as a hypodiploid peak (sub-G1 peak) because of nuclear fragmentation and loss of DNA, and we calculated number of cells included this peak and determined the ratio of apoptotic cells to total cells (10, 000). For statistical analysis, we measured three different samples in the same experiment. Total RNA was extracted from gastric tissues or AGS cells using the RNeasy kit according to the manufacturer's protocol. Samples (2.5 μg of RNA) were reverse-transcribed using the first strand cDNA synthesis kit according to the manufacturer's instructions. Synthesized cDNA was used in real time RT-PCR (Bio-Rad Chromo 4 system) experiments using iQ SYBR Green Supermix and analyzed with Opticon Monitor software according to the manufacturer's instructions. The real time PCR cycle conditions were 95 °C for 3 min, followed by 45 cycles at 95 °C for 10 s and at 60 °C for 60 s. Specificity was confirmed by electrophoretic analysis of the reaction products and by inclusion of template- or reverse transcriptase-free controls. To normalize the amount of total RNA present in each reaction, glyceraldehyde-3-phosphate dehydrogenase or actin cDNA was used as an internal standard. The primers were designed using the Primer3 website (Table 1).TABLE 1Primers usedGeneForward primerReverse primerhsp255′-cctcttccctatcccctgag-3′5′-ttggctccagactgttcaga-3′hsp605′-cgttgccaataacacaaacg-3′5′-cttcaggggttgtcacaggt-3′hsp705′-tggtgctgacgaagatgaag-3′5′-aggtcgaagatgagcacgtt-3′hsp90a5′-aaaggcagaggctgacaaga-3′5′-aggggaggcatttcttcagt-3′hsp90b5′-gcggcaaagacaagaaaaag-3′5′-gaagtggtcctcccagtcat-3′atf45′-tcaaacctcatgggttctcc-3′5′-gtgtcatccaacgtggtcag-3′chop5′-tgcctttctcttcggacact-3′5′-tgtgacctctgctggttctg-3′puma5′-gacgacctcaacgcacagta-3′5′-ggagtcccatgatgagattgt-3′actin5′-tgcctttctcttcggacact-3′5′-tgtgacctctgctggttctg-3′gapdh5′-tgcctttctcttcggacact-3′5′-tgtgacctctgctggttctg-3′ Open table in a new tab Total protein was extracted as described previously (30.Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (53) Google Scholar). The protein concentration of each sample was determined by the Bradford method (31.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar). Samples were applied to 8% (HSP70) or 10% (actin, Bax, and cytochrome c) polyacrylamide SDS gels and subjected to electrophoresis, after which the proteins were immunoblotted with appropriate antibodies. Gastric tissue samples were fixed in 4% buffered paraformaldehyde and embedded in paraffin before being cut into 4-μm sections. For histological examination (hematoxylin and eosin staining), the sections were stained first with Mayer's hematoxylin and then with 1% eosin alcohol solution. The samples were mounted with Malinol and inspected with the aid of an Olympus BX51 microscope. For immunohistochemical analysis, the sections were blocked with 2.5% goat serum for 10 min, incubated for 12 h with antibody against HSP70 (1:250 dilution) in the presence of 2.5% bovine serum albumin and finally incubated for 1 h with Alexa Fluor 488 goat anti-mouse immunoglobulin G in the presence of DAPI (5 μg/ml). The samples were mounted with VECTASHIELD and inspected using fluorescence microscopy (Olympus BX51). For TUNEL assay, the sections were incubated first with proteinase K (20 μg/ml) for 15 min at 37 °C, then with TdT and biotin 14-ATP for 1 h at 37 °C, and finally with Alexa Fluor 488 conjugated with streptavidin for 1 h. The samples were mounted with VECTASHIELD and inspected using fluorescence microscopy (Olympus BX51). The cells were cultured on four-well Lab-Tek II glass slides (Nunc). After fixation with 4% formaldehyde for 20 min and permeabilization with 0.5% Triton X-100 for 5 min, nonspecific binding sites were blocked with 3% bovine serum albumin for 30 min. Immunostaining to detect the active form of Bax was performed with a polyclonal antibody (Bax N20) and Alexa Fluor 488 goat anti-rabbit immunoglobulin G. The cells were simultaneously stained with DAPI (5 μg/ml). The cells were mounted with VECTASHIELD and inspected using fluorescence microscopy (Olympus BX51). All of the values are expressed as the means ± S.E. The Tukey test or the Student's t test for unpaired results was used to evaluate differences between more than three groups or between two groups, respectively. Differences were considered to be significant for values of p < 0.05. The development of gastric lesions following oral administration of indomethacin was compared between wild-type and HSF1-null mice. Administration of indomethacin produced gastric lesions in a dose-dependent manner, and this lesion production was significantly worse in HSF1-null mice relative to wild-type controls (Fig. 1A). This result shows that HSF1 plays an important role in protecting gastric mucosa from indomethacin-induced lesions. As mentioned above, both a decrease in PGE2 and mucosal apoptosis play important roles in NSAID-produced gastric lesions, and we have examined these processes in HSF1-null mice. As shown in Fig. 1B, there was no significant difference in the gastric level of PGE2 between the HSF1-null mice and wild-type mice in either the presence or absence of indomethacin treatment. The level of gastric mucosal apoptosis was determined by TUNEL assay. An increase in the number of TUNEL-positive (apoptotic) cells was observed after the indomethacin administration, and this increase was more apparent in HSF1-null mice than wild-type mice (Fig. 1, C and D). These results suggest that HSF1 protects the gastric mucosa from lesion formation by inhibiting indomethacin-induced apoptosis rather than by affecting the gastric PGE2 level. We monitored the expression of hsp mRNAs in gastric tissues by real time RT-PCR. Indomethacin administration up-regulated the expression of hsp70 mRNA, with this expression significantly lower in indomethacin-treated HSF1-null mice than in the wild-type mice (Fig. 2A). Lack of the hsf1 gene did not affect the background level of expression of hsp70 mRNA (Fig. 2A). In contrast, the expression of hsp25, hsp60, hsp90α, and hsp90β mRNAs was not affected by either indomethacin administration or lack of the hsf1 gene (Fig. 2A). Based on these results, we subsequently focused on HSP70. Immunoblotting and immunohistochemical analyses demonstrated that indomethacin administration increased the level of HSP70 in gastric mucosa in wild-type mice but not in HSF1-null mice (Fig. 2, B–D). Based on the results in Fig. 2, we proposed that the inability of HSF1-null mice to induce expression of HSP70 is responsible for their phenotypic sensitivity to indomethacin-induced gastric lesions and mucosal apoptosis. To test the idea described above, the level of indomethacin-induced gastric lesions was compared between transgenic mice expressing HSP70 and wild-type mice. Relative to control mice, formation of indomethacin-induced gastric lesions was significantly suppressed in transgenic mice expressing HSP70 (Fig. 3A). By immunoblotting and immunohistochemical analyses, we confirmed that HSP70 expression was much higher in the gastric tissues of the transgenic mice than in those of the wild-type mice, regardless of whether or not they were treated with indomethacin (Fig. 3, B–D). These results suggest that HSP70 expression somehow suppresses the formation of indomethacin-induced gastric lesions. As shown in Fig. 3E, there was no significant difference in the gastric level of PGE2 between transgenic mice expressing HSP70 and wild-type mice. On the other hand, the level of indomethacin-induced gastric mucosal apoptosis was lower in transgenic mice expressing HSP70 than in wild-type mice (Fig. 3, F and G). To understand the molecular mechanism for HSP70-conferred protection against indomethacin-induced cell death in vitro, we first examined the effect of siRNA specific for HSP70 on indomethacin-induced cell death in AGS cells. Transfection of cells with siRNA for HSP70 inhibited the expression of HSP70 both in the presence and absence of indomethacin (Fig. 4A). As shown in Fig. 4B, treatment of cells with indomethacin induced cell death in a dose-dependent manner. Transfection of cells with siRNA for HSP70 stimulated this cell death but did not affect cell viability in the absence of indomethacin (Fig. 4B). We also found that overexpression of HSP70 slightly inhibited indomethacin-induced cell death (Fig. 4, C and D). Based on results reported in one of our previous papers (8.Tomisato W. Tanaka K. Katsu T. Kakuta H. Sasaki K. Tsutsumi S. Hoshino T. Aburaya M. Li D. Tsuchiya T. Suzuki K. Yokomizo K. Mizushima T. Biochem. Biophys. Res. Commun. 2004; 323: 1032-1039Crossref PubMed Scopus (84) Google Scholar), we speculated that the cell death evidenced in Fig. 4 (B and D) is mediated by apoptosis. To confirm
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