Positive Role of CCAAT/Enhancer-Binding Protein Homologous Protein, a Transcription Factor Involved in the Endoplasmic Reticulum Stress Response in the Development of Colitis
2009; Elsevier BV; Volume: 174; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.080864
ISSN1525-2191
AutoresTakushi Namba, Ken‐ichiro Tanaka, Yosuke Ito, Tomoaki Ishihara, Tatsuya Hoshino, Tomomi Gotoh, Motoyoshi Endo, Keizo Sato, Tohru Mizushima,
Tópico(s)Phagocytosis and Immune Regulation
ResumoAlthough recent reports suggest that the endoplasmic reticulum (ER) stress response is induced in association with the development of inflammatory bowel disease, its role in the pathogenesis of inflammatory bowel disease remains unclear. The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a transcription factor that is involved in the ER stress response, especially ER stress-induced apoptosis. In this study, we found that experimental colitis was ameliorated in CHOP-null mice, suggesting that CHOP exacerbates the development of colitis. The mRNA expression of Mac-1 (CD11b, a positive regulator of macrophage infiltration), Ero-1α, and Caspase-11 (a positive regulator of interleukin-1β production) in the intestine was induced with the development of colitis, and this induction was suppressed in CHOP-null mice. ERO-1α is involved in the production of reactive oxygen species (ROS); an increase in ROS production, which is associated with the development of colitis in the intestine, was suppressed in CHOP-null mice. A greater number of apoptotic cells in the intestinal mucosa of wild-type mice were observed to accompany the development of colitis compared with CHOP-null mice, suggesting that up-regulation of CHOP expression exacerbates the development of colitis. Furthermore, this CHOP activity appears to involve various stimulatory mechanisms, such as macrophage infiltration via the induction of Mac-1, ROS production via the induction of ERO-1α, interleukin-1β production via the induction of Caspase-11, and intestinal mucosal cell apoptosis. Although recent reports suggest that the endoplasmic reticulum (ER) stress response is induced in association with the development of inflammatory bowel disease, its role in the pathogenesis of inflammatory bowel disease remains unclear. The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a transcription factor that is involved in the ER stress response, especially ER stress-induced apoptosis. In this study, we found that experimental colitis was ameliorated in CHOP-null mice, suggesting that CHOP exacerbates the development of colitis. The mRNA expression of Mac-1 (CD11b, a positive regulator of macrophage infiltration), Ero-1α, and Caspase-11 (a positive regulator of interleukin-1β production) in the intestine was induced with the development of colitis, and this induction was suppressed in CHOP-null mice. ERO-1α is involved in the production of reactive oxygen species (ROS); an increase in ROS production, which is associated with the development of colitis in the intestine, was suppressed in CHOP-null mice. A greater number of apoptotic cells in the intestinal mucosa of wild-type mice were observed to accompany the development of colitis compared with CHOP-null mice, suggesting that up-regulation of CHOP expression exacerbates the development of colitis. Furthermore, this CHOP activity appears to involve various stimulatory mechanisms, such as macrophage infiltration via the induction of Mac-1, ROS production via the induction of ERO-1α, interleukin-1β production via the induction of Caspase-11, and intestinal mucosal cell apoptosis. Inflammatory bowel disease (IBD), Crohn's disease, and ulcerative colitis, have become substantial health problems with an actual prevalence of 200 to 500 per 100,000 people in western countries, which almost doubles every 10 years.1Cuzzocrea S Emerging biotherapies for inflammatory bowel disease.Expert Opin Emerg Drugs. 2003; 8: 339-347Crossref PubMed Scopus (27) Google Scholar Although the etiology of IBD is not clear at present, recent studies suggest that IBD is a disorder involving activation of leukocytes (macrophages, lymphocytes, and neutrophils) and their infiltration into the inflamed intestine, and intestinal mucosal damage induced by reactive oxygen species (ROS).2Podolsky DK Inflammatory bowel disease.N Engl J Med. 2002; 347: 417-429Crossref PubMed Scopus (3163) Google Scholar To understand the molecular mechanism underlying the pathogenesis of IBD and to establish a clinical protocol for its treatment, it is important to identify proteins that are involved in the pathogenesis of IBD. For this purpose, various experimental animal models of colitis, in particular the dextran sulfate sodium (DSS)- and trinitrobenzene sulfonic acid (TNBS)-induced colitis models, are useful.3Jurjus AR Khoury NN Reimund JM Animal models of inflammatory bowel disease.J Pharmacol Toxicol Methods. 2004; 50: 81-92Crossref PubMed Scopus (246) Google Scholar Pro-inflammatory cytokines and cell adhesion molecules (CAMs) play an important role in the activation and infiltration of leukocytes that are associated with IBD. 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C/EBP homologous transcription factor (CHOP) is a transcription factor involved in the ER stress response, especially ER stress-induced apoptosis through various mechanisms such as down-regulation of Bcl-2 and up-regulation of Bim.16Puthalakath H O'Reilly LA Gunn P Lee L Kelly PN Huntington ND Hughes PD Michalak EM McKimm-Breschkin J Motoyama N Gotoh T Akira S Bouillet P Strasser A ER stress triggers apoptosis by activating BH3-only protein Bim.Cell. 2007; 129: 1337-1349Abstract Full Text Full Text PDF PubMed Scopus (1130) Google Scholar, 17Oyadomari S Mori M Roles of CHOP/GADD153 in endoplasmic reticulum stress.Cell Death Differ. 2004; 11: 381-389Crossref PubMed Scopus (2241) Google Scholar, 18Zinszner H Kuroda M Wang X Batchvarova N Lightfoot RT Remotti H Stevens JL Ron D CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum.Genes Dev. 1998; 12: 982-995Crossref PubMed Scopus (1683) Google Scholar A close relationship between inflammation and the ER stress response, especially the induction of CHOP, has been suggested. For example, TNF-α was reported to induce the ER stress response and expression of CHOP.19Xue X Piao JH Nakajima A Sakon-Komazawa S Kojima Y Mori K Yagita H Okumura K Harding H Nakano H Tumor necrosis factor alpha (TNFalpha) induces the unfolded protein response (UPR) in a reactive oxygen species (ROS)-dependent fashion, and the UPR counteracts ROS accumulation by TNFalpha.J Biol Chem. 2005; 280: 33917-33925Crossref PubMed Scopus (326) Google Scholar CREBH was recently identified as a factor connecting the ER stress response and the acute inflammatory response.20Zhang K Shen X Wu J Sakaki K Saunders T Rutkowski DT Back SH Kaufman RJ Endoplasmic reticulum stress activates cleavage of CREBH to induce a systemic inflammatory response.Cell. 2006; 124: 587-599Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar Therefore, it is reasonable to hypothesize that the ER stress response, and CHOP in particular, is involved in the pathogenesis of IBD. In fact, some recent reports support this idea; up-regulation of CHOP and GRP78 was observed in the inflamed intestine in both IBD.21Shkoda A Ruiz PA Daniel H Kim SC Rogler G Sartor RB Haller D Interleukin-10 blocked endoplasmic reticulum stress in intestinal epithelial cells: impact on chronic inflammation.Gastroenterology. 2007; 132: 190-207Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 22Burczynski ME Peterson RL Twine NC Zuberek KA Brodeur BJ Casciotti L Maganti V Reddy PS Strahs A Immermann F Spinelli W Schwertschlag U Slager AM Cotreau MM Dorner AJ Molecular classification of Crohn's disease and ulcerative colitis patients using transcriptional profiles in peripheral blood mononuclear cells.J Mol Diagn. 2006; 8: 51-61Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar However, the exact role (positive or negative) of the ER stress response (or CHOP) in the pathogenesis of IBD has remained unknown. The analysis of knockout mice is useful in addressing this type of question. For example, we recently suggested, through analysis of DSS-induced colitis in heat shock factor 1 (HSF1, a transcription factor involved in the heat shock response)-null mice, that HSF1 plays a protective role, inhibiting the development of IBD.23Tanaka K Namba T Arai Y Fujimoto M Adachi H Sobue G Takeuchi K Nakai A Mizushima T Genetic evidence for a protective role for heat shock factor 1 and heat shock protein 70 against colitis.J Biol Chem. 2007; 282: 23240-23252Crossref PubMed Scopus (120) Google Scholar In the present study, we compared the development of DSS- and TNBS-induced colitis between CHOP-null mice and wild-type (WT) mice and obtained genetic evidence that CHOP plays a positive role in the pathogenesis of experimental colitis. Furthermore, results in this study suggest that CHOP achieves this effect through various mechanisms such as stimulation of intestinal ROS production, sensitization of intestinal mucosal cells to ROS-induced apoptosis, stimulation of macrophage infiltration into the inflamed intestine, and stimulation of the intestinal production of IL-1β. Based on these findings, we propose that inhibitors for CHOP may be therapeutically beneficial for the treatment of IBD. Paraformaldehyde, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), menadione, fetal bovine serum, o-dianisidine, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF), and TNBS were obtained from Sigma (St. Louis, MO). Thiobarbituric acid (TBA), butylated hydroxytoluene (BHT), n-butanol, and pyridine were from Nacalai Tesque (Kyoto, Japan). DSS (M.W. 5000, 15 to 20% sulfur content) was from Wako Pure Chemicals (Tokyo, Japan). Proteose peptone was from Becton Dickinson (San Jose, CA). Lipopolysaccharide (LPS) was from List Biological Laboratories, Inc (Campbell, CA). Antioxidant Assay Kit was from Cayman (Ann Arbor, MI). An enzyme-linked immunosorbent assay kit for the detection of IL-1β was from Pierce Chemical (Rockford, IL). Optimal cutting temperature (O.C.T.) compound was from Sakura Finetek Japan (Tokyo, Japan). Mayer's hematoxylin, 1% eosin alcohol solution, and Malinol were from Muto Pure Chemicals (Tokyo, Japan). Terminal deoxynucleotidyl transferase (TdTase) was obtained from Toyobo (Osaka, Japan). The Envision kit was from DAKO (Carpinteria, CA). Biotin 14-ATP and Alexa Fluor 488 conjugated with streptavidin were purchased from Invitrogen (Carlsbad, CA). Vectashield was from Vector Laboratories (Burlingame, CA). HilyMax and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) were from Dojindo Laboratories (Kumamoto, Japan). The RNeasy kit and HiPerFect were obtained from Qiagen (Valencia, CA), the PrimeScript first strand cDNA synthesis kit was purchased from Takara Bio (Ohtsu, Japan), and iQ SYBR Green Supermix was from Bio-Rad (Hercules, CA). Antibodies against CHOP, actin, and GRP78 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and that against CD68 was from Dack Co. (Carpinteria, CA). α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) was from Alexis (San Diego, CA). HCT-15 and RAW264 cells were obtained from the Cell Resource Center for Biochemical Research at Tohoku University (Sendai, Japan) and RIKEN BioResource Center (Tsukuba, Japan), respectively. CHOP-null mice that had been backcrossed with WT mice (C57BL/6) for more than 10 times and the WT mice (5 to 7 weeks old, male) were prepared and there was no apparent phenotypes in CHOP-null mice as described previously.24Oyadomari S Takeda K Takiguchi M Gotoh T Matsumoto M Wada I Akira S Araki E Mori M Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway.Proc Natl Acad Sci USA. 2001; 98: 10845-10850Crossref PubMed Scopus (517) Google Scholar The experiments and procedures described here were performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health, and were approved by the Animal Care Committee of Kumamoto University. DSS-induced colitis was induced in mice by the addition of 3% DSS (w/v, final concentration) to their drinking water as described previously.23Tanaka K Namba T Arai Y Fujimoto M Adachi H Sobue G Takeuchi K Nakai A Mizushima T Genetic evidence for a protective role for heat shock factor 1 and heat shock protein 70 against colitis.J Biol Chem. 2007; 282: 23240-23252Crossref PubMed Scopus (120) Google Scholar The animals were allowed free access to the DSS-containing water for 7 days. For histopathological observation, measurement of myeloperoxidase (MPO), various mRNAs, ROS, thiobarbituric acid reactant substances (TBARS), as well as apoptosis, we used rectum and distal colon. After 7 days, animals were placed under deep ether anesthesia and sacrificed, the colons were dissected and their length measured from the ileocecal junction to the anal verge. The DAI was determined macroscopically by an observer unaware of the treatment the mice had received, according to previously reported criteria.23Tanaka K Namba T Arai Y Fujimoto M Adachi H Sobue G Takeuchi K Nakai A Mizushima T Genetic evidence for a protective role for heat shock factor 1 and heat shock protein 70 against colitis.J Biol Chem. 2007; 282: 23240-23252Crossref PubMed Scopus (120) Google Scholar, 25Rumi G Tsubouchi R Nishio H Kato S Mozsik G Takeuchi K Dual role of endogenous nitric oxide in development of dextran sodium sulfate-induced colitis in rats.J Physiol Pharmacol. 2004; 55: 823-836PubMed Google Scholar Briefly, the DAI was calculated as the sum of the diarrheal stool score (0: normal stool; 1: mildly soft stool; 2: very soft stool; 3: watery stool) and the bloody stool score (0: normal color stool; 1: brown color stool; 2: reddish color stool; 3: bloody stool). TNBS-induced colitis was produced by intrarectal administration of TNBS once as described previously.26Nishihara T Matsuda M Araki H Oshima K Kihara S Funahashi T Shimomura I Effect of adiponectin on murine colitis induced by dextran sulfate sodium.Gastroenterology. 2006; 131: 853-861Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar MPO activity in the colonic tissues was measured as previously described.23Tanaka K Namba T Arai Y Fujimoto M Adachi H Sobue G Takeuchi K Nakai A Mizushima T Genetic evidence for a protective role for heat shock factor 1 and heat shock protein 70 against colitis.J Biol Chem. 2007; 282: 23240-23252Crossref PubMed Scopus (120) Google Scholar, 27Krawisz JE Sharon P Stenson WF Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models.Gastroenterology. 1984; 87: 1344-1350Abstract Full Text PDF PubMed Scopus (1626) Google Scholar After DSS treatment, colons were dissected, rinsed with cold saline, and cut into small pieces. Samples were homogenized and protein concentrations of the supernatants were determined using the Bradford method.28Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar MPO activity was determined in 10 mmol/L phosphate buffer with 0.5 mmol/L o-dianidisine, 0.00005% (w/v) hydrogen peroxide, and 20 μg of protein. MPO activity was obtained from the slope of the reaction curve and its specific activity was expressed as the number of hydrogen peroxide molecules converted per minute per mg of protein. The amounts of TBARS in colonic tissues were measured as previously described.29Rumi G Tsubouchi R Okayama M Kato S Mozsik G Takeuchi K Protective effect of lactulose on dextran sulfate sodium-induced colonic inflammation in rats.Dig Dis Sci. 2004; 49: 1466-1472Crossref PubMed Scopus (74) Google Scholar, 30Ohkawa H Ohishi N Yagi K Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.Anal Biochem. 1979; 95: 351-358Crossref PubMed Scopus (23305) Google Scholar, 31Tanaka K Tsutsumi S Arai Y Hoshino T Suzuki K Takaki E Ito T Takeuchi K Nakai A Mizushima T Genetic evidence for a protective role of heat shock factor 1 against irritant-induced gastric lesions.Mol Pharmacol. 2007; 71: 985-993Crossref PubMed Scopus (51) Google Scholar After DSS treatment, colons were dissected, cut into small pieces and weighed. Samples were homogenized and centrifuged. Supernatants were mixed with 20 μl of 8.1% sodium dodecyl sulfate solution, 150 μl of 20% acetic acid solution, and 5 μl of 0.8% BHT solution, then with 150 μl of 0.8% TBA solution, and finally boiled for 1 hour. Samples were mixed with 500 μl of n-butanol/pyridine (15:1) and centrifuged. The absorbance of the supernatant was measured at 532 nm and the amount of TBARS expressed as the number of TBARS molecules per gram of tissue. Total protein was extracted from the colonic tissues as described previously.23Tanaka K Namba T Arai Y Fujimoto M Adachi H Sobue G Takeuchi K Nakai A Mizushima T Genetic evidence for a protective role for heat shock factor 1 and heat shock protein 70 against colitis.J Biol Chem. 2007; 282: 23240-23252Crossref PubMed Scopus (120) Google Scholar, 32Tsutsumi S Tomisato W Takano T Rokutan K Tsuchiya T Mizushima T Gastric irritant-induced apoptosis in guinea pig gastric mucosal cells in primary culture.Biochim Biophys Acta. 2002; 1589: 168-180Crossref PubMed Scopus (53) Google Scholar The protein concentration of the samples was determined by the Bradford method.28Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar Samples were applied to 8% (GRP78 and actin) or 12% (CHOP) polyacrylamide sodium dodecyl sulfate gels and subjected to electrophoresis, after which the proteins were immunoblotted with appropriate antibodies. Real-time RT-PCR was done as described33Mima S Tsutsumi S Ushijima H Takeda M Fukuda I Yokomizo K Suzuki K Sano K Nakanishi T Tomisato W Tsuchiya T Mizushima T Induction of claudin-4 by nonsteroidal anti-inflammatory drugs and its contribution to their chemopreventive effect.Cancer Res. 2005; 65: 1868-1876Crossref PubMed Scopus (64) Google Scholar with some modifications. Total RNA was extracted from intestinal tissues using an RNeasy kit according to the manufacturer's protocol. Samples (2.5 μg of RNA) were reverse-transcribed using a first-strand cDNA synthesis kit according to the manufacturer's instructions. Synthesized cDNA was used in real-time RT-PCR (Chromo 4 instrument, Bio-Rad) experiments using iQ SYBR Green Supermix and analyzed with Opticon Monitor Software (Bio-Rad, Hercules, CA). 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 (GAPDH) cDNA was used as an internal standard. Primers were designed using the Primer3 website (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The primers used were (name: forward primer, reverse primer): for mouse; Tnf-α: 5′-CGTCAGCCGATTTGCTATCT-3′, 5′-CGGACTCCGCAAAGTCTAAG-3′; Il-1β: 5′-GATCCCAAGCAATACCCAAA-3′, 5′-GGGGAACTCTGCAGACTCAA-3′; Il-6: 5′-CTGGAGTCACAGAAGGAGTGG-3′, 5′-GGTTTGCCGAGTAGATCTCAA-3′; Vcam-1 (vascular cell adhesion molecule): 5′-CTCCTGCACTTGTGGAAATG-3′, 5′-TGTACGAGCCATCCACAGAC-3′; Icam-1: 5′-TCGTGATGGCAGCCTCTTAT-3′, 5′-GGGCTTGTCCCTTGAGTTTT-3′; Madcam-1 (mucosal addressin cell adhesion molecule): 5′-GCAGGCTGGGAGCTACTCT-3′, 5′-TCCCTCTTGTGGTAGGTTGc-3′; CD49d: 5′-CAGAGCCACACCCAAAAGTT-3′, 5′-TGAAATGTCGTTTGGGTCTTt-3′; CD11b: 5′-TGTGAGCAGCACTGAGATCC-3′, 5′-ATGGCTCCACTTTGGTCTCT-3′; L-selectin: 5′-ATTCCTGTAGCCGTCATGGT-3′, 5′-CATCCTTTCTTGAGATTTCTTGC-3′; Il-10: 5′-GGCCCTTTGCTATGGTGTCC-3′, 5′-AAGCGGCTGGGGGATGAC-3′; Caspase-11: 5′-TGGAAGCTGATGCTGTCAAG-3′, 5′-GAGCCTCCTGTTTTGTCTCG-3′; endoplasmic reticulum oxidoreductin (Ero)-1α: 5′-TTAAGTCTGCGAGCTACAAGTATTC-3′, 5′-AGTAAGTCCACATACTCAGCATCG-3′; Bcl-2: 5′-CCTGTGGATGACTGAGTACC-3′, 5′-GAGACAGCCAGGAGAAAT-3′; Chop: 5′-ACAGAGGTCACACGCACATC-3′, 5′-GGGCACTGACCACTCTGTTT-3′; Grp78: 5′-GCTTCCGATAATCAGCCAAC-3′, 5′-GCAGGAGGAATTCCAGTCA-3′; C/ebp-β: 5′-GCAAGAGCCGCGACAAG-3′, 5′-GGCTCGGGCAGCTGCTT-3′. For human; CHOP: 5′-TGCCTTTCTCTTCGGACACT-3′, 5′-TGTGACCTCTGCTGGTTCTG-3′. Colonic tissue samples were fixed in 4% buffered paraformaldehyde, embedded in O.C.T. compound, and cryosectioned. For histological examination [hematoxylin and eosin (H&E) staining], sections were stained first with Mayer's hematoxylin and then with 1% eosin alcohol solution. Samples were mounted with Malinol and inspected with the aid of an Olympus (Tokyo, Japan) BX51 microscope. For histological evaluation of the tissue damage (damage score) and areas of lesions (extent of lesion), sections were evaluated microscopically by an observer unaware of the treatment the animals had received, and quantified as described.34Ohkawara T Nishihira J Takeda H Hige S Kato M Sugiyama T Iwanaga T Nakamura H Mizue Y Asaka M Amelioration of dextran sulfate sodium-induced colitis by anti-macrophage migration inhibitory factor antibody in mice.Gastroenterology. 2002; 123: 256-270Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 35Cooper HS Murthy SN Shah RS Sedergran DJ Clinicopathologic study of dextran sulfate sodium experimental murine colitis.Lab Invest. 1993; 69: 238-249PubMed Google Scholar Colonic damage (damage score) was categorized into six groups (0: normal mucosa; 1: infiltration of inflammatory cells; 2: shortening of the crypt by less than 50%; 3: shortening of the crypt by more than 50%; 4: crypt loss; 5: destruction of epithelial cells). The extent of lesions in the total colon was categorized into six grades (0: 0%; 1: 1 to 20%; 2: 21 to 40%; 3: 41 to 60%; 4: 61 to 80%; 5: 81 to 100%). For immunohistochemical analysis, sections were treated in a microwave oven with 0.01 mol/L citric acid buffer for antigen activation and incubated with 0.3% hydrogen peroxide in methanol for removal of endogenous peroxidase. Sections were blocked with 2.5% goat serum for 10 minutes, incubated for 12 hours with each antibody in the presence of 3% bovine serum albumin, and then incubated for 1 hour with peroxidase-labeled polymer conjugated to goat anti-rabbit immunoglobulins (Envision kit). Then, 3,3′-diaminobenzidine was applied to the sections and the sections were finally incubated with Mayer's hematoxylin. Samples were mounted with malinol and inspected using a fluorescence microscope (BX51; Olympus, Tokyo, Japan). The CHOP- and ERO-1α-specific siRNAs were purchased from Qiagen. A plasmid expressing CHOP or C/EBP-β was as described.36Nishiyori A Tashiro H Kimura A Akagi K Yamamura K Mori M Takiguchi M Determination of tissue specificity of the enhancer by combinatorial operation of tissue-enriched transcription factors. Both HNF-4 and C/EBP beta are required for liver-specific activity of the ornithine transcarbamylase enhancer.J Biol Chem. 1994; 269: 1323-1331Abstract Full Text PDF PubMed Google Scholar, 37Gotoh T Oyadomari S Mori K Mori M Nitric oxide-induced apoptosis in RAW 264.7 macrophages is mediated by endoplasmic reticulum stress pathway involving ATF6 and CHOP.J Biol Chem. 2002; 277: 12343-12350Crossref PubMed Scopus (218) Google Scholar HCT-15 and RAW264 cells were transfected with these siRNAs or plasmids using HiPerFect or HilyMax (transfection reagents) according to the manufacturer's instructions. Nonsilencing siRNA (5′-UUCUCCGAACGUGUCACGUDTdT-3′ and 5′-ACGUGACACGUUCGGAGAADTdT-3′) was used as a negative control. Mouse peritoneal macrophages were prepared as described previously.23Tanaka K Namba T Arai Y Fujimoto M Adachi H Sobue G Takeuchi K Nakai A Mizushima T Genetic evidence for a protective role for heat shock factor 1 and heat shock protein 70 against colitis.J Biol Chem. 2007; 282: 23240-23252Crossref PubMed Scopus (120) Google Scholar, 38Salimuddin Nagasaki A Gotoh T Isobe H Mori M Regulation of the genes for arginase isoforms and related enzymes in mouse macrophages by lipopolysaccharide.Am J Physiol. 1999; 277: E110-E117PubMed Google Scholar Mice were given 0.5 ml of 10% proteose peptone by intraperitoneal injection and peritoneal cells were harvested 3 days later. The cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. After incubation for 4 hours, nonadherent cells were removed and the adherent cells were cultured for use in the experiments. Virtually all of the adherent cells were macrophages, as previously described.38Salimuddin Nagasaki A Gotoh T Isobe H Mori M Regulation of the genes for arginase isoforms and related enzymes in mouse macrophages by lipopolysaccharide.Am J Physiol. 1999; 277: E110-E117PubMed Google Scholar Caspase-1 activity was measured as described.39Endo M Mori M Akira S Gotoh T C/EBP homologous protein (CHOP) is crucial for the induction of caspase-11 and the pathogenesis of lipopolysaccharide-induced inflammation.J Immunol. 2006; 176: 6245-6253PubMed Google Scholar The amounts of IL-1β secreted into the medium were measured by enzyme-linked immunosorbent assay according to the manufacturer's protocol. Colonic tissue samples were fixed in 4% buffered paraformaldehyde, embedded in O.C.T. com
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