STAT3 Down-regulates the Expression of Cyclin D during Liver Development
2002; Elsevier BV; Volume: 277; Issue: 39 Linguagem: Inglês
10.1074/jbc.m203184200
ISSN1083-351X
AutoresTakaaki Matsui, Taisei Kinoshita, Toshio Hirano, Takashi Yokota, Atsushi Miyajima,
Tópico(s)Digestive system and related health
ResumoAs the expression of cyclin D1 is induced during liver regeneration and also in hepatic tumor cells, cyclin D1 is likely to play an important role in the proliferation and transformation of hepatocytes. However, the role of cyclin D1 in liver development remains unknown. Here we show that the expression of D-type cyclins including cyclin D1, D2, and D3 is down-regulated along with liver development. In addition, oncostatin M (OSM), an interleukin-6 family cytokine, down-regulated the expression of cyclin D1 and D2 in a primary culture of fetal hepatocytes in which OSM induces hepatic differentiation. Ectopic expression of receptor mutants defective in the activation of either STAT3 or SHP-2/Ras indicated that the down-regulation of D1 and D2 cyclins by OSM was mediated by STAT3 but not by SHP-2/Ras. Consistently, expression of dominant negative STAT3 but not Ras relieved OSM-induced suppression of cyclin D expression. Activation of STAT3 in fetal hepatocytes of transgenic mice expressing the STAT3-estrogen receptor fusion protein by 4-hydroxytamoxifen resulted in the suppression of cyclin D1 and D2 expression. These results indicate that STAT3 activation is necessary and sufficient for down-regulation of D1 and D2 cyclins in fetal hepatocytes. Furthermore, STAT3-C, a constitutively active form of STAT3, suppressed transcription of the cyclin D1 promoter in fetal hepatocytes, whereas it activated the transcription in hepatic tumor cells, huH7 and HepG2. Thus, STAT3-mediated down-regulation of cyclin D expression is rather specific to fetal hepatocytes that are undergoing maturation processes including a reduction of their proliferation potential. As the expression of cyclin D1 is induced during liver regeneration and also in hepatic tumor cells, cyclin D1 is likely to play an important role in the proliferation and transformation of hepatocytes. However, the role of cyclin D1 in liver development remains unknown. Here we show that the expression of D-type cyclins including cyclin D1, D2, and D3 is down-regulated along with liver development. In addition, oncostatin M (OSM), an interleukin-6 family cytokine, down-regulated the expression of cyclin D1 and D2 in a primary culture of fetal hepatocytes in which OSM induces hepatic differentiation. Ectopic expression of receptor mutants defective in the activation of either STAT3 or SHP-2/Ras indicated that the down-regulation of D1 and D2 cyclins by OSM was mediated by STAT3 but not by SHP-2/Ras. Consistently, expression of dominant negative STAT3 but not Ras relieved OSM-induced suppression of cyclin D expression. Activation of STAT3 in fetal hepatocytes of transgenic mice expressing the STAT3-estrogen receptor fusion protein by 4-hydroxytamoxifen resulted in the suppression of cyclin D1 and D2 expression. These results indicate that STAT3 activation is necessary and sufficient for down-regulation of D1 and D2 cyclins in fetal hepatocytes. Furthermore, STAT3-C, a constitutively active form of STAT3, suppressed transcription of the cyclin D1 promoter in fetal hepatocytes, whereas it activated the transcription in hepatic tumor cells, huH7 and HepG2. Thus, STAT3-mediated down-regulation of cyclin D expression is rather specific to fetal hepatocytes that are undergoing maturation processes including a reduction of their proliferation potential. cyclin-dependent kinase interleukin oncostatin M mitogen-activated protein kinase tyrosine aminotransferase 4-hydroxytamoxifen Src homology signal transducers and activators of transcription The cell cycle is tightly regulated by cell cycle regulatory molecules including cyclins, cyclin-dependent kinases (CDKs),1 and CDK inhibitors. D-type cyclins including D1, D2, and D3 cyclins interact with CDKs (cdk4, cdk6, and cdk2) during the mid- to late-G1 phase and activate CDKs, the activity of which is negatively controlled by CDK inhibitors (p15INK4A, 16INK4B, 18INK4C, p19INK4D, p21WAF1, and p27Kip1) (1Sherr C.J. Stem Cells. 1994; 12: 47-55PubMed Google Scholar, 2Sherr C.J. Trends Biochem. Sci. 1995; 20: 187-190Abstract Full Text PDF PubMed Scopus (890) Google Scholar). Among D-type cyclins, D1 cyclin is known to be a key player in the regulation of progression through the G1 phase and of the transition from the G1 to S phase (3Baldin V. Lukas J. Marcote M.J. Pagano M. Draetta G. Genes Dev. 1993; 7: 812-821Crossref PubMed Scopus (1438) Google Scholar, 4Quelle D.E. Ashmun R.A. Shurtleff S.A. Kato J.Y. Bar-Sagi D. Roussel M.F. Sherr C.J. Genes Dev. 1993; 7: 1559-1571Crossref PubMed Scopus (980) Google Scholar). The expression of cyclin D1 is rapidly induced by mitogenic signals in the G1 phase and is also increased by gene amplification and by oncogene products such as Ras and Src in various tumor cells (5Bartkova J. Lukas J. Muller H. Lutzhoft D. Strauss M. Bartek J. Int. J. Cancer. 1994; 57: 353-361Crossref PubMed Scopus (476) Google Scholar, 6Gillett C. Fantl V. Smith R. Fisher C. Bartek J. Dickson C. Barnes D. Peters G. Cancer Res. 1994; 54: 1812-1817PubMed Google Scholar, 7Lee R.J. Albanese C. Stenger R.J. Watanabe G. Inghirami G. Haines III, G.K. Webster M. Muller W.J. Brugge J.S. Davis R.J. Pestell R.G. J. Biol. Chem. 1999; 274: 7341-7350Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 8Tetsu O. McCormick F. Nature. 1999; 398: 422-426Crossref PubMed Scopus (3265) Google Scholar, 9Wulf G.M. Ryo A. Wulf G.G. Lee S.W. Niu T. Petkova V. Lu K.P. EMBO J. 2001; 20: 3459-3472Crossref PubMed Scopus (486) Google Scholar). 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IL-6 family cytokines, which include IL-6, IL-11, BSF3, ciliary neurotrophic factor, leukemia inhibitory factor, and oncostatin M (OSM), play important roles in various cells; e.g. IL-6 induces differentiation of many cell types including myeloid cell lines, M1, Y6, and 1A9-M (13Hirano T. Yasukawa K. Harada H. Taga T. Watanabe Y. Matsuda T. Kashiwamura S. Nakajima K. Koyama K. Iwamatsu A. et al.Nature. 1986; 324: 73-76Crossref PubMed Scopus (1681) Google Scholar, 14Ishibashi T. Kimura H. Uchida T. Kariyone S. Friese P. Burstein S.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5953-5957Crossref PubMed Scopus (341) Google Scholar, 15Miyaura C. Onozaki K. Akiyama Y. Taniyama T. Hirano T. Kishimoto T. Suda T. FEBS Lett. 1988; 234: 17-21Crossref PubMed Scopus (127) Google Scholar, 16Oritani K. Kaisho T. Nakajima K. Hirano T. Blood. 1992; 80: 2298-2305Crossref PubMed Google Scholar, 17Oritani K. Tomiyama Y. Kincade P.W. Aoyama K. Yokota T. Matsumura I. Kanakura Y. Nakajima K. Hirano T. Matsuzawa Y. Blood. 1999; 93: 1346-1354Crossref PubMed Google Scholar). Leukemia inhibitory factor is essential for self-renewal of murine embryonic stem cells (18Smith A.G. Heath J.K. Donaldson D.D. Wong G.G. Moreau J. Stahl M. Rogers D. Nature. 1988; 336: 688-690Crossref PubMed Scopus (1483) Google Scholar, 19Williams R.L. Hilton D.J. Pease S. Willson T.A. Stewart C.L. Gearing D.P. Wagner E.F. Metcalf D. Nicola N.A. Gough N.M. Nature. 1988; 336: 684-687Crossref PubMed Scopus (1543) Google Scholar), and OSM induces differentiation of murine fetal hepatocytes (20Kamiya A. Kinoshita T. Ito Y. Matsui T. Morikawa Y. Senba E. Nakashima K. Taga T. Yoshida K. Kishimoto T. Miyajima A. EMBO J. 1999; 18: 2127-2136Crossref PubMed Scopus (365) Google Scholar) and growth arrest of human A375 melanoma cells (21McDonald V.L. Dick K.O. Malik N. Shoyab M. Growth Factors. 1993; 9: 167-175PubMed Google Scholar). These IL-6 family cytokines manifest their functions through receptors that consist of a ligand-specific subunit(s) and the common signal transducer, gp130 (22Hirano T. Nakajima K. Hibi M. Cytokine Growth Factor Rev. 1997; 8: 241-252Crossref PubMed Scopus (334) Google Scholar). Binding of a ligand to its cognate receptor induces activation of Janus kinases that are bound to the receptor, leading to phosphorylation of the docking sites in the receptor for SHP-2 and STAT3. Following the recruitment of signaling molecules, SHP-2 transmits the signal to the Ras-MAPK cascade and phosphorylated STAT3 forms a dimer and is translocated to the nucleus where it regulates gene expression by binding to its target sequences (22Hirano T. Nakajima K. Hibi M. Cytokine Growth Factor Rev. 1997; 8: 241-252Crossref PubMed Scopus (334) Google Scholar). Studies using receptor mutants and dominant negative mutants of signaling molecules have demonstrated that STAT3 is a key regulator of IL-6 function, such as macrophage differentiation and acute phase responses (22Hirano T. Nakajima K. Hibi M. Cytokine Growth Factor Rev. 1997; 8: 241-252Crossref PubMed Scopus (334) Google Scholar, 23Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). In addition, it was shown that IL-6 induces apoptosis in 1A9-M cells (17Oritani K. Tomiyama Y. Kincade P.W. Aoyama K. Yokota T. Matsumura I. Kanakura Y. Nakajima K. Hirano T. Matsuzawa Y. Blood. 1999; 93: 1346-1354Crossref PubMed Google Scholar), whereas it activates anti-apoptotic signals in IL-3-dependent Ba/F3 cells (24Fukada T. Hibi M. Yamanaka Y. Takahashi-Tezuka M. Fujitani Y. Yamaguchi T. Nakajima K. Hirano T. Immunity. 1996; 5: 449-460Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 25Fukada T. Ohtani T. Yoshida Y. Shirogane T. Nishida K. Nakajima K. Hibi M. Hirano T. EMBO J. 1998; 17: 6670-6677Crossref PubMed Scopus (215) Google Scholar). Thus, the intracellular signals activated by IL-6 lead to various outcomes depending on the target cells. The proliferation and differentiation of hepatocytes are regulated by various external signals, such as hormones, cytokines, extracellular matrix, and cell-cell contacts. Glucocorticoid modulates the proliferation and function of hepatocytes in vivo andin vitro and transforming growth factor-β is a potent inhibitor of hepatic growth (26Nakamura T. Tomita Y. Hirai R. Yamaoka K. Kaji K. Ichihara A. Biochem. Biophys. Res. Commun. 1985; 133: 1042-1050Crossref PubMed Scopus (305) Google Scholar, 27de Juan C. Benito M. Alvarez A. Fabregat I. Exp. Cell Res. 1992; 202: 495-500Crossref PubMed Scopus (67) Google Scholar). IL-6 family cytokines are involved in the development, function, and regeneration of the liver. For example, IL-6-deficient mice exhibit defects in liver regeneration after partial hepatectomy and CCl4 liver injury (28Cressman D.E. Greenbaum L.E. DeAngelis R.A. Ciliberto G. Furth E.E. Poli V. Taub R. Science. 1996; 274: 1379-1383Crossref PubMed Scopus (1325) Google Scholar). While the liver is apparently normal in gp130-deficient mice at the time of birth when mutant mice die, gp130-deficient liver exhibits metabolic defects such as a limited accumulation of glycogen and decreased expression of enzymes for metabolism (20Kamiya A. Kinoshita T. Ito Y. Matsui T. Morikawa Y. Senba E. Nakashima K. Taga T. Yoshida K. Kishimoto T. Miyajima A. EMBO J. 1999; 18: 2127-2136Crossref PubMed Scopus (365) Google Scholar). OSM is a paracrine factor produced by hematopoietic cells in fetal liver and induces differentiation of fetal hepatocytes (20Kamiya A. Kinoshita T. Ito Y. Matsui T. Morikawa Y. Senba E. Nakashima K. Taga T. Yoshida K. Kishimoto T. Miyajima A. EMBO J. 1999; 18: 2127-2136Crossref PubMed Scopus (365) Google Scholar). By using a primary culture of fetal hepatocytes derived from murine embryos at embryonic day 14.5 (E14.5), it was demonstrated that STAT3 and K-Ras mediate the OSM signaling for functional maturation of fetal hepatocytes into neonatal liver cells and morphological changes including the formation of E-cadherin-based adherens junctions, respectively (29Ito Y. Matsui T. Kamiya A. Kinoshita T. Miyajima A. Hepatology. 2000; 32: 1370-1376Crossref PubMed Scopus (35) Google Scholar, 30Matsui T. Kinoshita T. Morikawa Y. Tohya K. Katsuki M. Ito Y. Kamiya A. Miyajima A. EMBO J. 2002; 21: 1021-1030Crossref PubMed Scopus (69) Google Scholar). Differentiated hepatocytes in adult liver are quiescent and exhibit various metabolic functions; however, liver injury induces proliferation of hepatocytes at the expense of metabolic functions. Most hepatocellular carcinomas actively proliferate, but they lose many functions of mature hepatocytes, indicating that they are de-differentiated by transformation. Consistently, fetal hepatocytes proliferate vigorously but lack most mature liver functions. Thus, the proliferation and function of hepatocytes are inversely related. Consistent with these observations, evidence is accumulating that the proliferation of hepatocytes is correlated with the expression of D-type cyclins, particularly cyclin D1. The expression of cyclin D1 is up-regulated at the initial phase of liver regeneration induced by partial hepatectomy and CCl4 (31Albrecht J.H., Hu, M.Y. Cerra F.B. Biochem. Biophys. Res. Commun. 1995; 209: 648-655Crossref PubMed Scopus (80) Google Scholar,32Fausto N. J. Hepatol. 2000; 32: 19-31Abstract Full Text PDF PubMed Google Scholar). In addition, amplification of the cyclin D1 gene is found in 13% of hepatocellular carcinomas (33Joo M. Kang Y.K. Kim M.R. Lee H.K. Jang J.J. Liver. 2001; 21: 89-95Crossref PubMed Scopus (85) Google Scholar). These observations suggest that the regulation of cyclin D1 expression is critical for the proliferation and differentiation of hepatocytes. However, the regulation of cyclin D expression in liver development remains unexplored. In this study, we investigated the mechanism underlying the regulation of D-type cyclins during liver development. The expression of D-type cyclins was down-regulated along with liver development in vivo, and OSM suppressed the expression of cyclin D1 and D2 in fetal hepatocytes in vitro. We provide evidence that this negative regulation is specific to fetal hepatocytes and is mediated by STAT3 but not Ras. Fetal livers were isolated from E14.5 embryos of C57BL/6CrSlc mice or STAT3ER chimeric mice. Isolated livers were dissociated with a collagenase-based dissociation buffer (liver digest medium, Invitrogen) followed by hemolysis with a hypotonic buffer as described previously (20Kamiya A. Kinoshita T. Ito Y. Matsui T. Morikawa Y. Senba E. Nakashima K. Taga T. Yoshida K. Kishimoto T. Miyajima A. EMBO J. 1999; 18: 2127-2136Crossref PubMed Scopus (365) Google Scholar). Dissociated cells were inoculated at a density of 2 × 104 cells/cm2 in 0.1% gelatin-coated dishes (Costar) and then cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen), 2 mml-glutamine, minimum Eagle's medium non-essential amino acid solution (Invitrogen), insulin-transferrin-selenium X (Invitrogen), and 10−7m dexamethasone (Sigma). After 4 h of inoculation, the cells were washed extensively with phosphate-buffered saline to remove hematopoietic cells and cell debris. Culture media were changed every 2 days. Hepatic cell lines, huH7, huH2.2, and HepG2, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Total RNA from E12.5, E14.5, neonatal or adult liver, and cultured cells was extracted with Trizol solution (Invitrogen). A 10-μg aliquot of total RNA was separated on a 1.2% agarose gel containing 2% formaldehyde and transferred onto a positively charged nylon membrane. After UV cross-linking, the membrane was hybridized with digoxigenin-labeling probes (cyclin D1, cyclin D2, cyclin D3, tyrosine aminotransferase (TAT), tryptophan oxygenase, or glyceraldehyde-3-phosphate dehydrogenase). The blot was treated with anti-digoxigenin antibody conjugated with alkaline phosphatase and then developed with CDP-Star (New England BioLabs). The EcoRI-XbaI cDNA fragments encoding G-CSFR-G133, G-CSFR-G133F2, G-CSFR-G133F3, and G-CSFR-G133F2/3 were inserted into the retroviral vector pMXII. The pMX-IRES/GFP (pMIG) retroviral vectors carrying cDNAs for ΔSTAT3, ΔSTAT5, RasV12, and RasN17 were constructed previously (34Chida D. Miura O. Yoshimura A. Miyajima A. Blood. 1999; 93: 1567-1578Crossref PubMed Google Scholar). The expression vector pME18S/STAT3-C was constructed by inserting the NotI-ApaI fragment of STAT3-C (a gift from Dr. J. F. Bromberg) (35Bromberg J.F. Wrzeszczynska M.H. Devgan G. Zhao Y. Pestell R.G. Albanese C. Darnell Jr., J.E. Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2517) Google Scholar). The integrity of these vectors was determined by sequencing and by digestion with restriction enzymes. The retroviral vectors were transfected into the retrovirus-packaging cell line (BOSC23 cells) by lipofection as described previously (29Ito Y. Matsui T. Kamiya A. Kinoshita T. Miyajima A. Hepatology. 2000; 32: 1370-1376Crossref PubMed Scopus (35) Google Scholar, 36Kitamura T. Onishi M. Kinoshita S. Shibuya A. Miyajima A. Nolan G.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9146-9150Crossref PubMed Scopus (224) Google Scholar). After 48 h of culture, the virus particles were enriched by centrifugation at 6,000 × g for 16 h at 4 °C. Pellets of the particles were resuspended in culture medium for fetal hepatocytes, and then the suspension was filtered through a 0.45-μm filter. Fetal hepatocytes were incubated with the viral solution for 2 days, and then the medium was changed to hepatocyte culture medium. Cultured hepatocytes were lysed in cell lysis buffer (25 mm Hepes-KOH, pH 7.5, 150 mmNaCl, 5 mm EDTA, 2 mmNa3VO4, leupeptin, Pefa-block, 1% Triton X-100, and 0.5% Nonidet P-40). Ten micrograms of total cell lysate were used for SDS-PAGE, and proteins were then transferred to an Immobilon-P membrane (Millipore). The membrane was incubated with primary antibodies at room temperature for 2 h and washed with TBS-T (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 0.1% Tween 20). It was then incubated with anti-mouse or rat IgG conjugated with horseradish peroxidase (Amersham Biosciences) at room temperature for 1 h, and the immune complex was visualized with the ECL system (Amersham Biosciences). Fetal hepatocytes were transfected with pME18S/STAT3-C and the reporter carrying two tandem repeats of APRE of α2-macroglobulin using LipofectAMINE plus (Invitrogen). As an internal control, the plasmid pRL containing theRenilla luciferase gene was co-transfected. To examine the effect of STAT3 on the cyclin D1 promoter in hepatic cells, fetal hepatocyte, huH7, HepG2, and huH2.2 were transiently transfected with pME18S/STAT3-C and the luciferase reporter linked to the human cyclin D1 promoter (−1745-Luc) (a gift from Dr. Y. Kanakura). Cells were cultured in medium for each cell type for 24 h and then lysed with passive lysis buffer (Promega). Luciferase activity was measured according to the technical manual for the Dual-Luciferase Reporter Assay System (Promega). To establish STAT3-C stable transfectants of huH7, HepG2, and huH2.2, these cells were transfected with RcCMV-STAT3-C-neo (35Bromberg J.F. Wrzeszczynska M.H. Devgan G. Zhao Y. Pestell R.G. Albanese C. Darnell Jr., J.E. Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2517) Google Scholar), and stable transfectants of STAT3-C were selected by treatment of 800 μg/ml of G418 for 3 weeks. Since STAT3-C was tagged with FLAG, ectopic expression of STAT3-C was confirmed by Western blotting using anti-FLAG antibody (data not shown). As the expression of cyclin D1 is rapidly induced during liver injury, it is believed that D1 cyclin plays an essential role in the proliferation of hepatocytes during liver regeneration (32Fausto N. J. Hepatol. 2000; 32: 19-31Abstract Full Text PDF PubMed Google Scholar). However, neither the expression nor role of D-type cyclins in liver development had been clarified. We therefore first examined the expression of D-type cyclins during liver development by Northern analysis using RNA prepared from livers at different developmental stages (Fig. 1 A). While both D1 and D3 cyclins were highly expressed in fetal liver until E14.5, their expression decreased along with the development of the liver. The expression level of cyclin D2 was rather low in the E12.5 and E14.5 livers and also further decreased during the development. In contrast, the expression of metabolic enzymes, TAT and tryptophan oxygenase, was induced in neonatal and adult liver, respectively (Fig. 1 A). These results indicate that the expression of D-type cyclins is negatively regulated during liver development in vivo. We previously demonstrated that OSM in combination with dexamethasone induces hepatic differentiation in vitro (20Kamiya A. Kinoshita T. Ito Y. Matsui T. Morikawa Y. Senba E. Nakashima K. Taga T. Yoshida K. Kishimoto T. Miyajima A. EMBO J. 1999; 18: 2127-2136Crossref PubMed Scopus (365) Google Scholar). Because OSM induces the expression of differentiation markers of neonatal liver such as TAT (20Kamiya A. Kinoshita T. Ito Y. Matsui T. Morikawa Y. Senba E. Nakashima K. Taga T. Yoshida K. Kishimoto T. Miyajima A. EMBO J. 1999; 18: 2127-2136Crossref PubMed Scopus (365) Google Scholar, 29Ito Y. Matsui T. Kamiya A. Kinoshita T. Miyajima A. Hepatology. 2000; 32: 1370-1376Crossref PubMed Scopus (35) Google Scholar), it is possible that OSM also regulates the expression of D-type cyclins in liver development. To test this possibility, we examined the mRNA expression of D-type cyclins in a primary culture of fetal hepatocytes at E14.5 (Fig.1 B). Consistent with the expression of D-type cyclins in the E14.5 liver in vivo (Fig. 1 A), cyclin D1 was expressed abundantly in culture at day 0, and its expression was maintained in the absence of OSM for 7 days, while it was slightly decreased transiently at day 3 (Fig. 1 B). In contrast, in the presence of OSM the expression of cyclin D1 was almost completely abrogated within 3 days. Although cyclin D2 was not expressed in culture at day 0, its expression increased gradually in the absence of OSM. OSM also suppressed the expression of cyclin D2, indicating the expression to be negatively regulated by the OSM signaling. While cyclin D3 expression was down-regulated during liver developmentin vivo, it was not affected by OSM in vitro, indicating that the OSM signaling is not sufficient to down-regulate cyclin D3 expression. We also investigated the mRNA expression of other cell cycle regulators including cyclin A, CDK 4, c-Myc, p21, and p27 in fetal hepatocytes; however, in no cases was it affected by OSM (data not shown). These results suggest that OSM down-regulates the expression of D1 and D2 cyclins in liver development. OSM functions through the OSM receptor, which consists of the OSM specific subunit (OSMRβ) and gp130, the common signal transducing subunit of the IL-6 family cytokines (22Hirano T. Nakajima K. Hibi M. Cytokine Growth Factor Rev. 1997; 8: 241-252Crossref PubMed Scopus (334) Google Scholar, 37Lindberg R.A. Juan T.S. Welcher A.A. Sun Y. Cupples R. Guthrie B. Fletcher F.A. Mol. Cell. Biol. 1998; 18: 3357-3367Crossref PubMed Scopus (95) Google Scholar, 38Tanaka M. Hara T. Copeland N.G. Gilbert D.J. Jenkins N.A. Miyajima A. Blood. 1999; 93: 804-815Crossref PubMed Google Scholar). There are seven tyrosine residues in the cytoplasmic domain of gp130. Among them, the second (Tyr-757) and third (Tyr-765) tyrosine residues from the transmembrane domain are known to be the docking site for SHP-2 and STAT3, respectively (24Fukada T. Hibi M. Yamanaka Y. Takahashi-Tezuka M. Fujitani Y. Yamaguchi T. Nakajima K. Hirano T. Immunity. 1996; 5: 449-460Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar,39Stahl N. Boulton T.G. Farruggella T., Ip, N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. et al.Science. 1994; 263: 92-95Crossref PubMed Scopus (849) Google Scholar, 40Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell Jr., J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (869) Google Scholar, 41Yamanaka Y. Nakajima K. Fukada T. Hibi M. Hirano T. EMBO J. 1996; 15: 1557-1565Crossref PubMed Scopus (204) Google Scholar). To investigate the signaling pathways responsible for the suppression of cyclin D, we utilized chimeric receptors consisting of the extracellular domain of the G-CSF receptor with various mutants of the intracellular domain of gp130 (Fig.2) (24Fukada T. Hibi M. Yamanaka Y. Takahashi-Tezuka M. Fujitani Y. Yamaguchi T. Nakajima K. Hirano T. Immunity. 1996; 5: 449-460Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 25Fukada T. Ohtani T. Yoshida Y. Shirogane T. Nishida K. Nakajima K. Hibi M. Hirano T. EMBO J. 1998; 17: 6670-6677Crossref PubMed Scopus (215) Google Scholar). Because the chimeric receptor, G133, lacks 144 amino acid residues from the C terminus of gp130, but still has both docking sites for SHP-2 and STAT3, stimulation with G-CSF leads to the activation of SHP-2/Ras and STAT3 signaling pathways in a manner similar to the full-length gp130 (Fig.2 A) (24Fukada T. Hibi M. Yamanaka Y. Takahashi-Tezuka M. Fujitani Y. Yamaguchi T. Nakajima K. Hirano T. Immunity. 1996; 5: 449-460Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 25Fukada T. Ohtani T. Yoshida Y. Shirogane T. Nishida K. Nakajima K. Hibi M. Hirano T. EMBO J. 1998; 17: 6670-6677Crossref PubMed Scopus (215) Google Scholar). As the G133F2 receptor lacks the docking site of SHP-2 due to substitution of the second tyrosine (Tyr) with phenylalanine (Phe), it activates only the STAT3 pathway. Conversely, G133F3, which lacks the docking site of STAT3, activates the SHP-2/Ras signaling alone (Fig. 2 A). The G133F2/3 receptor that lacks both docking sites does not activate either signaling pathway. We constructed pMXII retroviral vectors carrying these chimeric receptors and expressed them in fetal hepatocytes as we previously reported. Fluorescence-activated cell sorter analysis using anti-G-CSFR antibody revealed that these receptors were expressed in 79–84% of fetal hepatocytes (Fig. 2 B). Expression of the G133 receptor slightly reduced the expression of cyclin D1 and D2 in response to G-CSF as compared with the control level (Fig. 2 C,lanes 3 and 4). Importantly, the G133F2 mutant receptor, which only activates the STAT3 signaling pathway, suppressed the expression of cyclin D1 and D2 in response to G-CSF to the same level as OSM (lanes 2 and 5). On the other hand, both G133F3 and G133F2/3, which do not activate STAT3 signaling, did not show such strong effect as G133F2 on cyclin expression (lanes 5, 6, and 7). These results suggest that the docking site of STAT3 in gp130 is essential for the down-regulation of cyclin D expression by OSM. To further confirm the role of STAT3 in the suppression of cyclin D expression, we next expressed STAT mutants using pMIG retroviral vectors. Ectopic expression of the dominant negative form of STAT3 (ΔSTAT3), which lacks the C-terminal transactivation domain, sustained the expression of D1 and D2 cyclins when OSM was present, indicating that ΔSTAT3 inhibits the negative effect of OSM on the expression of D1 and D2 cyclins (Fig.3 A, lane 3). In contrast, ΔSTAT5, which also lacks the C-terminal transactivation domain, had no inhibitory effect on their expression (Fig.3 A, lane 4). Although OSM also activates the SHP-2/Ras pathway, RasN17, a dominant negative form of Ras, did not affect the negative effect of OSM on the cyclin expression. Interestingly, however, activation of the Ras pathway by expression of RasV12, a constitutively active form, augmented the expression of cyclin D1 (Fig. 3 A, lane 5). These results suggest that oncogenic Ras contributes to the induction of cyclin D1 expression, as previously shown by Wulf et al.(9Wulf G.M. Ryo A. Wulf G.G. Lee S.W. Niu T. Petkova V. Lu K.P. EMBO J. 2001; 20: 3459-3472Crossref PubMed Scopus (486) Google Scholar). These results clearly demonstrate that the activation of STAT3 is indispensable for the suppression of the expression of D1 and D2 cyclins by OSM. The results described above clearly indicate that the STAT3 signaling pathway is necessary for the suppression of cyclin D expression during hepatic differentiation induced by OSM. To test whether the activation of STAT3 is sufficient for the down-regulation, we utilized transgenic mice that express STAT3ER, a fusion protein composed of the entire STAT3 with the modified ligand binding domain (G525R) of the estrogen receptor (Fig.4 A) (42Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (704) Google Scholar). STAT3ER dimerizes in response to the synthetic estrogen receptor ligand, 4-hydroxytamoxifen (4HT), leading to the activation of the STAT3 signaling pathway (42Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (704) Google Scholar). Recently, Matsuda et al. generated a chimeric mouse with embryonic stem cells expressing STAT3ER, which had been maintained in the presence of 4HT instead of leukemia inhibitory factor (43Matsuda T. Nakamura T. Nakao K. Arai T. Katsuki M. Heike T. Yokota T. EMBO J. 1999; 18: 4261-4269Crossref PubMed Scopus (733) Google Scholar). As the STAT3ER allele was transmitted to the germ line in this mouse, the offspring of the chimeric mouse expressed STAT3ER ubiquitously in almost all organs including the liver (data not shown). As E14.5 fetal
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