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• W2142823218 abstract "Inflammatory bowel disease is a chronic inflammatory condition of the intestinal mucosa whose etiology is unclear but is likely to be multifactorial. We have shown previously that an increased amount of hyaluronan (HA) is present both in the inflamed mucosa of inflammatory bowel disease patients and in isolated human cells after polyI:C treatment. The signal transducer and activator of transcription (STAT)1 protein plays an important role in many signaling pathways that are associated with inflammation. We therefore investigated the role of STAT1 in adhesive interactions that occur between leukocytes and polyI:C-induced mucosal smooth muscle cells (M-SMCs). Activation of STAT1 was observed after the polyI:C treatment of M-SMCs. Specific phosphorylation of tyrosine and serine residues of STAT1 was observed in polyI:C-treated, but not untreated, M-SMC cultures. To evaluate further the role of STAT1, a corresponding STAT-1-null mouse was used. PolyI:C-induced, HA-mediated leukocyte adhesion to colon SMCs from STAT1-null mice was significantly decreased compared with that from wild-type control mice. In vivo, using the dextran sulfate sodium-induced model of colon inflammation, both tissue damage and HA deposition were attenuated in STAT1-null mice compared with that in wild-type control mice. Additionally, the inter-α-trypsin inhibitor (IαI), a proteoglycan essential for facilitating leukocyte binding to the HA matrix, was reduced in STAT1-null mice. Together, these results demonstrate that STAT1 plays an important role in HA-mediated inflammatory processes. Inflammatory bowel disease is a chronic inflammatory condition of the intestinal mucosa whose etiology is unclear but is likely to be multifactorial. We have shown previously that an increased amount of hyaluronan (HA) is present both in the inflamed mucosa of inflammatory bowel disease patients and in isolated human cells after polyI:C treatment. The signal transducer and activator of transcription (STAT)1 protein plays an important role in many signaling pathways that are associated with inflammation. We therefore investigated the role of STAT1 in adhesive interactions that occur between leukocytes and polyI:C-induced mucosal smooth muscle cells (M-SMCs). Activation of STAT1 was observed after the polyI:C treatment of M-SMCs. Specific phosphorylation of tyrosine and serine residues of STAT1 was observed in polyI:C-treated, but not untreated, M-SMC cultures. To evaluate further the role of STAT1, a corresponding STAT-1-null mouse was used. PolyI:C-induced, HA-mediated leukocyte adhesion to colon SMCs from STAT1-null mice was significantly decreased compared with that from wild-type control mice. In vivo, using the dextran sulfate sodium-induced model of colon inflammation, both tissue damage and HA deposition were attenuated in STAT1-null mice compared with that in wild-type control mice. Additionally, the inter-α-trypsin inhibitor (IαI), a proteoglycan essential for facilitating leukocyte binding to the HA matrix, was reduced in STAT1-null mice. Together, these results demonstrate that STAT1 plays an important role in HA-mediated inflammatory processes. The pathogenesis of chronic inflammatory diseases such as inflammatory bowel disease (IBD) is not well understood. The origin of IBD is unknown and likely to be multifactorial. Environmental factors, including viruses or microbial infections, alter the normal immune balances in the intestine of genetically susceptible people that may lead to Crohn's disease and ulcerative colitis. Pathological changes in IBD include destruction of normal tissue architecture, increase of mononuclear leukocyte influx into the intestinal mucosa, and hyperplasia of the mucosal smooth muscle cells (M-SMCs). Alterations in cytokine levels are thought to have critical roles in the pathogenesis of IBD. The mucosal concentrations of several cytokines, including tumor necrosis factor-α, interleukin (IL)-12, IL-6, IL-23, IL-17, and interferon (IFN)-γ are increased in IBD1Beagley KW Elson CO Cells and cytokines in mucosal immunity and inflammation.Gastroenterol Clin North Am. 1992; 21: 347-366PubMed Google Scholar, 2Becker C Dornhoff H Neufert C Fantini MC Wirtz S Huebner S Nikolaev A Lehr HA Murphy AJ Valenzuela DM Yancopoulos GD Galle PR Karow M Neurath MF Cutting edge: IL-23 cross-regulates IL-12 production in T cell-dependent experimental colitis.J Immunol. 2006; 177: 2760-2764PubMed Google Scholar, 3Podolsky DK Inflammatory bowel disease.N Engl J Med. 2002; 347: 417-429Crossref PubMed Scopus (3163) Google Scholar, 4Fuss IJ Neurath M Boirivant M Klein JS de la Motte C Strong SA Fiocchi C Strober W Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn's disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5.J Immunol. 1996; 157: 1261-1270PubMed Google Scholar, 5Fiocchi C Fukushima K Strong SA Ina K Pitfalls in cytokine analysis in inflammatory bowel disease.Aliment Pharmacol Ther. 1996; 10: 61-70PubMed Google Scholar, 6Mudter J Neurath MF IL-6 signaling in inflammatory bowel disease: pathophysiological role and clinical relevance.Inflamm Bowel Dis. 2007; 13: 1016-1023Crossref PubMed Scopus (306) Google Scholar, 7Neurath MF IL-23: a master regulator in Crohn disease.Nat Med. 2007; 13: 26-28Crossref PubMed Scopus (197) Google Scholar, 8Hue S Ahern P Buonocore S Kullberg MC Cua DJ McKenzie BS Powrie F Maloy KJ Interleukin-23 drives innate and T cell-mediated intestinal inflammation.J Exp Med. 2006; 203: 2473-2483Crossref PubMed Scopus (682) Google Scholar, 9Kullberg MC Jankovic D Feng CG Hue S Gorelick PL McKenzie BS Cua DJ Powrie F Cheever AW Maloy KJ Sher A IL-23 plays a key role in Helicobacter hepaticus-induced T cell-dependent colitis.J Exp Med. 2006; 203: 2485-2494Crossref PubMed Scopus (474) Google Scholar and contribute to tissue damage and chronic disease. Janus kinases (Jak)-signal transducer and activator of transcription (STAT)-dependent pathways are major signaling pathways for biological functions of IFN-γ.10Darnell Jr, JE Kerr IM Stark GR Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5028) Google Scholar, 11Stark GR Kerr IM Williams BR Silverman RH Schreiber RD How cells respond to interferons.Annu Rev Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3380) Google Scholar, 12O'Shea JJ Gadina M Schreiber RD Cytokine signaling in 2002: new surprises in the Jak/Stat pathway.Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 13Schindler C Levy DE Decker T JAK-STAT signaling: from interferons to cytokines.J Biol Chem. 2007; 282: 20059-20063Crossref PubMed Scopus (958) Google Scholar However, STAT-independent pathways are also known.14Ramana CV Gil MP Han Y Ransohoff RM Schreiber RD Stark GR Stat1-independent regulation of gene expression in response to IFN-gamma.Proc Natl Acad Sci USA. 2001; 98: 6674-6679Crossref PubMed Scopus (209) Google Scholar, 15Ramana CV Gil MP Schreiber RD Stark GR Stat1-dependent and -independent pathways in IFN-gamma-dependent signaling.Trends Immunol. 2002; 23: 96-101Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar Jaks and STATs are present in the cytoplasm of most cell types. In general, cytokines and growth factors that signal through Jak-STAT-dependent pathways form specific Jaks and STAT-factor complexes with their respective receptors. Subsequently, Jaks are activated through auto- and trans-phosphorylation before phosphorylating the receptor at a specific residue near the C-terminus that creates a docking site on the receptor for STAT factors. Complex-bound STAT is then similarly phosphorylated by Jaks and dissociates from the receptor complex as a dimer. The liberated, phosphorylated STAT dimer then translocates to the nucleus and binds with the gamma-activating sequence present in many cytokine and growth factor-inducible gene promoters that stimulates the transcription and mediates the biological responses of these gene products.10Darnell Jr, JE Kerr IM Stark GR Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5028) Google Scholar, 11Stark GR Kerr IM Williams BR Silverman RH Schreiber RD How cells respond to interferons.Annu Rev Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3380) Google Scholar, 12O'Shea JJ Gadina M Schreiber RD Cytokine signaling in 2002: new surprises in the Jak/Stat pathway.Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 13Schindler C Levy DE Decker T JAK-STAT signaling: from interferons to cytokines.J Biol Chem. 2007; 282: 20059-20063Crossref PubMed Scopus (958) Google Scholar, 16Levy DE Darnell Jr, JE Stats: transcriptional control and biological impact.Nat Rev Mol Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar Acetylation of STATs also occurs after cytokine stimulation that promotes stable dimer formation and subsequent transcriptional stimulation of target genes.17Yuan ZL Guan YJ Chatterjee D Chin YE Stat3 dimerization regulated by reversible acetylation of a single lysine residue.Science. 2005; 307: 269-273Crossref PubMed Scopus (616) Google Scholar, 18O'Shea JJ Kanno Y Chen X Levy DE Cell signaling. Stat acetylation—a key facet of cytokine signaling?.Science. 2005; 307: 217-218Crossref PubMed Scopus (51) Google Scholar Hyaluronan (HA) is a glycosaminoglycan composed of glucuronic acid and N-acetyl-d-glucosamine. Our laboratory has established that virus or viral mimic double-stranded RNA (poly I:C) treatment increases cell surface HA deposition and formation of long cable-like structures of HA that are important for leukocyte attachment.19de la Motte CA Hascall VC Calabro A Yen-Lieberman B Strong SA Mononuclear leukocytes preferentially bind via CD44 to hyaluronan on human intestinal mucosal smooth muscle cells after virus infection or treatment with poly(I.C).J Biol Chem. 1999; 274: 30747-30755Crossref PubMed Scopus (171) Google Scholar In IBD patients, the association of viruses such as cytomegalovirus, respiratory syncytial virus, herpesvirus, mumps, and measles has been reported.20Bernstein CN Blanchard JF Viruses and inflammatory bowel disease: is there evidence for a causal association?.Inflamm Bowel Dis. 2000; 6: 34-39Crossref PubMed Scopus (8) Google Scholar, 21Rahbar A Bostrom L Lagerstedt U Magnusson I Soderberg-Naucler C Sundqvist VA Evidence of active cytomegalovirus infection and increased production of IL-6 in tissue specimens obtained from patients with inflammatory bowel diseases.Inflamm Bowel Dis. 2003; 9: 154-161Crossref PubMed Scopus (84) Google Scholar, 22Papadakis KA Tung JK Binder SW Kam LY Abreu MT Targan SR Vasiliauskas EA Outcome of cytomegalovirus infections in patients with inflammatory bowel disease.Am J Gastroenterol. 2001; 96: 2137-2142Crossref PubMed Google Scholar, 23Wong NA Herbst H Herrmann K Kirchner T Krajewski AS Moorghen M Niedobitek F Rooney N Shepherd NA Niedobitek G Epstein-Barr virus infection in colorectal neoplasms associated with inflammatory bowel disease: detection of the virus in lymphomas but not in adenocarcinomas.J Pathol. 2003; 201: 312-318Crossref PubMed Scopus (57) Google Scholar We also demonstrated that increased HA staining was observed in inflamed tissue sections from patients with ulcerative colitis or Crohn's disease compared to noninflamed and less inflamed areas from the same patient.19de la Motte CA Hascall VC Calabro A Yen-Lieberman B Strong SA Mononuclear leukocytes preferentially bind via CD44 to hyaluronan on human intestinal mucosal smooth muscle cells after virus infection or treatment with poly(I.C).J Biol Chem. 1999; 274: 30747-30755Crossref PubMed Scopus (171) Google Scholar In addition, we showed by immunohistochemical staining that inter-α-trypsin inhibitor (IαI), a HA-binding protein, is localized in these HA cable structures.24de la Motte CA Hascall VC Drazba J Bandyopadhyay SK Strong SA Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid:polycytidylic acid: inter-alpha-trypsin inhibitor is crucial to structure and function.Am J Pathol. 2003; 163: 121-133Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar Recent reports have demonstrated the activation of STAT1 and also STAT3 in IBD patients.25Schreiber S Rosenstiel P Hampe J Nikolaus S Groessner B Schottelius A Kuhbacher T Hamling J Folsch UR Seegert D Activation of signal transducer and activator of transcription (STAT) 1 in human chronic inflammatory bowel disease.Gut. 2002; 51: 379-385Crossref PubMed Scopus (170) Google Scholar, 26Plevy S A STAT need for human immunologic studies to understand inflammatory bowel disease.Am J Gastroenterol. 2005; 100: 73-74Crossref PubMed Scopus (8) Google Scholar, 27Pfitzner E Kliem S Baus D Litterst CM The role of STATs in inflammation and inflammatory diseases.Curr Pharm Des. 2004; 10: 2839-2850Crossref PubMed Scopus (129) Google Scholar, 28Mudter J Weigmann B Bartsch B Kiesslich R Strand D Galle PR Lehr HA Schmidt J Neurath MF Activation pattern of signal transducers and activators of transcription (STAT) factors in inflammatory bowel diseases.Am J Gastroenterol. 2005; 100: 64-72Crossref PubMed Scopus (183) Google Scholar Constitutive activation of STAT3 in IBD has also been reported.29Lovato P Brender C Agnholt J Kelsen J Kaltoft K Svejgaard A Eriksen KW Woetmann A Odum N Constitutive STAT3 activation in intestinal T cells from patients with Crohn's disease.J Biol Chem. 2003; 278: 16777-16781Crossref PubMed Scopus (158) Google Scholar In this study, we demonstrate that STAT1 has a role in the attachment of U937 cells to poly I:C-treated M-SMCs and STAT1 is required for dextran sulfate sodium (DSS)-induced intestinal damage in mice. Cell culture media and additives were obtained from our institute core facility. Fetal bovine serum was from Bio- Whitaker, Walkersville, MD, cycloheximide, poly I:C, and α-actin antibody were from Sigma, St. Louis, MO. STAT1 and STAT3 antibodies were from Cell Signaling Technology, Beverly, MA, and poly dI:dC, protease inhibitors, EGCG, and AG490 were from Calbiochem, La Jolla, CA. The techniques of cell isolation, culture, and adhesion assays of M-SMCs (obtained from either human colon or mouse specimens) were described in our earlier publication.19de la Motte CA Hascall VC Calabro A Yen-Lieberman B Strong SA Mononuclear leukocytes preferentially bind via CD44 to hyaluronan on human intestinal mucosal smooth muscle cells after virus infection or treatment with poly(I.C).J Biol Chem. 1999; 274: 30747-30755Crossref PubMed Scopus (171) Google Scholar Smooth muscle cell (SMC) cultures were derived from the large bowel of STAT-1 knockout and control wild-type mice by explant culture. Briefly, after isolation, M-SMCs were cultured in Dulbecco's modified Eagle's medium/F12 medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 0.25 μg/ml of fungizone in a humidified cell culture incubator at 37°C with 5% CO2 unless otherwise indicated. All experiments were done within passage 3 of cells in culture. Approximately 80% confluent cell cultures were either untreated or treated with 50 μg/ml of poly I:C in fresh medium unless otherwise indicated. Experiments were performed at least in triplicate and representative data from one such experiment are shown. Electrophoretic mobility shift assays were done with a synthetic double-stranded hSIE oligonucleotide DNA (labeled by kinase reaction using γ-32P-ATP) as a probe, using an equal amount of protein from whole cell extracts according to standard procedures. Equal amounts of extracts were incubated in 20-μl reaction volumes in binding buffer [20 mmol/L HEPES (pH 7.9), 5% glycerol, 50 mmol/L NaCl, 1 mmol/L MgCl2, 1 mmol/L dithiothreitol, and 0.5 mmol/L ethylenediaminetetraacetic acid] containing 2 μg of poly (dI-dC), and 0.2 ng of labeled probe. After 20 minutes of incubation at 22°C, the reaction mixture was resolved in a 5% nondenaturing polyacrylamide gel in TBE buffer (45 mmol/L Tris, 45 mmol/L boric acid, and 1 mmol/L ethylenediaminetetraacetic acid) at 180 V for 2 hours. The gel was dried and visualized by autoradiography.30Leaman DW Salvekar A Patel R Sen GC Stark GR A mutant cell line defective in response to double-stranded RNA and in regulating basal expression of interferon-stimulated genes.Proc Natl Acad Sci USA. 1998; 95: 9442-9447Crossref PubMed Scopus (31) Google Scholar, 31Bandyopadhyay SK de la Motte CA Williams BR Induction of E-selectin expression by double-stranded RNA and TNF-alpha is attenuated in murine aortic endothelial cells derived from double-stranded RNA-activated kinase (PKR)-null mice.J Immunol. 2000; 164: 2077-2083Crossref PubMed Scopus (23) Google Scholar Supershift analyses were done by preincubating extracts with the specific antibody for 10 minutes at 22°C in the reaction mixture before the addition of the probe. Western blots were done with equal amounts of protein for each lane, electrophoresed in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride-type transfer membrane at 4°C by a standard procedure.31Bandyopadhyay SK de la Motte CA Williams BR Induction of E-selectin expression by double-stranded RNA and TNF-alpha is attenuated in murine aortic endothelial cells derived from double-stranded RNA-activated kinase (PKR)-null mice.J Immunol. 2000; 164: 2077-2083Crossref PubMed Scopus (23) Google Scholar Both phosphorylated STAT1 and actin antibodies were used according to the manufacturers’ protocols. Binding of leukocytes to SMCs was measured with radiolabeled U937 cells according to our previously published procedure.19de la Motte CA Hascall VC Calabro A Yen-Lieberman B Strong SA Mononuclear leukocytes preferentially bind via CD44 to hyaluronan on human intestinal mucosal smooth muscle cells after virus infection or treatment with poly(I.C).J Biol Chem. 1999; 274: 30747-30755Crossref PubMed Scopus (171) Google Scholar, 24de la Motte CA Hascall VC Drazba J Bandyopadhyay SK Strong SA Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid:polycytidylic acid: inter-alpha-trypsin inhibitor is crucial to structure and function.Am J Pathol. 2003; 163: 121-133Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar Cells binding values were presented as the mean data from at least triplicate wells ± SE. All animal experiments were performed following guidelines and approvals from the institutional animal care and use committee. Age-matched, 8- to 10-week-old, 129S6/SvEv strain of STAT-1-null and wild-type mice (obtained from Taconic Farms, Germantown, NY) were treated with or without 5% DSS, average molecular mass 10,000 Da, in the drinking water. Mice were sacrificed after 4 or 7 days of treatment and their colons were removed and processed for immunohistochemical staining from paraffin sections. The sections were examined by Leica (Wetzlar, Germany) TCS-SP laser-scanning confocal microscopy.24de la Motte CA Hascall VC Drazba J Bandyopadhyay SK Strong SA Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid:polycytidylic acid: inter-alpha-trypsin inhibitor is crucial to structure and function.Am J Pathol. 2003; 163: 121-133Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar Detection of HA and heavy chains of IαI were performed using biotinylated HA binding protein (Seikagaku, Tokyo, Japan) and an IαI antibody, respectively.19de la Motte CA Hascall VC Calabro A Yen-Lieberman B Strong SA Mononuclear leukocytes preferentially bind via CD44 to hyaluronan on human intestinal mucosal smooth muscle cells after virus infection or treatment with poly(I.C).J Biol Chem. 1999; 274: 30747-30755Crossref PubMed Scopus (171) Google Scholar, 24de la Motte CA Hascall VC Drazba J Bandyopadhyay SK Strong SA Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid:polycytidylic acid: inter-alpha-trypsin inhibitor is crucial to structure and function.Am J Pathol. 2003; 163: 121-133Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar We used secondary fluorescein isothiocyanate-conjugated streptavidin for HA detection and secondary Texas Red-conjugated antibody for IαI detection, and also 4,6-diamidino-2-phenylindole for nuclei staining. Colon tissue was obtained directly after surgical resection from the Cleveland Clinic Surgery Department according to institutional review board guidelines with their approval. The colon tissues from noninflamed and inflamed areas were embedded in OCT medium and stored at −80°C until further processing for experiments.32Kristiansen G Pilarsky C Wissmann C Kaiser S Bruemmendorf T Roepcke S Dahl E Hinzmann B Specht T Pervan J Stephan C Loening S Dietel M Rosenthal A Expression profiling of microdissected matched prostate cancer samples reveals CD166/MEMD and CD24 as new prognostic markers for patient survival.J Pathol. 2005; 205: 359-376Crossref PubMed Scopus (148) Google Scholar A small portion of tissue was also obtained for homogenization in ice-cold buffer containing a cocktail of protease inhibitors. Tissue debris was then removed by low-speed centrifugation. Protein concentrations were determined and an equal amount of protein was loaded in each lane for gel electrophoresis. Sections of muscularis mucosae layers were selected and laser cuts were done similarly from inflamed and noninflamed areas. The cells in the selected regions were automatically acquired in a protein extraction buffer (Tris/HCl buffer containing all protease inhibitors). Extracts with an equal volume were loaded in SDS-PAGE gels for Western blots.31Bandyopadhyay SK de la Motte CA Williams BR Induction of E-selectin expression by double-stranded RNA and TNF-alpha is attenuated in murine aortic endothelial cells derived from double-stranded RNA-activated kinase (PKR)-null mice.J Immunol. 2000; 164: 2077-2083Crossref PubMed Scopus (23) Google Scholar We previously demonstrated that STAT1 is the most important factor among the cytoplasmic components of the Jak-STAT pathway for induction of IFN-inducible gene, p56, by poly I:C using mutant cells lacking individual components of the Jak-STAT pathway.33Bandyopadhyay SK Leonard Jr, GT Bandyopadhyay T Stark GR Sen GC Transcriptional induction by double-stranded RNA is mediated by interferon-stimulated response elements without activation of interferon-stimulated gene factor 3.J Biol Chem. 1995; 270: 19624-19629Crossref PubMed Scopus (126) Google Scholar Many reports have demonstrated the activation of STAT1 by cytokines and growth factors.10Darnell Jr, JE Kerr IM Stark GR Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5028) Google Scholar, 11Stark GR Kerr IM Williams BR Silverman RH Schreiber RD How cells respond to interferons.Annu Rev Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3380) Google Scholar, 12O'Shea JJ Gadina M Schreiber RD Cytokine signaling in 2002: new surprises in the Jak/Stat pathway.Cell. 2002; 109: S121-S131Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 13Schindler C Levy DE Decker T JAK-STAT signaling: from interferons to cytokines.J Biol Chem. 2007; 282: 20059-20063Crossref PubMed Scopus (958) Google Scholar, 16Levy DE Darnell Jr, JE Stats: transcriptional control and biological impact.Nat Rev Mol Cell Biol. 2002; 3: 651-662Crossref PubMed Scopus (2501) Google Scholar Because STAT1 is important for poly I:C-stimulated IFN-inducible gene induction,33Bandyopadhyay SK Leonard Jr, GT Bandyopadhyay T Stark GR Sen GC Transcriptional induction by double-stranded RNA is mediated by interferon-stimulated response elements without activation of interferon-stimulated gene factor 3.J Biol Chem. 1995; 270: 19624-19629Crossref PubMed Scopus (126) Google Scholar we investigated whether poly I:C activates STAT1 in primary human colon M-SMCs. Cultures of human M-SMCs were treated with or without poly I:C for 0, 2, and 4 hours, and STAT-DNA binding activity was measured by gel-shift assay. Activation of STAT is observed by 2 hours and the level of induction is greater at 4 hours after poly I:C treatment (Figure 1a). Control experiments using an excess amount of the same and different cold oligoneucleotides were performed and confirmed the specificity of DNA binding activity (data not shown). IL-6 primarily activates STAT3, but in many cells IL-6 also activates STAT1.34Kerr IM Costa-Pereira AP Lillemeier BF Strobl B Of JAKs, STATs, blind watchmakers, jeeps and trains.FEBS Lett. 2003; 546: 1-5Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 35Kishimoto T Interleukin-6: from basic science to medicine—40 years in immunology.Annu Rev Immunol. 2005; 23: 1-21Crossref PubMed Scopus (788) Google Scholar We determined whether STAT is induced by IL-6 in M-SMCs by treating primary M-SMCs with 10 ng/ml of IL-6 for 30 minutes. The results indicate that the DNA binding STAT protein is present in IL-6-treated extracts (Figure 1a). To characterize whether the poly I:C-induced DNA-binding protein band is STAT1 or STAT3, we used antibody supershift experiments using STAT1 and STAT3 antibodies. Extracts from poly I:C-treated (4 hours) and IL-6-treated (30 minutes) cells were incubated with STAT1 or STAT3 antibody. STAT1 antibody supershifted both bands almost completely whereas STAT3 antibody did not shift either band in extracts of poly I:C-treated cells. IL-6 activates both STAT1 and STAT3 as shown by band supershifting with both antibodies (Figure 1b) and primarily serves as a positive control for determining supershift of both STAT1 and STAT3 in this experiment. STAT1 activation was also observed in M-SMCs after IFN-γ treatment (data not shown). STAT1 was not activated at 30 minutes or 1 hour after poly I:C treatment and activation started to decline after 4 hours and was completely gone after 8 hours (data not shown). Maximum STAT1 activation was observed at 4 hours, although activation of STAT1 was also detected at the 2-hour time point (similar as Figure 1a). We investigated the requirement of ongoing protein synthesis for STAT1 activation by poly I:C. M-SMCs were treated with and without poly I:C in the presence or absence of cycloheximide. Poly I:C induces the STAT1 band in electrophoretic mobility shift assay, and induction of the STAT1 band is reduced in poly I:C plus cycloheximide treatment (Figure 1c). Partial activation of STAT1 in the presence of cycloheximide indicates that STAT1 is likely induced by poly I:C, and other secreted factors that might be induced by poly I:C treatment could contribute to full STAT1 activation. To further understand the role of secreted factors induced by poly I:C in STAT1 activation, we treated human M-SMCs with poly I:C for 4 hours and then collected the medium for subsequent treatment of untreated human M-SMCs. These cellular extracts were also subjected to a similar gel-shift assay to see whether STAT1 is activated by any secreted factors that are initially produced because of poly I:C treatment. Figure 1d shows no STAT1-activated band by treatment with supernatant obtained from poly I:C-treated cells. The results of STAT1 activation by poly I:C in the presence of cycloheximide and also the lack of STAT1 activation by medium obtained from poly I:C-stimulated cells indicate that STAT1 activation in M-SMCs by poly I:C is partially mediated by poly I:C and not by other secondary secreted factors induced by poly I:C. Because STAT1 is activated in human M-SMCs by poly I:C, we sought to determine whether STAT1 is phosphorylated at 701-tyrosine in primary human M-SMCs after poly I:C treatment. Because poly I:C activation of STAT1 was observed in the previous experiment in M-SMCs starting at 2 hours and achieving a maximum at 4 hours, we prepared cell extracts from M-SMCs treated with or without poly I:C for 2 hours and 4 hours for immunoblot analysis. Extracts were subjected to SDS-PAGE followed by immunoblot and phosphotyrosine analysis of STAT1 protein using a specific STAT1-phosphotyrosine antibody. Figure 2a shows the 701-tyrosine-phosphorylated STAT1 band in extracts prepared from cells treated with poly I:C for 2 hours and a more intense band at 4 hours. However, phosphorylated bands are not observed in extracts of cells not treated with poly I:C (designated medium 2 hours and medium 4 hours in Figure 2a). This result indicates that STAT1 is tyrosine-phosphorylated after poly I:C treatment in human M-SMCs. We also determined whether poly I:C treatment causes phosphorylation of the serine residue at the 727-amino acid position of STAT1, which is required for further transcriptional activation. Human M-SMCs were similarly treated with and without poly I:C for 2 hours and 4 hours and cell extracts were examined by SDS-PAGE followed by Western blots with a 727-serine-specific antibody. The results indicate that STAT1 serine phosphorylation is barely visible after 2 hours of poly I:C treatment but is easily visible at 4 hours after poly I:C treatment (Figure 2b). The same blots were also used for α-actin expression using a specific antibody to verify equal sample loading in each lane. Therefore, both critical 701-tyrosine and 727-serine residues of STAT1 are phosphorylated in M-SMCs after poly I:C treatment. We pr" @default.
• W2142823218 created "2016-06-24" @default.
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• W2142823218 date "2008-11-01" @default.
• W2142823218 modified "2023-10-16" @default.
• W2142823218 title "Hyaluronan-Mediated Leukocyte Adhesion and Dextran Sulfate Sodium-Induced Colitis Are Attenuated in the Absence of Signal Transducer and Activator of Transcription 1" @default.
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