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• W2020186183 abstract "Interleukin-4 (IL-4) activates Stat6 (signal transducer and activator of transcription 6) and plays multiple roles in regulation of the immune system. IL-4 also triggers phosphorylation of insulin receptor substrate (IRS), leading to stimulation of cell growth. Moreover, IL-4 inhibits proliferation of a variety of cells, but the molecular mechanism of its growth inhibitory effect is not understood. In this study, we demonstrated that IL-4 inhibited cell growth of colon carcinoma cell lines (HT29 and WiDr) but promoted cell growth of Burkitt's lymphoma cell lines (BL30 and BL41) in a dose-dependent manner. The growth inhibition was not dependent on Stat6 activation, because Stat6 was activated at similar levels in all cell lines in response to IL-4. Strikingly, IL-4 activated Stat1 in colon carcinoma cell lines but not in Burkitt's lymphoma cell lines. Therefore, these results suggest that IL-4 induced Stat1 activation, resulting in growth inhibition of colon carcinoma cell lines. Importantly, we present evidence that Stat1 is necessary for IL-4-mediated growth inhibition using Stat1-deficient and Stat1-reconstituted cells. The growth inhibitory effect of IL-4 was diminished in Stat1-deficient cells, whereas it was restored in Stat1-reconstituted cells. In addition, the expression of dominant-negative Stat1 in HT29 cells led to the loss of growth inhibition in response to IL-4. Taken together, our data suggest that IL-4 activates Stat1, leading to cell growth inhibition in colon cancer cells. Thus, this study demonstrates, for the first time, a molecular mechanism by which IL-4 inhibits cell growth. Interleukin-4 (IL-4) activates Stat6 (signal transducer and activator of transcription 6) and plays multiple roles in regulation of the immune system. IL-4 also triggers phosphorylation of insulin receptor substrate (IRS), leading to stimulation of cell growth. Moreover, IL-4 inhibits proliferation of a variety of cells, but the molecular mechanism of its growth inhibitory effect is not understood. In this study, we demonstrated that IL-4 inhibited cell growth of colon carcinoma cell lines (HT29 and WiDr) but promoted cell growth of Burkitt's lymphoma cell lines (BL30 and BL41) in a dose-dependent manner. The growth inhibition was not dependent on Stat6 activation, because Stat6 was activated at similar levels in all cell lines in response to IL-4. Strikingly, IL-4 activated Stat1 in colon carcinoma cell lines but not in Burkitt's lymphoma cell lines. Therefore, these results suggest that IL-4 induced Stat1 activation, resulting in growth inhibition of colon carcinoma cell lines. Importantly, we present evidence that Stat1 is necessary for IL-4-mediated growth inhibition using Stat1-deficient and Stat1-reconstituted cells. The growth inhibitory effect of IL-4 was diminished in Stat1-deficient cells, whereas it was restored in Stat1-reconstituted cells. In addition, the expression of dominant-negative Stat1 in HT29 cells led to the loss of growth inhibition in response to IL-4. Taken together, our data suggest that IL-4 activates Stat1, leading to cell growth inhibition in colon cancer cells. Thus, this study demonstrates, for the first time, a molecular mechanism by which IL-4 inhibits cell growth. interleukin-4 signal transducer and activator of transcriptions 1 and 6, respectively fetal bovine serum electrophoretic mobility shift assay Interleukin-4 (IL-4)1has pleiotropic effects on a wide variety of cell types of hematopoietic and nonhematopoietic origin (reviewed in Refs. 1.Banchereau J. Rybak M.E. Thomson A. The Cytokine Handbook. Academic Press, New York1994: 99-126Google Scholar, 2.Paul W.E. Blood. 1991; 77: 1859-1870Crossref PubMed Google Scholar, 3.Puri R.K. Kurzrock R. Talpaz M. Cytokines: Interleukins and Their Receptors. Kluwer Academic Publishers, Norwell, MA1995: 143-186Google Scholar, 4.Paul W.E. Seder R.A. Cell. 1994; 76: 241-251Abstract Full Text PDF PubMed Scopus (1695) Google Scholar). Mainly secreted by stimulated T cells, mast cells, and basophils (5.Brown M.A. Pierce J.H. Watson C.J. Falco J. Ihle J.N. Paul W.E. Cell. 1987; 50: 809-818Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 6.Howard M. Farrar J. Hilfiker M. Johnson B. Takatsu K. Hamaoka T. Paul W.E. J. Exp. Med. 1982; 155: 914-923Crossref PubMed Scopus (830) Google Scholar, 7.Seder R.A. Paul W.E. Ben-Sasson S.Z. LeGros G.S. Kagey-Sobotka A. Finkelman F.D. Pierce J.H. Plaut M. Int. Arch. Allergy Appl. Immunol. 1991; 94: 137-140Crossref PubMed Scopus (83) Google Scholar), IL-4 plays a significant role in controlling cell growth and regulating the immune system. Major functions of IL-4 in the immune system include induction of Th2 cell differentiation (8.Le Gros G. Ben-Sasson S.Z. Seder R. Finkelman F.D. Paul W.E. J. Exp. Med. 1990; 172: 921-929Crossref PubMed Scopus (845) Google Scholar, 9.Swain S.L. Weinberg A.D. English M. Huston G. J. Immunol. 1990; 145: 3796-3806PubMed Google Scholar), IgE and IgG1 class switching (10.Vitetta E.S. Ohara J. Myers C.D. Layton J.E. Krammer P.H. Paul W.E. J. Exp. Med. 1985; 162: 1726-1731Crossref PubMed Scopus (319) Google Scholar, 11.Coffman R.L. Ohara J. Bond M.W. Carty J. Zlotnik A. Paul W.E. J. Immunol. 1986; 136: 4538-4541PubMed Google Scholar), and induction of expression of surface molecules such as major histocompatibility complex class II, IL-4 receptor (12.Noelle R. Krammer P.H. Ohara J. Uhr J.W. Vitetta E.S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6149-6153Crossref PubMed Scopus (381) Google Scholar), and the low affinity IgE receptor, CD23 (13.Conrad D.H. Waldschmidt T.J. Lee W.T. Rao M. Keegan A.D. Noelle R.J. Lynch R.G. Kehry M.R. J. Immunol. 1987; 139: 2290-2296PubMed Google Scholar). IL-4 induces proliferation of T cells (14.Brown M. Hu-Li J. Paul W.E. J. Immunol. 1988; 141: 504-511PubMed Google Scholar, 15.Miller C.L. Hooton J.W. Gillis S. Paetkau V. J. Immunol. 1990; 144: 1331-1337PubMed Google Scholar, 16.Spits H. Yssel H. Takebe Y. Arai N. Yokota T. Lee F. Arai K. Banchereau J. de Vries J.E. J. Immunol. 1987; 139: 1142-1147PubMed Google Scholar, 17.Kaplan M.H. Daniel C. Schindler U. Grusby M.J. Mol. Cell. Biol. 1998; 18: 1996-2003Crossref PubMed Scopus (115) Google Scholar) and promotes growth of B cells costimulated by anti-IgM (6.Howard M. Farrar J. Hilfiker M. Johnson B. Takatsu K. Hamaoka T. Paul W.E. J. Exp. Med. 1982; 155: 914-923Crossref PubMed Scopus (830) Google Scholar). In contrast to its growth stimulatory effect on lymphocytes, IL-4 significantly inhibits proliferation of many kinds of cells, including those derived from human melanoma, colon, renal, and breast carcinoma (3.Puri R.K. Kurzrock R. Talpaz M. Cytokines: Interleukins and Their Receptors. Kluwer Academic Publishers, Norwell, MA1995: 143-186Google Scholar, 18.Hollingsworth S.J. Darling D. Gaken J. Hirst W. Patel P. Kuiper M. Towner P. Humphreys S. Farzaneh F. Mufti G.J. Br. J. Cancer. 1996; 74: 6-15Crossref PubMed Scopus (23) Google Scholar, 19.Toi M. Bicknell R. Harris A.L. Cancer Res. 1992; 52: 275-279PubMed Google Scholar, 20.Tepper R.I. Pattengale P.K. Leder P. Cell. 1989; 57: 503-512Abstract Full Text PDF PubMed Scopus (761) Google Scholar). However, the molecular mechanism of the antiproliferative effect of IL-4 remains to be determined. The IL-4 receptor is composed of a cytokine-specific α chain and the common γc chain shared by IL-2, IL-7, IL-9, and IL-15 receptors in hematopoietic cells (reviewed in Ref. 21.Leonard W.J. Annu. Rev. Med. 1996; 47: 229-239Crossref PubMed Scopus (153) Google Scholar). Binding of IL-4 to the IL-4 receptor triggers phosphorylation of Janus kinases JAK1 and JAK3 (22.Johnston J.A. Kawamura M. Kirken R.A. Chen Y.Q. Blake T.B. Shibuya K. Ortaldo J.R. McVicar D.W. O'Shea J.J. Nature. 1994; 370: 151-153Crossref PubMed Scopus (507) Google Scholar, 23.Miyazaki T. Kawahara A. Fujii H. Nakagawa Y. Minami Y. Liu Z.J. Oishi I. Silvennoinen O. Witthuhn B.A. Ihle J.N. et al.Science. 1994; 266: 1045-1047Crossref PubMed Scopus (503) Google Scholar, 24.Russell S.M. Johnston J.A. Noguchi M. Kawamura M. Bacon C.M. Friedmann M. Berg M. McVicar D.W. Witthuhn B.A. Silvennoinen O. et al.Science. 1994; 266: 1042-1045Crossref PubMed Scopus (589) Google Scholar, 25.Witthuhn B.A. Silvennoinen O. Miura O. Lai K.S. Cwik C. Liu E.T. Ihle J.N. Nature. 1994; 370: 153-157Crossref PubMed Scopus (535) Google Scholar), leading to activation of two major signaling pathways that are known as Stat6 (signal transducer and activator of transcription 6) (26.Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (727) Google Scholar, 27.Kotanides H. Reich N.C. Science. 1993; 262: 1265-1267Crossref PubMed Scopus (230) Google Scholar) and insulin receptor substrate (IRS), which includes IRS-1 and IRS-2/4PS (28.Keegan A.D. Nelms K. White M. Wang L.M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 29.Sun X.J. Wang L.M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (765) Google Scholar). IRS-1 and IRS-2 regulate cell proliferation in the myeloid cell line 32D in response to IL-4 (28.Keegan A.D. Nelms K. White M. Wang L.M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 29.Sun X.J. Wang L.M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (765) Google Scholar, 30.Wang L.M. Myers Jr., M.G. Sun X.J. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (371) Google Scholar). Recent studies on Stat6 (−/−) mice have shown that Stat6 is essential for T-cell differentiation, IgE class switching, and expression of CD23 and major histocompatibility complex class II in response to IL-4 (31.Shimoda K. van Deursen J. Sangster M.Y. Sarawar S.R. Carson R.T. Tripp R.A. Chu C. Quelle F.W. Nosaka T. Vignali D.A. Doherty P.C. Grosveld G. Paul W.E. Ihle J.N. Nature. 1996; 380: 630-633Crossref PubMed Scopus (1108) Google Scholar, 32.Takeda K. Tanaka T. Shi W. Matsumoto M. Minami M. Kashiwamura S. Nakanishi K. Yoshida N. Kishimoto T. Akira S. Nature. 1996; 380: 627-630Crossref PubMed Scopus (1269) Google Scholar, 33.Kaplan M.H. Schindler U. Smiley S.T. Grusby M.J. Immunity. 1996; 4: 313-319Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar). To understand the molecular mechanism of cell growth controlled by IL-4, we studied the effect of IL-4 on cell proliferation and STAT activation. We demonstrated that IL-4 induced differential Stat1 activation and growth inhibition in different cell lines. In addition, Stat6 was not essential for IL-4-induced cell growth inhibition. These results indicate that IL-4-mediated Stat1 activation plays a critical role in cell growth inhibition. Human and mouse IL-4 were kindly provided by Schering-Plow Research Institute (Kenilworth, NJ). Antibodies against Stat6 (S-20 and M-200) and Stat1 (N terminus, catalog no. G16930) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Transduction Laboratories (Lexington, KY), respectively. Anti-Stat3 antibody was kindly provided by Dr. Zhong and purchased from Santa Cruz Biotechnology. Human colon carcinoma cell lines HT29 and WiDr were purchased from the American Type Culture Collection (Manassas, VA). Burkitt's lymphoma cell lines BL30 and BL41 were kindly provided by Dr. I. George Miller (Yale University). HT29 cells were cultured in McCoy's 5A medium containing 10% FBS (Life Technologies, Inc). WiDr cells were cultured in Dulbecco's modified Eagle's medium with 10% FBS, and Burkitt's lymphoma cell lines were grown in RPM I1640 containing 10% FBS. To determine cell proliferation by [3H]thymidine incorporation assay, cells at 5 × 103 per well in a 96-well plate were treated with IL-4 at different concentrations for 3 days before pulsing with 1 mCi of [3H]thymidine for 4 h. Cells were then harvested onto glass filters using a microtiter plate cell harvester (Tomtec, CT), and incorporation of radiolabeled thymidine into DNA was determined by scintillation counting. All assays were done in triplicate, and the mean and standard deviations were calculated. EMSA was performed as described previously by Levy et al. (34.Levy D.E. Kessler D.S. Pine R. Darnell Jr., J.E. Genes Dev. 1989; 3: 1362-1371Crossref PubMed Scopus (364) Google Scholar) and Chin et al. (35.Chin Y.E. Kitagawa M. Su W.C. You Z.H. Iwamoto Y. Fu X.Y. Science. 1996; 272: 719-722Crossref PubMed Scopus (730) Google Scholar) with some modifications. Briefly, whole cell extracts were prepared by lysis of cells in 20 mmHEPES buffer (pH 7.9) with 0.2% Nonidet P-40; 10% glycerol; 400 mm NaCl; 0.1 mm EDTA; 1 mmdithiothreitol; 1 mm sodium orthovanadate; 0.5 mm phenylmethylsulfonyl fluoride; and aprotinin, leupeptin, and pepstatin at 1 mg/ml each. Binding reactions were carried out at room temperature for 30 min in a 15-μl total volume containing 13 mm HEPES (pH 7.9), 185 mm NaCl, 0.15 mm EDTA, 8% glycerol, 1 μg of poly(dI-dC):poly(dI-dC), 1 μg of single-stranded DNA, whole cell extracts (15 μg of proteins), and 5′-end 32P-labeled double stranded oligonucleotide (0.1 ng; ∼1 × 104 cpm). Samples were then fractionated in a 5% polyacrylamide gel. For supershift experiments, antibodies (0.1 μg) were added to the reaction after 20-min incubation and the reaction was further incubated for 20 min at room temperature before electrophoresis. The nucleotide sequences of probes were 5′-GTGCATTTCCCGTAATCTTGTCTACAATTC-3′ for m67-SIE (36.Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Crossref PubMed Scopus (640) Google Scholar) and 5′-TACAACAGCCTGATTTCCCCGAAATGACGGC-3′ for IRF-1-GAS (37.Bovolenta C. Driggers P.H. Marks M.S. Medin J.A. Politis A.D. Vogel S.N. Levy D.E. Sakaguchi K. Appella E. Coligan J.E. et al.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5046-5050Crossref PubMed Scopus (163) Google Scholar), spanning from −137 to −107 of the IRF-1 promoter (38.Harada H. Takahashi E. Itoh S. Harada K. Hori T.A. Taniguchi T. Mol. Cell. Biol. 1994; 14: 1500-1509Crossref PubMed Scopus (287) Google Scholar, 39.Pine R. Nucleic Acids Res. 1997; 25: 4346-4354Crossref PubMed Scopus (142) Google Scholar). The STAT binding core sequence is underlined in each nucleotide. HT29 cells (1 × 107) were transfected with 50 μg of linearized pEFneo, or pEFneo-Stat1Y701F (CYF) by electroporation. Cells were selected with G418 (Life Technologies, Inc.) at 750 μg/ml for 7 days. Individual colonies were cloned, expanded, and analyzed by Western blot analysis and EMSA. To determine the effect of IL-4 on proliferation of HT29, WiDr (colon carcinoma), and BL41 and BL30 (Burkitt's lymphoma) cell lines, cells were treated with IL-4 at different concentrations for 3 days and cell proliferation was determined by [3H]thymidine incorporation assay. As shown in Fig. 1, IL-4 inhibited cell growth of HT29 and WiDr cells but promoted cell growth of BL30 and BL41 cells in a dose-dependent manner. In comparison with nontreated cells, [3H]thymidine incorporation was reduced to approximately 60% in HT29 and WiDr cells with treatment of IL-4 at 1 ng/ml. In contrast, [3H]thymidine incorporation was increased to 3- to 4-fold in BL30 and BL41 cells treated with 10–50 ng/ml of IL-4. Furthermore, the lack of growth inhibition of BL30 and BL40 cells in response to IL-4 was not due to the possibility that these cells were generally refractory to inhibition to cytokines, because IFN-γ was able to inhibit these cells effectively (data not shown). To elucidate whether the cell growth inhibition by IL-4 was caused by an impaired IL-4 signaling pathway in BL30 and BL41 cell lines, Stat6 DNA binding activity was examined by electrophoretic mobility shift assay. Cells were treated with IL-4 at 20 ng/ml for 30 min, and whole cell extracts were prepared. Using a probe containing a STAT binding site within the IRF-1 promoter (40.Sims S.H. Cha Y. Romine M.F. Gao P.Q. Gottlieb K. Deisseroth A.B. Mol. Cell. Biol. 1993; 13: 690-702Crossref PubMed Scopus (249) Google Scholar), Stat6 DNA binding activity was present in BL30, BL41, HT29, and WiDr cell lines (Fig.2 A). The probe was used with over 10-fold excesses. The Stat6 activities were measured qualitatively but not quantitatively. In additional experiments, the Stat6 activity induced in WiDr cells (lane 4) was at the same level of other cell lines (Fig. 2 B). The Stat6 major complexes were confirmed by supershifting experiment using antibodies against Stat6 (Fig. 2 A, lane 9). The band just below the Stat6 complex was partially degraded Stat6 protein that could also be supershifted by anti-Stat6 (data not shown). Thus, these results indicated that the differential effects on cell growth in response to IL-4 in these cells were not caused by differential Stat6 activation. We then asked whether other STATs in addition to Stat6 were differentially activated in WiDr, HT29, BL30, and BL41 cell lines in response to IL-4. Whole cell extracts prepared from these cell lines after stimulation with IL-4 for 30 min were incubated with 32P-labeled m67-SIE oligonucleotide, which contains a high affinity DNA binding site for Stat1 and Stat3 (36.Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Crossref PubMed Scopus (640) Google Scholar, 41.Wagner B.J. Hayes T.E. Hoban C.J. Cochran B.H. EMBO J. 1990; 9: 4477-4484Crossref PubMed Scopus (554) Google Scholar). Surprisingly, we found that IL-4 induced Stat1 DNA binding activities in WiDr and HT29 but not in BL30 and BL41 cells (Fig.3 A). A weak Stat3 activity was also observed. Stat1 and Stat3 complexes were confirmed by supershift analysis using anti-Stat1 or anti-Stat3 antibodies (Fig. 3 A,lanes 9 and 10). Because it was shown that Stat1 has growth inhibitory effect, the finding that IL-4 induced Stat1 activation might provide a mechanism of growth inhibition in HT29 and WiDr cells but not in BL30 and BL41 cells. To examine further whether Stat1 activation by IL-4 is only unique for colon cancer cells or a normal physiological response, mouse splenocytes were isolated and treated with IL-4, and the Stat1 activation was analyzed under the same conditions. Both Stat1 and Stat3 were activated in IL-4-treated splenocytes (Fig. 3 B), indicating that the activation of Stat1 and Stat3 by IL-4 was not an artifact of colon cancer cells. In the above experiments, our results indicated that Stat1 activation correlated with the growth inhibition in response to IL-4. To establish direct evidence demonstrating that Stat1 was involved in IL-4-mediated growth inhibition, we used the Stat1-deficient cell line U3A (42.McKendry R. John J. Flavell D. Muller M. Kerr I.M. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11455-11459Crossref PubMed Scopus (229) Google Scholar) and Stat1α-reconstituted U3A-Stat1α cells (35.Chin Y.E. Kitagawa M. Su W.C. You Z.H. Iwamoto Y. Fu X.Y. Science. 1996; 272: 719-722Crossref PubMed Scopus (730) Google Scholar) for further analyses. In addition, 2fTGH cells (the parental cell line of U3A) were included. 2fTGH, U3A, and U3A-Stat1α cells were treated with IL-4 at different concentrations for 3 days, and cell proliferation was analyzed by measuring [3H]thymidine incorporation. As shown in Fig.4 A, proliferation of 2fTGH was inhibited by IL-4 to 50% with a concentration of 5 ng/ml or greater. In contrast, no significant effect on proliferation of Stat1-deficient U3A cells was observed even at a concentration of 100 ng/ml of IL-4. If the lack of the growth inhibitory effect of IL-4 was caused by Stat1 deficiency, one would expect that the reintroduction of Stat1 to U3A cells should restore the IL-4-mediated growth inhibitory effect. Indeed, the growth inhibitory effect of IL-4 was restored in Stat1α-reconstituted U3A-Stat1α cells (Fig. 4 A), indicating that Stat1 played a critical role in IL-4-mediated growth inhibition. To confirm whether Stat1 was differentially activated in these cells in response to IL-4, 2fTGH, U3A, and U3A-Stat1α cell lines were treated with 20 ng/ml IL-4 for 30 min. As a control, these cells were also treated with 10 ng/ml IFN-γ. Whole cell extracts were prepared, and STAT protein DNA binding activity was analyzed by EMSA. As shown in Fig. 4 B, IL-4 induced Stat1 and Stat3 DNA binding activities in 2fTGH and U3A-Stat1α cells but not in Stat1-deficient U3A cells. The complexes were confirmed by supershifting experiments using antibodies against Stat1 and Stat3 specifically (data not shown). In contrast to differential Stat1/Stat3 DNA binding activities, Stat6 DNA binding activities induced by IL-4 were similar in all three cell lines (data not shown). As expected, IFN-γ induced Stat1 DNA binding activities in 2fTGH and U3A-Stat1α cells but not in U3A cells (Fig.4 B, lanes 7–12) (35.Chin Y.E. Kitagawa M. Su W.C. You Z.H. Iwamoto Y. Fu X.Y. Science. 1996; 272: 719-722Crossref PubMed Scopus (730) Google Scholar). To further study the function of Stat1 in IL-4-mediated cell growth inhibition in colon cancer cells, we established stable HT29 cells expressing a dominant negative Stat1 protein (Y701F), Stat1-CYF. As shown in Fig. 5 A, IL-4-mediated Stat1/Stat3 DNA binding activities were abolished in cells expressing Stat1-CYF. As expected, Stat1 DNA binding activity induced by IFN-γ was also diminished in Stat1-CYF cells (Fig.5 B). We then asked whether Stat1-mediated gene induction is affected by expression of dominant negative Stat1. Cells were treated with or without IL-4 at 20 ng/ml for 1 h. In addition, cells were also treated with IFN-γ as a control. Total RNA was prepared, andIRF-1 gene induction was analyzed by Northern blot analysis. As shown in Fig. 6 A,IRF-1 mRNA was induced by either IFN-γ or IL-4 in HT29 cells within a 1-h stimulation, whereas it was not induced in cells expressing Stat1-CYF. Moreover, IFN-γ is a stronger inducer forIRF-1 mRNA than IL-4 in HT29 cells (Fig. 6 A,lanes 2 and 3). Interestingly, we also found that IFN-γ had a greater growth inhibitory effect on HT29 cells than IL-4 (data not shown). To examine IL-4-mediated cell growth inhibition in cells expressing Stat1-CYF, cells were treated with IL-4 at different concentrations for 3 days and cell proliferation was analyzed by measuring [3H]thymidine incorporation. Consistent with the hypothesis that Stat1 is required for growth inhibition, HT29 cells expressing Stat1-CYF were no longer inhibited by IL-4 treatment even at a concentration as high as to 100 ng/ml, whereas the growth of parental HT29 cells was significantly inhibited by IL-4 (Fig. 6 B). Taken together, these results indicated that Stat1 played a crucial role in cell growth inhibition in response to IL-4. A growth factor or cytokine can have dual effects on cell proliferation: It may stimulate cell growth of one type of cells but inhibit cell growth of another type of cells (43.Sporn M.B. Roberts A.B. Nature. 1988; 332: 217-219Crossref PubMed Scopus (657) Google Scholar). However, the molecular mechanism of such differential effects on cell growth is not well defined. In the case of IL-4, it has been shown that activation of IRS is involved in promoting cell growth; however, little is known on how IL-4 inhibits cell growth. In this study, we have demonstrated that IL-4 induces Stat1 activation that is associated with growth inhibition in colon carcinoma cells. Activation of Stat1/Stat3 by IL-4 was observed not only in colon carcinoma cell lines but also in mouse splenocytes (Fig. 3 B) and other cells such as HeLa cells (data not shown), suggesting that Stat1/Stat3 activation is a common pathway in IL-4 signaling. Therefore, IL-4-mediated Stat1/Stat3 activation may serve as an additional route besides IRS (44.Keegan A.D. Nelms K. Wang L.M. Pierce J.H. Paul W.E. Immunol. Today. 1994; 15: 423-432Abstract Full Text PDF PubMed Scopus (115) Google Scholar) and Stat6 pathways (Fig. 7). It has been shown that Stat1 activation leads to cell growth inhibition (35.Chin Y.E. Kitagawa M. Su W.C. You Z.H. Iwamoto Y. Fu X.Y. Science. 1996; 272: 719-722Crossref PubMed Scopus (730) Google Scholar, 45.Bromberg J.F. Horvath C.M. Wen Z. Schreiber R.D. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7673-7678Crossref PubMed Scopus (444) Google Scholar). The first link between Stat1 activation and the growth inhibitory effect of IL-4 was observed by comparing colon carcinoma cells with lymphoma cells. We have further shown evidence that Stat1 is necessary for IL-4-mediated growth inhibition using Stat1-positive cells versus Stat1-deficient cells. In particular, in Stat1-deficient U3A cells, IL-4 could neither activate Stat1 nor induce cell growth inhibition. However, in U3A-Stat1α and the parental 2fTGH cells, IL-4 induced Stat1 activation and inhibited cell growth. Because Stat6 was activated in all these cell types, we conclude that Stat1, rather than Stat6, is involved in IL-4-mediated growth inhibition. We further confirmed that Stat1 is directly involved in IL-4-mediated cell growth inhibition in HT29 cells by introducing a dominant negative Stat1. Our results suggest that Stat1 alone plays a crucial role in growth inhibition by IL-4. Due to lack of Stat3-deficient cells, the exact function of Stat3 in IL-4-mediated growth inhibition is not yet clear. We have shown that IL-4 induces IRF-1 gene expression and growth inhibition of colon carcinoma cell lines but not in BL30 and BL41 cells (data not shown). It has been shown that IRF-1gene induction depends on Stat1 activation (Fig. 6 A) (46.Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5026) Google Scholar), and the IRF-1 gene may play a significant role in negatively controlling cell growth (47.Taniguchi T. Lamphier M.S. Tanaka N. Biochim. Biophys. Acta. 1997; 1333: M9-M17Crossref PubMed Scopus (136) Google Scholar). However, we do not know whetherIRF-1 gene induction is the only factor involved in observed growth inhibition. It has been shown that Stat1 regulates p21CIP/WAF1, leading to cell growth arrest (35.Chin Y.E. Kitagawa M. Su W.C. You Z.H. Iwamoto Y. Fu X.Y. Science. 1996; 272: 719-722Crossref PubMed Scopus (730) Google Scholar). Our recent data indicate that IL-4 also induces p21CIP/WAF1 in 2fTGH cells but not in Stat1-deficient U3A cells (data not shown). Therefore, several downstream genes such as IRF-1 and CDK inhibitors may jointly play roles in IL-4-mediated cell growth inhibition. Furthermore, we have also examined whether apoptosis is a factor for reduced cell growth of these colon cancer cells. We have examined cells that were treated with IL-4 for 4 days and did not observe an apoptosis increase (data not shown). Thus, we believe that the observed 50% growth inhibition (Fig. 1) after IL-4 treatment was not due to apoptosis of these colon cancer cells. It remains unclear how IL-4 differentially activates Stat1 in different cell lines. It has been shown that HT29 and WiDr cells do not express IL-2R common γ chain (48.Murata T. Noguchi P.D. Puri R.K. J. Biol. Chem. 1995; 270: 30829-30836Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). IL-4 induces phosphorylation of JAK1, JAK2, and Tyk2 but not JAK3, and only JAK2 is associated with the IL-4 receptor in these colon carcinoma cells (48.Murata T. Noguchi P.D. Puri R.K. J. Biol. Chem. 1995; 270: 30829-30836Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 49.Murata T. Noguchi P.D. Puri R.K. J. Immunol. 1996; 156: 2972-2978PubMed Google Scholar). However, IL-4 induces JAK1 and JAK3 that are associated with IL-4 receptor and the common γ chain, respectively, in lymphocytes. Our preliminary results indicated that IRF-1 gene expression was induced by IL-4 in splenocytes from JAK3 (−/−) mice, suggesting that IL-4-mediatedIRF-1 gene induction did not require JAK3. Thus, determination of which Janus kinases activate Stat1 in immune cells and tumor cells may help to understand contrasting effects of IL-4 on cell proliferation. Based on our results presented here and previous findings, we propose a model for IL-4 signaling in regulation of cell growth and the immune system (Fig. 7). Binding of IL-4 to its receptor can trigger at least three signaling pathways mediated through IRS proteins, Stat6, and Stat1/Stat3. Activated IRS-1 and IRS-2 associate with proteins containing Src homology 2 domains, including phosphatidylinositol 3-kinase, growth factor receptor-bound protein 2, and SH2-containing protein tyrosine phosphatase-2, leading to cell proliferation (28.Keegan A.D. Nelms K. White M. Wang L.M. Pierce J.H. Paul W.E. Cell. 1994; 76: 811-820Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 29.Sun X.J. Wang L.M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (765) Google Scholar,31.Shimoda K. van Deursen J. Sangster M.Y. Sarawar S.R. Carson R.T. Tripp R.A. Chu C. Quelle F.W. Nosaka T. Vignali D.A. Doherty P.C. Grosveld G. Paul W.E. Ihle J.N. Nature. 1996; 380: 630-633Crossref PubMed Scopus (1108) Google Scholar, 50.Backer J.M. Schroeder G.G. Kahn C.R. Myers Jr., M.G. Wilden P.A. Cahill D.A. White M.F. J. Biol. Chem. 1992; 267: 1367-1374Abstract Full Text PDF PubMed Google Scholar, 51.Skolnik E.Y. Batzer A. Li N. Lee C.H. Lowenstein E. Mohammadi M. Margolis B. Schlessinger J. Science. 1993; 260: 1953-1955Crossref PubMed Scopus (504) Google Scholar). Results from Stat6-deficient mice (31.Shimoda K. van Deursen J. Sangster M.Y. Sarawar S.R. Carson R.T. Tripp R.A. Chu C. Quelle F.W. Nosaka T. Vignali D.A. Doherty P.C. Grosveld G. Paul W.E. Ihle J.N. Nature. 1996; 380: 630-633Crossref PubMed Scopus (1108) Google Scholar, 32.Takeda K. Tanaka T. Shi W. Matsumoto M. Minami M. Kashiwamura S. Nakanishi K. Yoshida N. Kishimoto T. Akira S. Nature. 1996; 380: 627-630Crossref PubMed Scopus (1269) Google Scholar, 33.Kaplan M.H. Schindler U. Smiley S.T. Grusby M.J. Immunity. 1996; 4: 313-319Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar) show that Stat6 activation is mainly involved in regulation of the immune system such as T helper cell differentiation, IgE class switching, and CD23 induction. Our data indicate that IL-4 induces Stat1 activation and up-regulates additional gene expression (such asIRF-1), resulting in growth inhibition of colon carcinoma cells. This model provides an explanation for the differential effects of IL-4 on cell growth in BL30 and HT29 cell lines. Analysis of IRS-1 phosphorylation indicated that IRS-1 was activated in BL30 cells in response to IL-4 (data not shown). Thus, when a positive signal (IRS-1) is activated and no negative signal (Stat1) is present, IL-4 promotes cell growth. However, once a negative signal is present, which may override the positive signal, IL-4 inhibits cell growth. Taken together, this study provides a molecular basis for the inhibition of cell growth in response to IL-4. We are grateful to J. N. Ihle for providing Stat6 (−/−) mice, R. Pine for anti-IRF-1 antibodies, I. G. Miller for B-lymphoma cells, and Schering-Plow Research Institute (Kenilworth, NJ) for IL-4. We thank R. Pine, H. Asao, M. Kitagawa, W.-C. S. Su, T. Welte, and B. Xie for helpful discussions, and O. Eickelberg, P. Lengyel, U. Schindler, and F. Seebach for critical reading of the manuscript." @default.
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