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• W2023694130 abstract "Interleukin (IL)-15 is able to regulate tight junction formation in intestinal epithelial cells. However, the mechanisms that regulate the intestinal barrier function in response to IL-15 and the involved subunits of the IL-15 ligand-receptor system are unknown. We determined the IL-2Rβ subunit and IL-15-dependent regulation of tight junction-associated proteins in the human intestinal epithelial cell line T-84. The IL-2Rβ subunit was expressed and induced signal transduction in caveolin enriched rafts in intestinal epithelial cells. IL-15-mediated tightening of intestinal epithelial monolayers correlated with the enhanced recruitment of tight junction proteins into Triton X-100-insoluble protein fractions. IL-15-mediated up-regulation of ZO-1 and ZO-2 expression was independent of the IL-2Rβ subunit, whereas the phosphorylation of occludin and enhanced membrane association of claudin-1 and claudin-2 by IL-15 required the presence of the IL-2Rβ subunit. Recruitment of claudins and hyperphosphorylated occludin into tight junctions resulted in a more marked induction of tight junction formation in intestinal epithelial cells than the up-regulation of ZO-1 and ZO-2 by itself. The regulation of the intestinal epithelial barrier function by IL-15 involves IL-2Rβ-dependent and -independent signaling pathways leading to the recruitment of claudins, hyperphosphorylated occludin, ZO-1, and ZO-2 into the tight junctional protein complex. Interleukin (IL)-15 is able to regulate tight junction formation in intestinal epithelial cells. However, the mechanisms that regulate the intestinal barrier function in response to IL-15 and the involved subunits of the IL-15 ligand-receptor system are unknown. We determined the IL-2Rβ subunit and IL-15-dependent regulation of tight junction-associated proteins in the human intestinal epithelial cell line T-84. The IL-2Rβ subunit was expressed and induced signal transduction in caveolin enriched rafts in intestinal epithelial cells. IL-15-mediated tightening of intestinal epithelial monolayers correlated with the enhanced recruitment of tight junction proteins into Triton X-100-insoluble protein fractions. IL-15-mediated up-regulation of ZO-1 and ZO-2 expression was independent of the IL-2Rβ subunit, whereas the phosphorylation of occludin and enhanced membrane association of claudin-1 and claudin-2 by IL-15 required the presence of the IL-2Rβ subunit. Recruitment of claudins and hyperphosphorylated occludin into tight junctions resulted in a more marked induction of tight junction formation in intestinal epithelial cells than the up-regulation of ZO-1 and ZO-2 by itself. The regulation of the intestinal epithelial barrier function by IL-15 involves IL-2Rβ-dependent and -independent signaling pathways leading to the recruitment of claudins, hyperphosphorylated occludin, ZO-1, and ZO-2 into the tight junctional protein complex. membrane-associated guanylate kinase-like homologue interleukin transepithelial electrical resistance polymerase chain reaction phosphate-buffered saline 4-morpholineethanesulfonic acid Madin-Darby canine kidney The regulation of the intestinal barrier is determined by the assembly of tight junctions (1Madara J. Annu. Rev. Physiol. 1998; 60: 143-159Crossref PubMed Scopus (464) Google Scholar). Tight junctions not only create a primary barrier to prevent paracellular transport of solutes, but they also restrict the lateral diffusion of membrane lipids and proteins to maintain cellular polarity (1Madara J. Annu. Rev. Physiol. 1998; 60: 143-159Crossref PubMed Scopus (464) Google Scholar, 2Schneeberger E.E. Lynch R.D. Am. J. Physiol. 1992; 262: L647-L661PubMed Google Scholar, 3Gumbiner B.M. J. Cell Biol. 1993; 123: 1631-1633Crossref PubMed Scopus (341) Google Scholar, 4Anderson J.M. Van Itallie C.M. Am. J. Physiol. 1995; 269: G467-G475Crossref PubMed Google Scholar). Tight junctions are macromolecular assemblies that form a specialized membrane domain at the most apical region of polarized epithelial cells. Tight junctions have a highly dynamic structure whose permeability, assembly, and/or disassembly can be regulated by a variety of cellular and metabolic mediators including cytokines, which have major functions in the immune system (5Madara J.L. Cell. 1988; 53: 497-498Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 6Nusrat A. Parkos C.A. Bacarra A.E. Godowski P.J. Delp-Archer C. Rosen E.M. Madara J.L. J. Clin. Invest. 1994; 93: 2056-2065Crossref PubMed Scopus (200) Google Scholar, 7Madara J.L. Stafford J. J. Clin. Invest. 1989; 83: 724-727Crossref PubMed Scopus (652) Google Scholar, 8Colgan S.P. Resnick M.B. Parkos C.A. Delp-Archer C. McGuirk D. Bacarra A.E. Weller P.F. Madara J.L. J. Immunol. 1994; 153: 2122-2129PubMed Google Scholar, 9Stevens A.C. Matthews J. Andres P. Baffis V. Zheng X.X. Chae D.W. Smith J. Strom T.B. Maslinski W. Am. J. Physiol. 1997; 272: G1201-G1208PubMed Google Scholar, 10Kinugasa T. Sakaguchi T. Gu X. Reinecker H.C. Gastroenterology. 2000; 118: 1001-1011Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). Immune modulators therefore may control tight junction-dependent intestinal barrier function during development, wound healing, and pathological processes such as cancer or chronic inflammation. To date, the claudins (11Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1730) Google Scholar) and occludin (12Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2143) Google Scholar,13Ando-Akatsuka Y. Saitou M. Hirase T. Kishi M. Sakakibara A. Itoh M. Yonemura S. Furuse M. Tsukita S. J. Cell Biol. 1996; 133: 43-47Crossref PubMed Scopus (287) Google Scholar) have been identified as tight junction-specific integral membrane proteins. Occludin and claudins can interact with the PDZ domains of zonula occludens proteins ZO-1 and ZO-2 (14Furuse M. Itoh M. Hirase T. Nagafuchi A. Yonemura S. Tsukita S. J. Cell Biol. 1994; 127: 1617-1626Crossref PubMed Scopus (809) Google Scholar, 15Itoh M. Furuse M. Morita K. Kubota K. Saitou M. Tsukita S. J. Cell Biol. 1999; 147: 1351-1363Crossref PubMed Scopus (915) Google Scholar). ZO-1 and ZO-2 are membrane-associated guanylate kinase-like homologues (MAGUKs)1 that may play a general role in creating and maintaining specialized membrane domains by cross-linking multiple integral membrane proteins at the cytoplasmic surface of plasma membranes in various cells types (16Woods D.F. Bryant P.J. Mech. Dev. 1993; 44: 85-89Crossref PubMed Scopus (190) Google Scholar, 17Jesaitis L.A. Goodenough D.A. J. Cell Biol. 1994; 124: 949-961Crossref PubMed Scopus (389) Google Scholar, 18Anderson J.M. Curr. Biol. 1996; 6: 382-384Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Furthermore, ZO-1 and ZO-2 bind directly to actin filaments at their COOH-terminal regions, suggesting that these molecules function as cross-linkers between tight junction strands and actin filaments (19Itoh M. Nagafuchi A. Moroi S. Tsukita S. J. Cell Biol. 1997; 138: 181-192Crossref PubMed Scopus (572) Google Scholar, 20Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1114) Google Scholar, 21Itoh M. Morita K. Tsukita S. J. Biol. Chem. 1999; 274: 5981-5986Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 22Wittchen E.S. Haskins J. Stevenson B.R. J. Biol. Chem. 1999; 274: 35179-35185Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). In addition, ZO-2 has been reported to associate with ZO-1 directly (20Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1114) Google Scholar, 21Itoh M. Morita K. Tsukita S. J. Biol. Chem. 1999; 274: 5981-5986Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Therefore zonula occludens proteins may establish a framework to combine the functional components of tight junctions during the generation of the intestinal barrier. Disruption of the intestinal epithelial cell barrier function may play an important role in the pathogenesis of inflammatory bowel disease (23Peeters M. Geypens B. Claus D. Nevens H. Ghoos Y. Verbeke G. Baert F. Vermeire S. Vlietinck R. Rutgeerts P. Gastroenterology. 1997; 113: 802-807Abstract Full Text PDF PubMed Scopus (222) Google Scholar, 24Hilsden R.J. Meddings J.B. Sutherland L.R. Gastroenterology. 1996; 110: 1395-1403Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 25Schmitz H. Barmeyer C. Fromm M. Runkel N. Foss H.D. Bentzel C.J. Riecken E.O. Schulzke J.D. Gastroenterology. 1999; 116: 301-309Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). Although it is not clear whether intestinal barrier dysfunction is involved in the initiation or is the result of intestinal inflammation, it is likely that dysfunction of the intestinal barrier would perpetuate intestinal inflammatory responses. Cytokines, which regulate intestinal immune response, may perturb the intestinal barrier or could contribute to mechanisms that may have evolved to rapidly seal the intestinal monolayer to limit the influx of highly antigenic substances into the intestinal lamina propria. The IL-15 cytokine receptor complex consists of the IL-15Rα, the IL-2Rβ, and the γc receptor subunit, which is common to several cytokine receptor complexes (26Grabstein K.H. Eisenman J. Shanebeck K. Rauch C. Srinivasan S. Fung V. Beers C. Richardson J. Schoenborn M.A. Ahdieh M. Johnson L. Alderson M.R. Watson J.D. Anderson D.M. Giri J.G. Science. 1994; 264: 965-968Crossref PubMed Scopus (1337) Google Scholar, 27Bamford R.N. Grant A.J. Burton J.D. Peters C. Kurys G. Goldman C.K. Brennan J. Roessler E. Waldmann T.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4940-4944Crossref PubMed Scopus (383) Google Scholar, 28Giri J.G. Ahdieh M. Eisenman J. Shanebeck K. Grabstein K. Kumaki S. Namen A. Park L.S. Cosman D. Anderson D. EMBO J. 1994; 13: 2822-2830Crossref PubMed Scopus (967) Google Scholar, 29Burton J.D. Bamford R.N. Peters C. Grant A.J. Kurys G. Goldman C.K. Brennan J. Roessler E. Waldmann T.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4935-4939Crossref PubMed Scopus (341) Google Scholar). Because the IL-15 receptor complex shares the IL-2Rβ and the γc receptor subunits with IL-2, much attention has focused on the overlapping functional effects of IL-15 and IL-2 in the regulation of T- and B-lymphocytes (26Grabstein K.H. Eisenman J. Shanebeck K. Rauch C. Srinivasan S. Fung V. Beers C. Richardson J. Schoenborn M.A. Ahdieh M. Johnson L. Alderson M.R. Watson J.D. Anderson D.M. Giri J.G. Science. 1994; 264: 965-968Crossref PubMed Scopus (1337) Google Scholar, 27Bamford R.N. Grant A.J. Burton J.D. Peters C. Kurys G. Goldman C.K. Brennan J. Roessler E. Waldmann T.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4940-4944Crossref PubMed Scopus (383) Google Scholar, 28Giri J.G. Ahdieh M. Eisenman J. Shanebeck K. Grabstein K. Kumaki S. Namen A. Park L.S. Cosman D. Anderson D. EMBO J. 1994; 13: 2822-2830Crossref PubMed Scopus (967) Google Scholar, 29Burton J.D. Bamford R.N. Peters C. Grant A.J. Kurys G. Goldman C.K. Brennan J. Roessler E. Waldmann T.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4935-4939Crossref PubMed Scopus (341) Google Scholar). However, the expression of the IL-15Rα subunit in a wide variety of different tissues suggests that the functional role of IL-15 is not limited to the regulation of lymphoid cells (30Anderson D.M. Kumaki S. Ahdieh M. Bertles J. Tometsko M. Loomis A. Giri J. Copeland N.G. Gilbert D.J. Jenkins N.A. Valentine V. Shapiro D.N. Morris S.W. Park L.S. Cosman D. J. Biol. Chem. 1995; 270: 29862-29869Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Recently IL-15 has been recognized as a cytokine up-regulated during inflammatory bowel disease as well as during the colitis in IL-2-deficient mice (31Sakai T. Kusugami K. Nishimura H. Ando T. Yamaguchi T. Ohsuga M. Ina K. Enomoto A. Kimura Y. Yoshikai Y. Gastroenterology. 1998; 114: 1237-1243Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 32Liu Z. Geboes K. Colpaert S. D'Haens G.R. Rutgeerts P. Ceuppens J.L. J. Immunol. 2000; 164: 3608-3615Crossref PubMed Scopus (164) Google Scholar, 33Meijssen M.A. Brandwein S.L. Reinecker H.C. Bhan A.K. Podolsky D.K. Am. J. Physiol. 1998; 274: G472-G479Crossref PubMed Google Scholar). Nevertheless, it is not clear whether IL-15 contributes to the perpetuation of inflammation or the regulation of intestinal tissue repair. Furthermore, IL-15 may be an important regulator of intestinal intraepithelial lymphocytes. IL-15 is a potent activator of intestinal intraepithelial lymphocytes (34Ebert E.C. Gastroenterology. 1998; 115: 1439-1445Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 35Inagaki-Ohara K. Nishimura H. Mitani A. Yoshikai Y. Eur. J. Immunol. 1997; 27: 2885-2891Crossref PubMed Scopus (130) Google Scholar, 36Lai Y.G. Gelfanov V. Gelfanova V. Kulik L. Chu C.L. Jeng S.W. Liao N.S. J. Immunol. 1999; 163: 5843-5850PubMed Google Scholar), and the number of intestinal intraepithelial lymphocytes is reduced in mice lacking the IL-15Rα subunit (37Lodolce J.P. Boone D.L. Chai S. Swain R.E. Dassopoulos T. Trettin S. Ma A. Immunity. 1998; 9: 669-676Abstract Full Text Full Text PDF PubMed Scopus (1099) Google Scholar). T-84 cells provide a well established model for the assembly of intercellular junctions and the development of apical-basolateral polarity (5Madara J.L. Cell. 1988; 53: 497-498Abstract Full Text PDF PubMed Scopus (166) Google Scholar). T-84 cells express the IL-15Rα subunit (9Stevens A.C. Matthews J. Andres P. Baffis V. Zheng X.X. Chae D.W. Smith J. Strom T.B. Maslinski W. Am. J. Physiol. 1997; 272: G1201-G1208PubMed Google Scholar) and the common γc receptor subunits (9Stevens A.C. Matthews J. Andres P. Baffis V. Zheng X.X. Chae D.W. Smith J. Strom T.B. Maslinski W. Am. J. Physiol. 1997; 272: G1201-G1208PubMed Google Scholar, 38Reinecker H.C. Podolsky D.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8353-8357Crossref PubMed Scopus (145) Google Scholar). However, T-84 cells lack, in contrast to primary intestinal epithelial cells, the expression of the IL-2Rβ subunit (9Stevens A.C. Matthews J. Andres P. Baffis V. Zheng X.X. Chae D.W. Smith J. Strom T.B. Maslinski W. Am. J. Physiol. 1997; 272: G1201-G1208PubMed Google Scholar, 38Reinecker H.C. Podolsky D.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8353-8357Crossref PubMed Scopus (145) Google Scholar). Nevertheless, T-84 cells respond to stimulation with IL-15 with an increase in transepithelial electrical resistance (TER) (9Stevens A.C. Matthews J. Andres P. Baffis V. Zheng X.X. Chae D.W. Smith J. Strom T.B. Maslinski W. Am. J. Physiol. 1997; 272: G1201-G1208PubMed Google Scholar). However, the mechanisms responsible for the regulation of the intestinal barrier function by IL-15 have not been determined. To study the regulation of tight junction formation mediated through the IL-15 ligand receptor system in intestinal epithelial cells, we stable transfected T-84 cells with IL-2Rβ and characterized the subsequent basal and IL-15-mediated regulation of tight junction-associated proteins. Human recombinant IL-15 was obtained from R & D Systems (Minneapolis, MN). Rabbit polyclonal antibodies against ZO-1, ZO-2, occludin, claudin-1, and claudin-2 were from Zymed Laboratories Inc. (San Francisco, CA). Anti-phosphotyrosine antibody (PY-20) was obtained from Transduction Laboratories (Lexington, KY). Rabbit polyclonal antibody against the human IL-2Rβ was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Blocking antibodies directed against the IL-2Rβ subunit (MIK1β) were purchased from Accurate Chemical & Scientific Co. (Westbury, NY), and control mouse IgG2A was obtained from Sigma. Fluorescein isothiocyanate-labeled anti-rabbit secondary antibodies were obtained from Vector Laboratories (Burlingame, CA). For Western blot analysis, horseradish peroxidase conjugated anti-rabbit or anti-mouse antibodies were purchased from Amersham Pharmacia Biotech. To generate an expression vector encoding the IL-2Rβ subunit, cDNAs encoding extracellular and intracellular regions were subcloned separately. Reverse transcriptase-PCR to generate the cDNA for the NH2-terminal region of the IL-2Rβ subunit was carried out with reverse transcribed RNA isolated from peripheral blood mononuclear cells with the primer pair 5′-CAG CAC CGG GGA GGA CTG GA-3′ and 5′-CGC CAG GGC TGA AGG ACG AT-3′ for 40 cycles at 94 °C for 1 min, 70 °C for 1 min, and 72 °C for 2 min. The PCR product was cloned into pBluescript KS+ (Stratagene, La Jolla, CA), and theBamHI/SacI fragment was released for ligation. To generate the COOH-terminal region of the IL-2Rβ cDNA, nested PCR was carried out for 20 cycles at 94 °C for 1 min, 70 °C for 1 min, and 72 °C for 2 min with first sets of primers of 5′-CTG CAA GGC GAG TTC ACG AC-3′ and 5′-AGC AGC AGT GGA GGT TTG GA-3′. A second PCR was carried out using a 1:100 dilution of the first PCR product as a template for 30 cycles with the second set of primers (5′-GGC TTT TGG CTT CAT CT-3′ and 5′-AGC TGC AAC TGG ACA CTG AG-3′) at 94 °C for 1 min, 70 °C for 1 min, and 72 °C for 2 min. The PCR product was cloned into pBluescript KS+, and the SacI/XhoI fragment was released. Extracellular and intracellular parts of IL-2Rβ cDNA were ligated at the SacI site and ligated into a BamHI/XhoI-digested pcDNA3.1+ vector (Invitrogen, San Diego, CA). Sequence was confirmed in both directions using the dideoxy termination method. The human colon cancer derived cell lines T-84 and Caco-2 were obtained from American Type Culture Collection (Manassas, VA). T-84 cells were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Cellgro, Mediatech Inc., Herndon, VA) with 100 IU/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated fetal calf serum (Sigma) in a humidified 5% CO2 atmosphere at 37 °C. The cells were transfected using LipofectAMINE (Life Technologies, Inc.) with 1 μg of pcDNA3.1+/IL-2Rβ construct. IL-2Rβ subunit stable transfectants, designated T-84β, were established by selection in medium supplemented with 1 mg/ml G418 (Life Technologies, Inc.) for 5 weeks. T-84β cells and parental T-84 cells were seeded into 96-well plates at a density of 5 × 103 cells/well and were cultured in serum-free Dulbecco's modified Eagle's medium in the presence of various concentration of recombinant human IL-15 at 37 °C for 24–72 h. Proliferation was assessed by MTS assay using CellTiter 96 Aqueous kit (Promega, Madison, WI) according to the manufacturer's instructions. Each assay was performed in triplicate. 20 μl of a fresh mixture of MTS tetrazolium compound and phenazine methosulfate was added to each well and incubated at 37 °C for 1 h. The amount of formazan corresponds to the number of viable cells and was measured at an absorbance of 490 nm by an enzyme-linked immunosorbent assay reader. Wells containing medium, but no cells, were subtracted as background from the raw absorbance values. For sequential TER measurements, T-84 monolayers were maintained on 24-well Transwell collagen-treated permeable supports (Corning Coster, Cambridge, MA). We determined the IL-2Rβ-dependent regulation of TER during the assembly of tight junctions after replating T-84 cells to model the reconstitution of the intestinal epithelial barrier function during in intestinal wound healing. T-84 cells were plated at a concentration of 1.5 × 106/well. The cells were stimulated with 100 ng/ml IL-15 24 h later in the presence or absence of anti-IL-2Rβ or control antibodies, and the TER measurements were carried out at the indicated time points in triplicate samples. Millicell-ERS epithelial volt-ohmmeter (World Precision Instruments, New Haven, CT) was utilized under temperature-controlled conditions at 37 °C with electrodes reproducibly placed. TER values were calculated by subtracting the contribution of the bare filter and medium. Statistical analysis was performed by Student's t test. To isolate Triton X-100-soluble and -insoluble protein fractions, confluent T-84 cell monolayers grown on 10-cm dishes were washed three times with ice-cold PBS, lysed in Triton X-100 buffer (1% Triton X-100, 100 mm NaCl, 10 mm HEPES, pH 7.6, 2 mm EDTA, 1 mm phenylmethylsulfonylfluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 4 mm sodium orthovanadate, 40 mm sodium fluoride), and then passed through a 21-gauge needle ten times. The lysates were then centrifuged at 15,000 × g for 30 min at 4 °C. The resulting supernatant was considered the Triton X-100-soluble fraction. The pellet was solubilized in Triton X-100 buffer containing 1% SDS using an ultrasonic disintegrator, cleared by centrifugation at 15,000 × g for 5 min at 4 °C, and referred to as the Triton X-100-insoluble fraction. The protein concentration of each sample was quantified by the Bradford method. Glycolipid-enriched membrane microdomains, or detergent-insoluble glycolipid rafts, were isolated as described before with minor modifications (39Brown D.A. London E. Biochem. Biophys. Res. Commun. 1997; 240: 1-7Crossref PubMed Scopus (464) Google Scholar, 40Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar, 41Muehlhoefer A. Saubermann L.J. Gu X. Luedtke-Heckenkamp K. Xavier R. Blumberg R.S. Podolsky D.K. MacDermott R.P. Reinecker H.C. J. Immunol. 2000; 164: 3368-3376Crossref PubMed Scopus (211) Google Scholar). Four dishes of T-84 or T-84β96 cells (diameter, 10 cm) were washed three times with ice-cold PBS, and the cells were solubilized in 1 ml of lysis buffer (25 mmMES, pH 6.8, 150 mm NaCl, 1% Triton X-100 supplemented with protease/phosphatase inhibitor mixture). After incubation for 30 min on ice, the lysate was gently processed in a tight fitting Dounce homogenizer 10 times and cleared by centrifugation for 5 min at 1000 × g. The supernatant was adjusted to 40% sucrose with an equal volume of 80% sucrose in MBS (25 mm MES, pH 6.8, 150 mm NaCl supplemented with the protease/phosphatase inhibitor mixture) and transferred to the bottom of a centrifugation tube. A sucrose step gradient with 30, 25, 20, 15, and 5% in MBS (2 ml each) was layered on top. After centrifugation in a SW 41 rotor (Beckman) at 39,000 rpm for 14–16 h at 4 °C, fractions of 1 ml each were taken, starting from the top and going to the bottom. Protein measurement of the cellular fractions was performed with BCA protein assay reagent, according to the manufacturer's instructions (Pierce). The samples were electrophoresed through a 4–20% gradient SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). After 1 h of blocking (Tris-buffered saline, 0.1% Tween 20, 1% bovine serum albumin), the blots were incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. After washing in Tris-buffered saline, 0.1% Tween 20, the membrane was incubated with appropriate secondary antibody diluted in blocking buffer for 60 min at room temperature. The hybridized band was detected by an ECL kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Immunoblots were stripped with 62.5 mm Tris, pH 6.8, 2% SDS containing 10 mm β-mercaptoethanol at 50 °C for 30 min. Total RNA was isolated from cultured cells using Trizol reagent (Life Technologies, Inc.). Thirty micrograms of total RNA were electrophoresed in a 1% agarose formaldehyde gel and then transferred onto a nylon membrane (Magna NT, MicroSeparations Inc., Westbrough, MA) by capillary blotting. The probes were labeled with [α-32P]dCTP using the Rediprime Random Primer Labeling Kit (Amersham Pharmacia Biotech). Membranes were hybridized with radiolabeled cDNA probes in Quickhyb solution (Stratagene, La Jolla, CA) at 65 °C for 1 h. The membranes were washed with 1% SDS, 2× sodium chloride sodium citrate buffer. The blots were analyzed by autoradiography. The blots were sequentially hybridized with IL-15 and glyceraldehyde-3-phosphate dehydrogenase probes as described previously (42Reinecker H.C. MacDermott R.P. Mirau S. Dignass A. Podolsky D.K. Gastroenterology. 1996; 111: 1706-1713Abstract Full Text PDF PubMed Scopus (210) Google Scholar). T-84 and T-84β96 cells were grown for 3 days with or without 100 ng/ml IL-15 on chamber slide culture chambers (Nunc Inc., Naperville, IL). T-84 cells or T-84β96 cells were washed three times with Dulbecco's PBS (Life Technologies, Inc.) and were fixed for 10 min in 2% paraformaldehyde for immunostaining with antibodies recognizing occludin or for 30 s in aceton at −20 °C for the detection of claudin-1, claudin-2, ZO-1, and ZO-2. Thereafter, the cells were blocked with 1:200 PBS-diluted normal donkey serum for 1 h at 20 °C and incubated with 1:200 in PBS-diluted primary antibodies overnight at 4 °C. After three washes with PBS, the monolayers were incubated at room temperature with anti-mouse or anti-rabbit IgG fluorescein isothiocyanate-conjugated antibody (1:500 in PBS) for 1 h in the dark at room temperature and analyzed with a confocal immunofluorescent microscope (Bio-Rad). An initial screen of 98 IL-2Rβ-transfected T-84 clones yielded two clones with a high expression of functional IL-2Rβ subunits. The low number of stable transfected clones reflects the difficulties in stably transfecting T-84 cells. Both IL-2Rβ subunit expressing T-84 cell clones (T-84β29 and T-84β96) exhibited a similar phenotype and were characterized by an increased resistance to separation by a 1 mm EDTA, 0.25% trypsin solution. T-84β29 and T-84β96 monolayers required 15–20 min of incubation to obtain single-cell suspensions instead of the 5–7 min of incubation time required for the parental T-84 monolayers. Both T-84β29 and T-84β96 cells form uniform, tightly packed monolayers within 3 days after separation at a 1:4 split ratio. Expression levels and membrane targeting of the IL-2Rβ subunit in stable transfected T-84 cells was analyzed in Triton X-100-soluble and -insoluble protein fractions (Fig.1 A). As demonstrated in Fig.1 A, T-84β29 and T-84β96 cells expressed the IL-2Rβ subunit, whereas the IL-2Rβ chain was not detectable in parental T-84 cells. When analyzed densitometrically, the expression of the IL-2Rβ was 3-fold higher in T-84β29 cells than in T-84β96 cells (Fig.1 A). In both T-84β29 and T-84β96 cells, the IL-2Rβ subunit was highly enriched in Triton X-100-insoluble protein fractions, demonstrating that the stable expressed cytokine receptor was integrated into T-84 cell membranes (Fig. 1 A). Overexpression of cytokine receptors may lead to autonomous activation of signal transduction events. Therefore, we determined the basal activation of tyrosine phosphorylation in parental T-84 cells, T-84β29, and T-84β96 cells. As demonstrated in Fig. 1 B, T-84β29 cells demonstrated an enhanced basal level of phosphotyrosine kinase activity compared with the parental T-84 cells and T-84β96 cells when membrane-associated Triton X-100-insoluble protein fractions were analyzed (Fig. 1 B, lane 4 compared withlanes 2 and 6). The elevation of tyrosine phosphorylation in the T-84β29 cells correlated with the high level of IL-2Rβ subunit expression in Triton X-100-insoluble membrane fraction in this T-84 cell clone. To further characterize the membrane compartment containing the IL-2Rβ subunit, we separated postnuclear membrane protein fractions by sucrose density gradient centrifugation. This method separates detergent-insoluble membrane fractions characterized by their specific lipid composition (40Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar, 43Lisanti M.P. Tang Z. Scherer P.E. Kubler E. Koleske A.J. Sargiacomo M. Mol. Membr. Biol. 1995; 12: 121-124Crossref PubMed Scopus (134) Google Scholar, 44Song S.K. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar). As demonstrated in Fig. 2 A, the IL-2Rβ subunit was present in low sucrose gradient fractions of T-84β96 cells. IL-2Rβ was present in membrane fractions corresponding to sucrose concentrations of 20–24%, with the highest concentration in the 20% sucrose gradient fraction (Fig.2 A). These low sucrose gradient fractions have been associated with detergent-insoluble glycolipid-enriched membrane fractions or rafts (45Parton R.G. Simons K. Science. 1995; 269: 1398-1399Crossref PubMed Scopus (296) Google Scholar). In epithelial cells, these membrane fractions are characterized by the presence of the scaffolding protein caveolin (46Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar). As demonstrated in Fig. 2 A, Western blot analysis of these cellular compartments revealed that the expression of caveolin-1 was restricted to the same sucrose gradients as that of the IL-2Rβ. In the next set of experiments, we determined whether the expression of the IL-2Rβ subunit enhanced the responsiveness of T-84 cells to IL-15. In these experiments, we used T-84β96 cells, which had a base-line activation of phosphotyrosine kinase comparable with T-84 cells (Fig. 1 B). T-84 and T-84β96 cells were cultured for 15 min with or without IL-15. As demonstrated in Fig. 2 B, IL-15 stimulation was able to induce a strong up-regulation of phosphotyrosine kinase activity in T-84β96 cells after 15 min (Fig.2 B, lane 10). IL-15 induced tyrosine phosphorylation of proteins with a molecular mass of 100 kDa, as well as a number of proteins with molecular masses between 42 and 54 kDa in T-84β96 cells (Fig. 2 B, lane 10). In contrast, IL-15 induced only a weak tyrosine phosphoryl" @default.
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• W2023694130 date "2001-09-01" @default.
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• W2023694130 title "Interleukin-2 Receptor β Subunit-dependent and -independent Regulation of Intestinal Epithelial Tight Junctions" @default.
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