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• W1966776937 abstract "In inflammatory bowel diseases (IBD), intestinal barrier function is impaired as a result of deteriorations in epithelial tight junction (TJ) structure. IL-6, a pleiotropic cytokine, is elevated in IBD patients, although the role of IL-6 in barrier function remains unknown. We present evidence that IL-6 increases TJ permeability by stimulating the expression of channel-forming claudin-2, which is required for increased caudal-related homeobox (Cdx) 2 through the MEK/ERK and PI3K pathways in intestinal epithelial cells. IL-6 increases the cation-selective TJ permeability without any changes to uncharged dextran flux or cell viability in Caco-2 cells. IL-6 markedly induces claudin-2 expression, which is associated with increased TJ permeability. The colonic mucosa of mice injected with IL-6 also exhibits an increase in claudin-2 expression. The claudin-2 expression and TJ permeability induced by IL-6 are sensitive to the inhibition of gp130, MEK, and PI3K. Furthermore, expression of WT-MEK1 induces claudin-2 expression in Caco-2 cells. Claudin-2 promoter activity is increased by IL-6 in a MEK/ERK and PI3K-dependent manner, and deletion of Cdx binding sites in the promoter sequence results in a loss of IL-6-induced promoter activity. IL-6 increases Cdx2 protein expression, which is suppressed by the inhibition of MEK and PI3K. These observations may reveal an important mechanism by which IL-6 can undermine the integrity of the intestinal barrier. In inflammatory bowel diseases (IBD), intestinal barrier function is impaired as a result of deteriorations in epithelial tight junction (TJ) structure. IL-6, a pleiotropic cytokine, is elevated in IBD patients, although the role of IL-6 in barrier function remains unknown. We present evidence that IL-6 increases TJ permeability by stimulating the expression of channel-forming claudin-2, which is required for increased caudal-related homeobox (Cdx) 2 through the MEK/ERK and PI3K pathways in intestinal epithelial cells. IL-6 increases the cation-selective TJ permeability without any changes to uncharged dextran flux or cell viability in Caco-2 cells. IL-6 markedly induces claudin-2 expression, which is associated with increased TJ permeability. The colonic mucosa of mice injected with IL-6 also exhibits an increase in claudin-2 expression. The claudin-2 expression and TJ permeability induced by IL-6 are sensitive to the inhibition of gp130, MEK, and PI3K. Furthermore, expression of WT-MEK1 induces claudin-2 expression in Caco-2 cells. Claudin-2 promoter activity is increased by IL-6 in a MEK/ERK and PI3K-dependent manner, and deletion of Cdx binding sites in the promoter sequence results in a loss of IL-6-induced promoter activity. IL-6 increases Cdx2 protein expression, which is suppressed by the inhibition of MEK and PI3K. These observations may reveal an important mechanism by which IL-6 can undermine the integrity of the intestinal barrier. One of the most important functions of gastrointestinal epithelial cells is to provide a physical barrier to the diffusion of pathogens, toxins, and antigens from the luminal environment into the circulation. This depends on the coordinated expression and interaction of proteins in cell-cell junctional complexes, including the tight junction (TJ) 2The abbreviations used are: TJtight junctionAPDCammonium pyrrolidinedithiocarbamateCdxcaudal-related homeoboxIBDinflammatory bowel diseaseJAMjunctional adhesion moleculeMLCKmyosin light chain kinaseTERtransepithelial electrical resistanceZOzonula occludens. (1González-Mariscal L. Betanzos A. Nava P. Jaramillo B.E. Prog. Biophys. Mol. Biol. 2003; 81: 1-44Crossref PubMed Scopus (916) Google Scholar). The TJ is a multi-protein complex, located around the apical end of the lateral membrane of polarized epithelial cells, which selectively regulates the paracellular passage of ions, molecules and water. Four types of integral transmembrane proteins, occludin (2Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2117) Google Scholar), the claudins (3Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1705) Google Scholar), junctional adhesion molecule (JAM) (4Martìn-Padura I. Lostaglio S. Schneemann M. Williams L. Romano M. Fruscella P. Panzeri C. Stoppacciaro A. Ruco L. Villa A. Simmons D. Dejana E. J. Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1146) Google Scholar), and tricellulin (5Ikenouchi J. Furuse M. Furuse K. Sasaki H. Tsukita S. J. Cell Biol. 2005; 171: 939-945Crossref PubMed Scopus (591) Google Scholar), have been identified so far. These transmembrane proteins interact with intracellular plaque proteins such as zonula occludens (ZO) proteins and cingluin, which in turn anchor the transmembrane proteins to the perijunctional actin cytoskeleton (1González-Mariscal L. Betanzos A. Nava P. Jaramillo B.E. Prog. Biophys. Mol. Biol. 2003; 81: 1-44Crossref PubMed Scopus (916) Google Scholar). The interaction of TJ proteins with the actin cytoskeleton is vital for maintaining TJ structure and function. Numerous studies have reported that TJ permeability and TJ protein expression/cytoskeletal association are dynamically regulated by various intracellular signaling molecules, such as PKC (6Suzuki T. Seth A. Rao R. J. Biol. Chem. 2008; 283: 3574-3583Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 7Suzuki T. Elias B.C. Seth A. Shen L. Turner J.R. Giorgianni F. Desiderio D. Guntaka R. Rao R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 61-66Crossref PubMed Scopus (168) Google Scholar), MAPK (8Basuroy S. Seth A. Elias B. Naren A.P. Rao R. Biochem. J. 2006; 393: 69-77Crossref PubMed Scopus (205) Google Scholar), PI3K (9Sheth P. Basuroy S. Li C. Naren A.P. Rao R.K. J. Biol. Chem. 2003; 278: 49239-49245Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), and protein phosphatases (10Nunbhakdi-Craig V. Machleidt T. Ogris E. Bellotto D. White 3rd, C.L. Sontag E. J. Cell Biol. 2002; 158: 967-978Crossref PubMed Scopus (223) Google Scholar). tight junction ammonium pyrrolidinedithiocarbamate caudal-related homeobox inflammatory bowel disease junctional adhesion molecule myosin light chain kinase transepithelial electrical resistance zonula occludens. Under pathophysiological conditions, the disturbance of the epithelial barrier allows contact with, or even penetration of, noxious luminal contents such as antigens or bacteria into the intestinal immune system resulting in mucosal inflammation, as is the case in inflammatory bowel diseases (IBD). Although the pathogenesis of the barrier defect in IBD is still unclear, studies conducted in cell cultures and animal models demonstrate that some proinflammatory cytokines such as TNF-α (11Wang F. Graham W.V. Wang Y. Witkowski E.D. Schwarz B.T. Turner J.R. Am. J. Pathol. 2005; 166: 409-419Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar), IFN-γ (11Wang F. Graham W.V. Wang Y. Witkowski E.D. Schwarz B.T. Turner J.R. Am. J. Pathol. 2005; 166: 409-419Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar, 12Bruewer M. Luegering A. Kucharzik T. Parkos C.A. Madara J.L. Hopkins A.M. Nusrat A. J. Immunol. 2003; 171: 6164-6172Crossref PubMed Scopus (700) Google Scholar), IL-1β (13Al-Sadi R. Ye D. Dokladny K. Ma T.Y. J. Immunol. 2008; 180: 5653-5661Crossref PubMed Scopus (297) Google Scholar), and IL-13 (14Heller F. Florian P. Bojarski C. Richter J. Christ M. Hillenbrand B. Mankertz J. Gitter A.H. Bürgel N. Fromm M. Zeitz M. Fuss I. Strober W. Schulzke J.D. Gastroenterology. 2005; 129: 550-564Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 15Weber C.R. Raleigh D.R. Su L. Shen L. Sullivan E.A. Wang Y. Turner J.R. J. Biol. Chem. 2010; 285: 12037-12046Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) impair barrier function by decreasing TJ protein expression, stimulating cytoskeletal contraction, and inducing epithelial apoptosis. However, changes in cytokine profile in inflammatory conditions are complicated, and the effects of other cytokines on barrier function require further investigation. IL-6 is a pleiotropic cytokine whose expression is important for the host response to a number of infections, exerts antigen-specific immune responses, and has both pro- as well as anti-inflammatory effects (16Alonzi T. Fattori E. Lazzaro D. Costa P. Probert L. Kollias G. De Benedetti F. Poli V. Ciliberto G. J. Exp. Med. 1998; 187: 461-468Crossref PubMed Scopus (515) Google Scholar, 17Tilg H. Dinarello C.A. Mier J.W. Immunol. Today. 1997; 18: 428-432Abstract Full Text PDF PubMed Scopus (543) Google Scholar). In pathological states, excessive secretion and dysregulation of IL-6 and its signaling pathway may play a major role in the pathogenesis of many diseases, including IBD (18Kusugami K. Fukatsu A. Tanimoto M. Shinoda M. Haruta J. Kuroiwa A. Ina K. Kanayama K. Ando T. Matsuura T. Dig. Dis. Sci. 1995; 40: 949-959Crossref PubMed Scopus (141) Google Scholar, 19Louis E. Belaiche J. van Kemseke C. Franchimont D. de Groote D. Gueenen V. Mary J.Y. Eur. J. Gastroenterol. Hepatol. 1997; 9: 939-944Crossref PubMed Scopus (99) Google Scholar, 20Reinisch W. Gasché C. Tillinger W. Wyatt J. Lichtenberger C. Willheim M. Dejaco C. Waldhör T. Bakos S. Vogelsang H. Gangl A. Lochs H. Am. J. Gastroenterol. 1999; 94: 2156-2164Crossref PubMed Scopus (124) Google Scholar). IL-6 is produced in substantially higher amounts in both the serum and tissues of IBD patients. In mice, the knock-out and blockade of IL-6 and IL-6 receptor suppress experimental colitis, indicating an important contribution of IL-6 and the IL-6 trans-signaling pathways for progression of IBD (21Yamamoto M. Yoshizaki K. Kishimoto T. Ito H. J. Immunol. 2000; 164: 4878-4882Crossref PubMed Scopus (355) Google Scholar, 22Atreya R. Mudter J. Finotto S. Müllberg J. Jostock T. Wirtz S. Schutz M. Bartsch B. Holtmann M. Becker C. Strand D. Czaja J. Schlaak J.F. Lehr H.A. Autschbach F. Schürmann G. Nishimoto N. Yoshizaki K. Ito H. Kishimoto T. Galle P.R. Rose-John S. Neurath M.F. Nat. Med. 2000; 6: 583-588Crossref PubMed Scopus (1063) Google Scholar, 23Suzuki A. Hanada T. Mitsuyama K. Yoshida T. Kamizono S. Hoshino T. Kubo M. Yamashita A. Okabe M. Takeda K. Akira S. Matsumoto S. Toyonaga A. Sata M. Yoshimura A. J. Exp. Med. 2001; 193: 471-481Crossref PubMed Scopus (421) Google Scholar, 24Sander L.E. Obermeier F. Dierssen U. Kroy D.C. Singh A.K. Seidler U. Streetz K.L. Lutz H.H. Müller W. Tacke F. Trautwein C. J. Immunol. 2008; 181: 3586-3594Crossref PubMed Scopus (29) Google Scholar). The major source of IL-6 seen in IBD has been shown to be intestinal epithelial cells and lamina propria mononuclear cells (18Kusugami K. Fukatsu A. Tanimoto M. Shinoda M. Haruta J. Kuroiwa A. Ina K. Kanayama K. Ando T. Matsuura T. Dig. Dis. Sci. 1995; 40: 949-959Crossref PubMed Scopus (141) Google Scholar). However, the role of IL-6 in the regulation of intestinal barrier function remains poorly understood. The claudin family consists of at least 24 members (1González-Mariscal L. Betanzos A. Nava P. Jaramillo B.E. Prog. Biophys. Mol. Biol. 2003; 81: 1-44Crossref PubMed Scopus (916) Google Scholar). In contrast to their structural similarities, the claudins perform different functions and can roughly be divided into two types, those involved in barrier formation, and those important in channel formation (25Colegio O.R. Van Itallie C.M. McCrea H.J. Rahner C. Anderson J.M. Am. J. Physiol. Cell Physiol. 2002; 283: C142-C147Crossref PubMed Scopus (432) Google Scholar). For example, claudin-1 is crucial for tightening the barrier in renal epithelial cells and mammalian epidermis (26Furuse M. Hata M. Furuse K. Yoshida Y. Haratake A. Sugitani Y. Noda T. Kubo A. Tsukita S. J. Cell Biol. 2002; 156: 1099-1111Crossref PubMed Scopus (1185) Google Scholar, 27Inai T. Kobayashi J. Shibata Y. Eur. J. Cell Biol. 1999; 78: 849-855Crossref PubMed Scopus (221) Google Scholar). In contrast, claudin-2 expression decreases the tightness of the epithelial barrier, which, it has been suggested, is mediated by a paracellular channel for small cations (28Amasheh S. Meiri N. Gitter A.H. Schöneberg T. Mankertz J. Schulzke J.D. Fromm M. J. Cell Sci. 2002; 115: 4969-4976Crossref PubMed Scopus (623) Google Scholar). It has been reported that claudin-2 expression is markedly increased in the colons of patients suffering from Crohn disease and ulcerative colitis, and it seems to have an important role in the pathogenesis of IBD (29Prasad S. Mingrino R. Kaukinen K. Hayes K.L. Powell R.M. MacDonald T.T. Collins J.E. Lab. Invest. 2005; 85: 1139-1162Crossref PubMed Scopus (354) Google Scholar). In the present study, we provide evidence that IL-6 has a role in the regulation of intestinal epithelial TJs. Our results demonstrate that IL-6 induces claudin-2 expression through signaling pathways involving MEK/ERK and PI3K, and transcriptional factor Cdx2 expression. The following antibodies were used in this study; rabbit polyclonal anti-claudin-1, anti-claudin-2, anti-claudin-3, anti-JAM-1, anti-ZO-1, and anti-ZO-2, mouse anti-claudin-4, and HRP-conjugated anti-occludin (Life Technologies); rabbit polyclonal anti-Cdx2 and rabbit monoclonal anti-pERK1/2 (Thr-202/Tyr-204), pSTAT3 (Tyr-705), pAkt (Ser-473), and mouse monoclonal anti-ERK1/2 and HA-Tag (Cell Signaling Technology); mouse monoclonal anti-gp130 (R&D Systems); mouse anti-β-actin and HRP-conjugated anti-mouse and -rabbit IgG (Sigma); AlexaFluor 488 conjugated goat polyclonal anti-rabbit IgG and AlexaFluor 546 conjugated goat polyclonal anti-mouse IgG (Life Technologies). The following signaling inhibitors were used in this study; U0126 (a MEK inhibitor), LY294002 (a PI3K inhibitor), PP2 (a Src inhibitor), AG490 (a JAK inhibitor), ammonium pyrrolidinedithiocarbamate (APDC, a NFκB inhibitor) were purchased from Calbiochem. All other chemicals were obtained from Wako Pure Chemical Industries. Caco-2 (HTB-37) and T84 intestinal epithelial cells (CCL-248) purchased from American Type Cell Culture were grown under standard cell culture conditions as described (6Suzuki T. Seth A. Rao R. J. Biol. Chem. 2008; 283: 3574-3583Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 7Suzuki T. Elias B.C. Seth A. Shen L. Turner J.R. Giorgianni F. Desiderio D. Guntaka R. Rao R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 61-66Crossref PubMed Scopus (168) Google Scholar). Cells were grown on polyester membranes in Transwell and Snapwell inserts (12-mm; Corning) for 14 (Caco-2) or 7 (T84) days prior to experiments. Recombinant human IL-6 (0∼100 ng/ml; Miltenyie Biotech) was applied to the basal aspect of cells. Cell monolayers were incubated with anti-gp130 (5 and 10 μg/ml), AG490 (1 and 3 μm), U0126 (5 and 10 μm), LY294002 (12 and 25 μm), PP2 (3 and 10 μm), and APDC (10 and 30 μm) 0.5 h before IL-6 administration. Transepithelial electrical resistance (TER) as an indicator of TJ permeability to ionic solutes, and unidirectional flux of FITC-conjugated dextran as an indicator of TJ permeability to uncharged macromolecules were assessed in Caco-2 and T84 cell monolayers. TER was measured as described previously (6Suzuki T. Seth A. Rao R. J. Biol. Chem. 2008; 283: 3574-3583Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 7Suzuki T. Elias B.C. Seth A. Shen L. Turner J.R. Giorgianni F. Desiderio D. Guntaka R. Rao R. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 61-66Crossref PubMed Scopus (168) Google Scholar) using a Millicell-ERS Electrical Resistance System (Millipore) at varying times during the experiment. Cell monolayers were incubated in the presence of FITC-dextran (4 kDa, 100 μm) in the apical well and the fluorescence in the basal well was determined using a fluorescence plate reader (ARVO X4, Perkin Elmer). The NaCl dilution potential in Caco-2 cells on Snapwell inserts were measured using a vertical diffusion chamber system (Harvard Apparatus). Each chamber was filled with 6 ml of Hanks' balanced salt solution supplemented with 5.6 mm d-glucose and 4 mm l-glutamine. The chambers were kept at 37 °C, and 100% O2 was bubbled through the solutions. The transepithelial potential was measured using 3 m-KCl electrode and voltage-clamping device (CEZ9100, Nihon Kohden). The NaCl dilution potential was determined from the shift in the reversal potential after replacing basal solution with one in which 130 mm mannitol was substituted for the 65 mm NaCl (15Weber C.R. Raleigh D.R. Su L. Shen L. Sullivan E.A. Wang Y. Turner J.R. J. Biol. Chem. 2010; 285: 12037-12046Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). The permeability ratio, PNa+/PCl−, was determined using Godlmann-Hodgkin-Katz equation as described previously (30Günzel D. Stuiver M. Kausalya P.J. Haisch L. Krug S.M. Rosenthal R. Meij I.C. Hunziker W. Fromm M. Müller D. J. Cell Sci. 2009; 122: 1507-1517Crossref PubMed Scopus (139) Google Scholar). Liquid junction potentials measured using empty Snapwell inserts were <0.1 mV. Cell viability was monitored by assaying mitochondrial dehydrogenase activity (WST-8, Dojindo) after IL-6 treatment. A MEK1WT-HA in pMCL vector was kindly provided from Dr Natalie Ahn (University of Colorado) (31Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1259) Google Scholar). Caco-2 cells were seeded on 6-well plates a day before transfection. The cells were transfected using 1 ml of antibiotics-free DMEM containing 10% FBS, 1 μg of DNA plasmid (empty and MEK1WT-HA vectors), 1 μl of Plus reagent, and 3 μl of Lipofectamine-LTX (Life Technologies) for each well. After 20 h, the cell monolayers were trypsinized and seeded in Transwell inserts. Male BALB/c mice (6-week-old) were purchased from Clea Japan. Recombinant mouse IL-6 (6 μg/mouse; Biolegend) was injected intraperitoneally 16 h prior to tissue harvest. PBS was used as the control treatment. The animal study was approved by the Hiroshima University Animal Committee, and the mice were maintained in accordance with the Hiroshima University guidelines for the care and use of laboratory animals. Detergent-insoluble fractions and whole cell extracts were prepared as previously described. The detergent insoluble fraction corresponds to the actin cytoskeleton-associated proteins. For preparations of detergent-insoluble fractions, Caco-2 cell monolayers were washed with ice-cold PBS and incubated for 5 min at 4 °C with 200 μl of lysis buffer-CS (1% Triton X-100, 5 mm EGTA in 50 mm Tris containing protease inhibitors (5 mg/liter aprotinin, 3 mg/liter leupeptin hemisulfate, 5 mm benzamidine hydrochloride, and 1 mm PMSF) and phosphatase inhibitors (25 mm glycerol-2-phosphate, 2 mm sodium orthovanadate, and 10 mm sodium fluoride, pH 7.4). Cell lysates were centrifuged at 15,600 × g for 10 min at 4 °C to precipitate the high-density actin-rich fraction. Pellets were resuspended in 100 μl of lysis buffer D (0.3% SDS, 10 mm Tris, and the protease and phosphatase inhibitors described above, pH 7.4). For preparation of the whole Caco-2 cell extracts, 200 μl of lysis buffer D was used after washing cell monolayers with ice-cold PBS. Mouse colon tissue (50 mg) was homogenized in 1 ml lysis buffer D using a polytron-type homogenizer. Protein concentrations in the different fractions were measured using the BCA method (Pierce Biotechnology). Cell extracts were mixed with a half volume of Laemmli sample buffer (3× concentrated; 6% (w:v) SDS, 30% (v:v) glycerol, 15% (v:v) 2-β-mercaptoethanol, and 0.02% (w:v) bromphenol blue in 188 mm Tris, pH 6.8) and heated to 100 °C for 5 min. Proteins (20 μg) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blotted for ZO-1, ZO-2, JAM-1, claudin-1, claudin-2, claudin-3, claudin-4, Cdx2, pSTAT3, pERK1/2, pAkt, and β-actin, using specific antibodies in combination with HRP-conjugated anti-mouse IgG or HRP-conjugated anti-rabbit IgG antibodies. HRP-conjugated anti-occludin antibody was used for immunoblot analysis of occludin. The blots were developed using the ECL chemiluminescence method (GE Healthcare). Quantification was performed by densitometric analysis of specific bands on the immunoblots using Image J software. Caco-2 cell monolayers were washed with ice-cold PBS, fixed in methanol at 0 °C for 5 min, and permeabilized with 0.2% Triton X-100 in PBS for 5 min. Cell monolayers were blocked in 4% nonfat milk in TBST (20 mm Tris, 150 mm NaCl, 0.05% Tween-20, pH7.4) and incubated for 1 h with rabbit polyclonal anti-claudin-2, ZO-1, Cdx2, and mouse monoclonal anti-HA tag, gp130, and gp80 antibodies followed by incubation for 1 h with secondary antibodies (goat AlexaFluor 488-conjugated anti-rabbit IgG and AlexaFluor 546-conjugated anti-mouse IgG) with DAPI. Mouse colon tissue was embedded in OCT compound (Sakura Finetek Japan) after fixation with 3.7% paraformaldehyde in PBS. Frozen sections (8 μm in thickness) were prepared on glass slides and washed with PBS. The sections were blocked in 5% normal goat serum and incubated for 1 h with rabbit polyclonal anti-claudin-2, followed by incubation for 1 h with goat AlexaFluor 488-conjugated anti-rabbit IgG, rhodamine-conjugated phalloidine and DAPI. The specimens were preserved in a mounting medium, and the fluorescence was visualized using Nikon ECLIPSE E600 fluorescence microscope (Nikon) and Olympus FW1000 confocal microscope (Olympus). The total RNA of Caco-2 cells was isolated using TRI reagent (Sigma), and reverse-transcribed with ReverTra Ace® qPCR RT kit (TOYOBO) according to the manufacturer's instructions. Quantitative real-time PCR was performed using an ABI PRISM 7700 Sequence Detection System (Life Technologies) and KAPA SYBR FAST qPCR kit (KAPA BIOSYSTEMS). The primer sequences used for PCR are shown in supplemental Table S1. Reactions were performed at 95 °C for 2 min, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. The dissociation stage was analyzed at 95 °C for 15 s, followed by one cycle of 60 °C for 15 s and 95 °C for 15 s. The fluorescence of the SYBR green dye was determined as a function of the PCR cycle number, giving the threshold cycle number at which amplification reached a significant threshold. Data were analyzed by the ΔΔCt method and presented as fold changes in gene expression after normalization to the internal control β-actin gene expression level. Genomic DNA was purified from Caco-2 cells using TRI reagent. The 5′ region (−1067 to −1) of the human Claudin-2 gene was amplified by PCR with gene-specific primers (forward/reverse) 5′-GAATCTTGGCAACACCGAGG-3′/5′-GGCAGACCTCTCAGTAGAAG-3′, inserted into the pGEM-T vector (Promega), and sequenced. The Claudin-2 promoter fragment with MluI and BglII sites was subcloned into the pGL3 Basic vector (Promega). In the promoter sequence, 6 putative transcription factor binding sites (4 sites for Cdx, a site for STAT, and a site for NFκB) were detected by TFSEARCH (Real World Computing Partnership). Deletion mutants for the putative transcription factor binding sites (Cdx, STAT, and NFκB) were introduced into the WT-claudin-2 promoter sequence using the KOD-Plus Mutagenesis kit (TOYOBO). The primer sequences used for mutagenesis are shown in supplemental Table S2. An hClaudin-2 promoter in a pGL3 vector driving luciferase expression was transfected to Caco-2 cells. Luciferase activity in cell lysates was assayed by the Luciferase assay system (Promega). Statistical analyses were performed using a one-way ANOVA approach followed by a Tukey-Kramer multiple range test using the general linear models procedure of the Statistical Analysis Systems program (version 6.07; SAS Institute Inc). A difference of p < 0.05 was considered significant. The degree of tightness of the TJ can be evaluated by measuring the transepithelial electrical resistance (TER, an indicator of TJ permeability to ionic solutes) and dextran flux (an indicator of TJ permeability to uncharged macromolecules). In Caco-2 cell monolayers, IL-6 decreased the TER in both a dose-dependent and time-dependent manner, indicating increased TJ permeability to ionic solutes (Fig. 1, A and B), without eliciting any changes to the permeance of the nonionic macromolecule 4 kDa dextran (Fig. 1C). Fig. 1A shows that the addition of ≥ 2.5 ng/ml IL-6 to the culture medium for 48 h induced significant decreases in the TER, and that the TER decreased to ∼70% of pretreatment level in the presence of 10 ng/ml IL-6. Fig. 1B shows that the TER readings taken after treatment with 5 ng/ml IL-6 at 24, 48, and 72 h, and after treatment with 10 ng/ml IL-6 at 12, 24, 48, and 72 h, were lower than the control values at each time point. Although the induction of apoptosis is known to decrease the TER, IL-6 (∼50 ng/ml) did not affect the cell viability (Fig. 1D). IL-6 also decreased the TER in another intestinal epithelial T84 cells in a similar manner to the Caco-2 cells, indicating that the IL-6-induced effect is not confined to the Caco-2 cells (supplemental Fig. S1). To determine the ion selectivity of Caco-2 cell monolayers incubated without or with IL-6, the dilution potentials were measured under an apicobasal chemical gradient of NaCl (Fig. 1, E–G). Fig. 1, E and F show that the treatment with 10 ng/ml IL-6 increased the NaCl dilution potential in the Caco-2 cells. As calculated from the dilution potential by the Goldman-Hodgkin-Katz equation, permeability of Na+ relative to Cl− (PNa+/PCl−) was increased by IL6 treatment (Fig. 1G). Because intestinal TJ permeability is regulated by TJ structure, we examined the effect of IL-6 on both the total expression of TJ proteins and on TJ proteins associated with the actin cytoskeleton in Caco-2 cell monolayers. Treatment of cells with 10 ng/ml IL-6 for 48 h increased claudin-2 protein expression in whole cell extracts and detergent-insoluble fractions (which corresponds to the actin cytoskeletal fraction) but did not affect the levels of other TJ proteins (ZO-1, ZO-2, occludin, JAM-1, claudin-1, claudin-3, or claudin-4) (Fig. 2A). It is known that claudin-2 expression decreases the tightness of the intestinal TJ barrier, due to the fact that claudin-2 forms paracellular cation channels. Furthermore, IL-6 had no effect on the expression of E-cadherin, MLCK, or on MLC phosphorylation (Fig. 2B), indicating that IL-6 is unlikely to regulate adherence junctions or cytoskeletal contraction. Fig. 2, C and D show increases in total claudin-2 protein levels in response to IL-6, occurring both dose-dependently and time-dependently. Treatment with 2.5, 5.0, 10, and 50 ng/ml IL-6 for 48 h significantly elevated claudin-2 protein expression (Fig. 2C). Increases in claudin-2 expression in response to 10 ng/ml IL-6 became apparent 6 h after treatment, but the most pronounced increases were observed at 24 and 48 h (Fig. 2D). The IL-6-induced claudin-2 expression was also observed in the T84 cell monolayers (supplemental Fig. S1). Increases in claudin-2 protein expression in response to IL-6 were confirmed using immunofluorescence microscopy (Fig. 2E). IL-6 enhanced the signal intensity of immunolabeled claudin-2, but not of ZO-1, in the junctional region of Caco-2 cell monolayers. To assess transcriptional regulation, claudin-2 mRNA was quantified by qPCR. Fig. 2E demonstrates that IL-6 treatment led to an increase in claudin-2 mRNA levels. These results show that the IL-6-induced increase in TJ permeability to Na+ is due to elevated claudin-2 expression in Caco-2 cells. To investigate whether IL-6 induced claudin-2 expression in vivo, we evaluated claudin-2 expression in mouse colons 16 h after IL-6 injection. Immunoblot analysis revealed that claudin-2 levels in the colons of mice injected with IL-6 were higher than those of the control mice (Fig. 3A). Immunofluorescence microscopy showed that claudin-2 expression was concentrated in the crypt epithelial cells of control mice, as previously reported (15Weber C.R. Raleigh D.R. Su L. Shen L. Sullivan E.A. Wang Y. Turner J.R. J. Biol. Chem. 2010; 285: 12037-12046Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), and the signal intensity was markedly increased after IL-6 injection (Fig. 3B). We then explored possible signaling pathways involved in IL-6-mediated increases in TJ permeability and claudin-2 expression. It has been reported that the binding of IL-6 to IL-6 receptor (gp80) induces the homodimerization of gp130, a signal-transducing subunit, which triggers several different signaling pathways including JAK/STAT, MEK/ERK, PI3K/Akt, cSrc, and NFκB. Fig. 4A shows that the IL-6-mediated decrease in TER was suppressed by anti-gp130, U0126 (a MEK inhibitor), LY294002 (a PI3K inhibitor), and PP2 (a cSrc inhibitor), but not by AG490 (a JAK inhibitor) or APDC (a NFκB inhibitor) in Caco-2 cell monolayers. Immunoblot analysis (Fig. 4B) further demonstrated that IL-6-induced claudin-2 expression was highly sensitive to the inhibition of gp130, MEK, and PI3K, but not cSrc. The results show that IL-6 induced the phosphorylation of STAT3, ERK1/2, and Akt (Fig. 4C), which were specifically or totally prevented by each inhibitor or anti-gp130 (Fig. 4D). Because of the possibility that MEK activation may have a primary role in IL-6-induced claudin-2 expression, Caco-2 cells were transiently transfected with MEK1wt-HA plasmid and triple-labeled for claudin-2 (green), MEK1-HA (red), and total DNA (blue), as shown in Fig. 4E. Cells expressing MEK1wt-HA demonstrated both enhanced expression and junctional distribution of claudin-2, while transfection of the control vector did not affect claudin-2 expression. These results indicate that the IL-6-mediated activation of the MEK/ERK and PI3K pathways, via gp130 subunit/IL-6Rα interaction, leads to enhanced claudin-2 expression res" @default.
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• W1966776937 title "Interleukin-6 (IL-6) Regulates Claudin-2 Expression and Tight Junction Permeability in Intestinal Epithelium" @default.
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