Title: Stat6 and Jak1 Are Common Elements in Platelet-derived Growth Factor and Interleukin-4 Signal Transduction Pathways in NIH 3T3 Fibroblasts
Abstract: Both platelet-derived growth factor (PDGF) and interleukin-4 (IL-4) play major roles in cell proliferation, differentiation, chemotaxis, and other functional responses. Here, we demonstrate that Stat6, previously shown to be activated by only IL-4 and IL-3, becomes activated after PDGF stimulation of NIH 3T3 fibroblasts. PDGF BB, and to a lesser extent PDGF AA, rapidly induced DNA binding activity from NIH 3T3 cell lysates utilizing the immunoglobulin heavy chain germ line ϵ promoter (Iϵ) that specifically binds to Stat6 in an electrophoretic mobility shift assay. DNA binding activity could be detected within 5 min and reached maximum levels at approximately 20 min in parental NIH 3T3 cells. An identical mobility shift and time course of PDGF-mediated Iϵ binding activity was more pronounced in lysates of NIH 3T3 transfectants overexpressing human Stat6 (NIH 3T3-Stat6). The observed radiolabeled Iϵ mobility shift was competed by unlabeled Iϵ as well as by the β-casein gene promoter but not by the interferon-α-stimulated response element or the interferon-γ response region of the guanylate-binding protein gene. A Stat6-specific polyclonal antisera also supershifted the PDGF-induced Iϵ mobility shift. After PDGF BB treatment, a 100-kDa tyrosine phosphorylated species was detected in anti-Stat6 immunoprecipitates. Cycloheximide had little effect on Stat6 tyrosine phosphorylation. In addition to Stat6, Stat5a, and Stat5b, PDGF BB also induced Jak1 tyrosine phosphorylation suggesting a potential pathway for Stat activation. Strikingly, the concurrent addition of IL-4 enhanced PDGF BB-induced Iϵ binding activity, Jak1 tyrosine phosphorylation, and [3H]thymidine incorporation. These results provide evidence that Stat6 and Jak1 are common elements in PDGF and IL-4 signaling pathways and suggest that IL-4 could play a role in potentiating certain known PDGF-induced biological responses. Both platelet-derived growth factor (PDGF) and interleukin-4 (IL-4) play major roles in cell proliferation, differentiation, chemotaxis, and other functional responses. Here, we demonstrate that Stat6, previously shown to be activated by only IL-4 and IL-3, becomes activated after PDGF stimulation of NIH 3T3 fibroblasts. PDGF BB, and to a lesser extent PDGF AA, rapidly induced DNA binding activity from NIH 3T3 cell lysates utilizing the immunoglobulin heavy chain germ line ϵ promoter (Iϵ) that specifically binds to Stat6 in an electrophoretic mobility shift assay. DNA binding activity could be detected within 5 min and reached maximum levels at approximately 20 min in parental NIH 3T3 cells. An identical mobility shift and time course of PDGF-mediated Iϵ binding activity was more pronounced in lysates of NIH 3T3 transfectants overexpressing human Stat6 (NIH 3T3-Stat6). The observed radiolabeled Iϵ mobility shift was competed by unlabeled Iϵ as well as by the β-casein gene promoter but not by the interferon-α-stimulated response element or the interferon-γ response region of the guanylate-binding protein gene. A Stat6-specific polyclonal antisera also supershifted the PDGF-induced Iϵ mobility shift. After PDGF BB treatment, a 100-kDa tyrosine phosphorylated species was detected in anti-Stat6 immunoprecipitates. Cycloheximide had little effect on Stat6 tyrosine phosphorylation. In addition to Stat6, Stat5a, and Stat5b, PDGF BB also induced Jak1 tyrosine phosphorylation suggesting a potential pathway for Stat activation. Strikingly, the concurrent addition of IL-4 enhanced PDGF BB-induced Iϵ binding activity, Jak1 tyrosine phosphorylation, and [3H]thymidine incorporation. These results provide evidence that Stat6 and Jak1 are common elements in PDGF and IL-4 signaling pathways and suggest that IL-4 could play a role in potentiating certain known PDGF-induced biological responses. INTRODUCTIONInflammation, immune responses, and wound repair involve the interplay of multiple growth factors and cytokines. Among these modulators and promoters of growth are platelet-derived growth factor (PDGF) 1The abbreviations used are: PDGFplatelet-derived growth factorILinterleukinEMSAelectrophoretic mobility shift assayGRRinterferon-γ, response regionβCASβ-casein gene promoterISREinterferon-α-stimulated response elementGASinterferon γ-activated sitesIRSinsulin receptor substratemAbmonoclonal antibodyRreceptorPAGEpolyacrylamide gel electrophoresis. and interleukin-4 (IL-4). PDGF is a major mitogen and chemotactic factor for mesenchymal cells such as fibroblasts and smooth muscle cells (1Aaronson S.A. Science. 1991; 254: 1146-1153Crossref PubMed Scopus (1150) Google Scholar, 2Heldin C.-H. Westermark B. Crit. Rev. Oncog. 1991; 2: 109-124PubMed Google Scholar). Although PDGF plays an important role in normal development, accumulating evidence suggests that its abnormal expression also contributes to a variety of diseases including cancer (1Aaronson S.A. Science. 1991; 254: 1146-1153Crossref PubMed Scopus (1150) Google Scholar, 2Heldin C.-H. Westermark B. Crit. Rev. Oncog. 1991; 2: 109-124PubMed Google Scholar). PDGF exists as disulfide-linked homo- or heterodimers composed of two polypeptide chains encoded by distinct genes, designated PDGF A and PDGF B. PDGF BB is the human homologue of the v-sis oncogene product (3Devare S.G. Reddy E.P. Law D.J. Robbins K.C. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 731-735Crossref PubMed Scopus (189) Google Scholar, 4Doolittle R.F. Hunkapiller M.W. Hood L.E. Devare S.G. Robbins K.C. Aaronson S.A. Antoniades H.M. 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Paul W.E. Pierce J.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4032-4036Crossref PubMed Scopus (166) Google Scholar), and Stat6, also termed IL-4 Stat, IL-4 NAF, or STF-IL-4 (28Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (725) Google Scholar, 29Kotanides H. Reich N.C. Science. 1993; 262: 1265-1267Crossref PubMed Scopus (230) Google Scholar, 30Quelle F.W. Shimoda K. Thierfelder W. Fischer C. Kim A. Ruben S.M. Cleveland J.L. Pierce J.H. Keegan A.D. Nelms K. Paul W.E. Ihle J.E. Mol. Cell. Biol. 1995; 15: 3336-3343Crossref PubMed Scopus (301) Google Scholar, 31Schindler C. Kashleva H. Pernis A. Pine R. Rothman P. EMBO J. 1994; 13: 1350-1356Crossref PubMed Scopus (169) Google Scholar). Phosphorylation of specific tyrosine residues within the two GYKXF motifs present in IL-4Rα has been proposed to be crucial for binding to and activating Stat6 (28Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (725) Google Scholar, 32Pernis A. Witthuhn B. Keegan A.D. Nelms K. Garfein E. Ihle J.N. Paul W.E. Pierce J.H. Rothman P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7971-7975Crossref PubMed Scopus (82) Google Scholar).The Stats (signal transducers and activators of transcription) were recently identified as a novel family of transcription factors (33Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4948) Google Scholar, 34Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 3951-3963Abstract Full Text PDF Scopus (818) Google Scholar, 35Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1640) Google Scholar). Stat activation represents a novel signaling pathway that has been correlated with mitogenic and pleiotropic functional responses induced by a variety of growth factors, cytokines and interferons (33Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4948) Google Scholar, 35Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1640) Google Scholar, 36Shuai K. Horvath C.M. Huang L.H.T. Qureshi S. Cowburn D. Darnell Jr., J.E. Cell. 1994; 76: 821-828Abstract Full Text PDF PubMed Scopus (677) Google Scholar). Selective activation of a Stat, presumably through tyrosine and serine (37David M. Petricoin E. Benjamin C. Pine R. Weber M.J. Larner A.C. Science. 1995; 269: 1721-1723Crossref PubMed Scopus (528) Google Scholar, 38Zhang X. Blenis J. Li H.-C. Schindler C. Chen-Kiang S. Science. 1995; 267: 1990-1994Crossref PubMed Scopus (521) Google Scholar) phosphorylation and dimerization, results in its translocation to the nucleus where it activates transcription (36Shuai K. Horvath C.M. Huang L.H.T. Qureshi S. Cowburn D. Darnell Jr., J.E. Cell. 1994; 76: 821-828Abstract Full Text PDF PubMed Scopus (677) Google Scholar). The Stats have been shown to bind with different affinities to a variety of DNA motifs related to interferon γ-activated sites (GAS). Therefore, it is likely that these differences as well as differences in Stat expression determine the transcriptional sequence that is activated and subsequent proliferation or differentiation events that occur in a given cell in response to this activation (39Schindler U. Wu P. Rothe M. Brasseur M. McKnight S.L. Immunity. 1995; 2: 689-697Abstract Full Text PDF PubMed Scopus (231) Google Scholar). The role of Stats in signaling through noncytokine receptors such as the tyrosine kinase receptors remains to be determined. However, it has been demonstrated that epidermal growth factor activates Stats 1, 3, and 5 (40Coffer P.J. Kruijer W. Biochem. Biophys. Res. Commun. 1995; 210: 74-81Crossref PubMed Scopus (70) Google Scholar, 41Quelle F.W. Thierfelder W. Witthuhn B.A. Tang B. Cohen S. Ihle J.N. J. Biol. 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Furthermore, our results provide evidence that IL-4 can act to enhance PDGF-induced Iϵ binding activity, Jak1 activation, and fibroblast proliferation, suggesting that IL-4 might synergize with PDGF to enhance PDGF-mediated biological responses in vivo.DISCUSSIONThe Stats selectively bind to DNA transcriptional elements and activate the transcription of genes that are often associated with differentiation functions and phenotypic changes (33Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4948) Google Scholar, 34Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 3951-3963Abstract Full Text PDF Scopus (818) Google Scholar, 35Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1640) Google Scholar). It has recently been demonstrated that IL-4 activates Stat6 which induces transcription of unrearranged constant regions for IgE (7Coffman R.L. Ohara J. Bond W. Carty J. Zlotnick E. Paul W.E. J. Immunol. 1986; 136: 4538-4541PubMed Google Scholar) and IgG1 (8Vitetta E.S. Ohara J. Myers C. Layton J. Krammer P.H. Paul W.E. J. Exp. Med. 1985; 162: 1726-1732Crossref PubMed Scopus (317) Google Scholar) in B cells, leading to immunoglobulin class switching and production of IgE and IgG1. Prior to this study, Stat6 activation was thought to be limited to IL-4 and IL-3 (30Quelle F.W. Shimoda K. Thierfelder W. Fischer C. Kim A. Ruben S.M. Cleveland J.L. Pierce J.H. Keegan A.D. Nelms K. Paul W.E. Ihle J.E. Mol. Cell. Biol. 1995; 15: 3336-3343Crossref PubMed Scopus (301) Google Scholar) and appeared to be more discriminating in its induction of DNA binding than many other Stat molecules which suggested more restricted functions (48Pernis A. Gupta S. Yopp J. Garfein E. Kashleva H. Schindler C. Rothman P. J. Biol. Chem. 1995; 270: 14517-14522Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar).In the present study, we demonstrated that PDGF is able to activate Stat6 in nonhematopoietic cells. However, we were unable to show a direct association of PDGFRs with Stat6, 4P. Kriebel and W. J. LaRochelle, unpublished observations. suggesting that an intermediate tyrosine kinase activated by the PDGFR may induce tyrosine phosphorylation of Stat6 in NIH 3T3 fibroblasts. Moreover, the PDGFRs do not contain the GYKXF motif thought to be utilized by the IL-4R for Stat6 binding (28Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (725) Google Scholar, 32Pernis A. Witthuhn B. Keegan A.D. Nelms K. Garfein E. Ihle J.N. Paul W.E. Pierce J.H. Rothman P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7971-7975Crossref PubMed Scopus (82) Google Scholar). We were able to demonstrate that the PDGFRs activate Jak1 which is activated by the IL-4R as well. Additionally, we were able to demonstrate a direct association between Jak1 and PDGFRs in Jak1 immunoprecipitates.4 Recent evidence suggests that none of the Jaks appear to be individually required for PDGFR activation of Stat1 or Stat3 (50Vignais M.-L. Sadowski H.B. Watling D. Rogers N.C. Gilman M. Mol. Cell. Biol. 1996; 16: 1759-1769Crossref PubMed Scopus (218) Google Scholar) and that Src kinases are also able to constitutively activate the DNA binding activity of Stat3 (51Yu C.-L. Meyer D.J. Campbell G.S. Larner A.C. Carter-Su C. Schwartz J. Jove R. Science. 1995; 269: 81-83Crossref PubMed Scopus (818) Google Scholar). c-Src has also been identified as a PDGFR substrate (52Kypta R.M. Goldberg Y. Ulug E.T. Courtneidge S.A. Cell. 1990; 62: 481-492Abstract Full Text PDF PubMed Scopus (478) Google Scholar). Whether Jak1, c-Src, or another intracellular kinase (53Danial N.N. Pernis A. Rothman P.B. Science. 1995; 269: 1875-1877Crossref PubMed Scopus (321) Google Scholar) mediate PDGFR activation of Stat6 is currently under investigation.We demonstrated that induction of Stat6 Iϵ binding activity, Jak1 tyrosine phosphorylation, and mitogenesis are enhanced when NIH 3T3 cells are treated with both IL-4 and PDGF. Whether these observed synergies are coincidental or directly related remains to be determined. However, the ability of IL-4 to synergize mitogenically with PDGF AA and BB was comparable, while PDGF BB induced a much more pronounced Stat6 binding activity than PDGF AA. Additionally, IL-4 activates Stat6 in NIH 3T3 fibroblasts but induces only a minor increase in [3H]thymidine uptake. Furthermore, PDGF/IL-4 mitogenic synergy was not significantly increased in NIH 3T3-Stat6 transfectants compared with parental NIH 3T3, although these transfectants possessed a higher background as measured by [3H]thymidine incorporation. 5B. K. R. Patel and W. J. LaRochelle, unpublished observations. Whether this result reflects lack of Stat6 involvement in mitogenesis or saturation of Stat6 signaling pathways involved in mitogenesis remains to be resolved.During the course of these studies two publications have strongly implicated Stat6 in T lymphocyte proliferation (54Kaplan M.H. Schindler U. Smiley S.T. Grusby M.J. Immunity. 1996; 4: 1-20Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar, 55Takeda K. Tanaka T. Wei S. Matsumoto M. Minami M. Kashiwamura S.-I. Nakanish K. Yoshida N. Kishimoto T. Akira S. Nature. 1996; 380: 627-630Crossref PubMed Scopus (1262) Google Scholar) as well as amplification of the proliferative response (54Kaplan M.H. Schindler U. Smiley S.T. Grusby M.J. Immunity. 1996; 4: 1-20Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar), while another concluded that proliferation was only partly affected (56Shimoda 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.A. Doherty P.C. Grosveld G. Paul W.E. Ihle J.N. Nature. 1996; 380: 630-633Crossref PubMed Scopus (1104) Google Scholar). Taken together, these observations suggest that the activation of Stat6 may be necessary but not sufficient for mediating optimal mitogenesis (30Quelle F.W. Shimoda K. Thierfelder W. Fischer C. Kim A. Ruben S.M. Cleveland J.L. Pierce J.H. Keegan A.D. Nelms K. Paul W.E. Ihle J.E. Mol. Cell. Biol. 1995; 15: 3336-3343Crossref PubMed Scopus (301) Google Scholar). Certainly, other signaling molecules (37David M. Petricoin E. Benjamin C. Pine R. Weber M.J. Larner A.C. Science. 1995; 269: 1721-1723Crossref PubMed Scopus (528) Google Scholar) are potential candidates for mediating the observed biological synergy. IL-4 has been demonstrated to induce potent tyrosine phosphorylation of IRS-1 and IRS-2 in hematopoietic cells, leading to the activation of phosphatidylinositol 3-kinase, GRB2, and several other SH2-containing molecules (27Wang L.-M. Keegan A.D. Li W. Lienhard G.E. Pacini S. Gutkind J.S. Myers Jr., M.G. Sun X.-J. White M.F. Aaronson S.A. Paul W.E. Pierce J.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4032-4036Crossref PubMed Scopus (166) Google Scholar, 57Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar). Our previous data also demonstrated that expression of either IRS-1 or IRS-2 in the IL-3-dependent hematopoietic 32D cell line was required for eliciting IL-4-induced mitogenesis (27Wang L.-M. Keegan A.D. Li W. Lienhard G.E. Pacini S. Gutkind J.S. Myers Jr., M.G. Sun X.-J. White M.F. Aaronson S.A. Paul W.E. Pierce J.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4032-4036Crossref PubMed Scopus (166) Google Scholar). However, we observed extremely weak IL-4-mediated tyrosine phosphorylation of IRS-1 and no IRS-2 phosphorylation in NIH 3T3 cells, and PDGF did not stimulate any detectable IRS-1 or IRS-2 phosphorylation. 6L. M. Wang and C. C. Lee, unpublished observations. Thus, our preliminary data suggest that IRS-1 and IRS-2 tyrosine phosphorylation do not contribute to PDGF-induced DNA synthesis or the observed IL-4-mediated synergy in fibroblasts.In addition to Stat6, the PDGF-mediated signal transduction pathway has been linked to Stat1 and 3 activation. Stat1 is also activated by a variety of other cytokines and growth factors including interferon, IL-3, IL-5, IL-6, IL-10, growth hormone, epidermal growth factor, and GM-CSF, suggesting that Stat1 binds multiple transcriptional targets and results in a plethora of effects (reviewed in Refs. 34Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 3951-3963Abstract Full Text PDF Scopus (818) Google Scholar, 57Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar, 58Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Silvennoinen O. Annu. Rev. Immunol. 1995; 13: 369-398Crossref PubMed Scopus (545) Google Scholar). Stat3 or SIF is activated by IL-2, IL-6, interferon-α, and G-CSF leading to transcription of a set of genes that include the serum response element of the c-fos promoter (57Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar, 58Ihle J.N. Witthuhn B.A. Quelle F.W. Yamamoto K. Silvennoinen O. Annu. Rev. Immunol. 1995; 13: 369-398Crossref PubMed Scopus (545) Google Scholar). While Stat1 and -3 also appear to be activated in some breast cancer tissues (59Watson C.J. Miller W.R. Br. J. Cancer. 1995; 71: 840-844Crossref PubMed Scopus (208) Google Scholar), the role of the Stats in carcinogenesis is actively under investigation.In summary, our results provide direct evidence that Stat6 and Jak1 can be activated in nonhematopoietic cells by a tyrosine kinase-containing receptor. While the biological role of Jak1 and Stat6 activation in fibroblasts remains to be determined, IL-4 and PDGF both elevate the expression of extracellular matrix proteins including collagen (13Postlethwaite A.E. Holness M.A. Katai H. Raghow R. J. Clin. Invest. 1992; 90: 1479-1485Crossref PubMed Scopus (424) Google Scholar, 60Narayanan A.S. Page R.C. J. Biol. Chem. 1983; 258: 11694-11699Abstract Full Text PDF PubMed Google Scholar) and promote chemotaxis (14Postlethwaite A.E. Seyer J.M. J. Clin. Invest. 1991; 87: 2147-2152Crossref PubMed Scopus (135) Google Scholar, 61Seppa H. Grotendorst G. Seppa S. Schiffmann E. Martin G.R. J. Cell Biol. 1982; 92: 584-588Crossref PubMed Scopus (555) Google Scholar). Moreover, the levels of IL-4 and PDGF are coincidently increased in the fibrotic disease scleroderma (16Needleman B.W. Wigley F.M. Stair R.W. Arthritis & Rheum. 1991; 35: 67-72Crossref Scopus (291) Google Scholar, 62Gay S. Jones R.E. Guo-qiang H. Gay R.E. J. Invest. Dermatol. 1989; 92: 301-303Abstract Full Text PDF PubMed Google Scholar). Elucidation of the genes that are transcriptionally regulated in this context by Stat6 should reveal potential roles in the proliferation, differentiation, and functional responses in cells of fibroblast origin. INTRODUCTIONInflammation, immune responses, and wound repair involve the interplay of multiple growth factors and cytokines. Among these modulators and promoters of growth are platelet-derived growth factor (PDGF) 1The abbreviations used are: PDGFplatelet-derived growth factorILinterleukinEMSAelectrophoretic mobility shift assayGRRinterferon-γ, response regionβCASβ-casein gene promoterISREinterferon-α-stimulated response elementGASinterferon γ-activated sitesIRSinsulin receptor substratemAbmonoclonal antibodyRreceptorPAGEpolyacrylamide gel electrophoresis. and interleukin-4 (IL-4). PDGF is a major mitogen and chemotactic factor for mesenchymal cells such as fibroblasts and smooth muscle cells (1Aaronson S.A. Science. 1991; 254: 1146-1153Crossref PubMed Scopus (1150) Google Scholar, 2Heldin C.-H. Westermark B. Crit. Rev. Oncog. 1991; 2: 109-124PubMed Google Scholar). Although PDGF plays an important role in normal development, accumulating evidence suggests that its abnormal expression also contributes to a variety of diseases including cancer (1Aaronson S.A. 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Publication Year: 1996
Publication Date: 1996-09-01
Language: en
Type: article
Indexed In: ['crossref']
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Cited By Count: 75
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