The Carboxyl-terminal Region of STAT3 Controls Gene Induction by the Mouse Haptoglobin Promoter
1997; Elsevier BV; Volume: 272; Issue: 23 Linguagem: Inglês
10.1074/jbc.272.23.14571
ISSN1083-351X
Autores Tópico(s)Erythrocyte Function and Pathophysiology
ResumoHaptoglobin (HP) is one of the major acute phase plasma proteins in the mouse, and its synthesis is additively induced by interleukin (IL)-6 and glucocorticoids. STAT3 serves as the mediator of the IL-6 receptor signal and appears to contribute to the transcriptional induction of acute phase protein genes. The carboxyl-terminal region of STAT3, consisting of an acidic domain and containing a serine phosphorylation site, has been proposed to contribute to the induction process. To assess the role of STAT3 in the transcriptional control of the HP promoter, we applied two mutant forms of STAT3: one with a deletion of the carboxyl-terminal 55 amino acid residues, STAT3Δ55C, and the other with a substitution of serine 727 to alanine, STAT3SA. Like the wild-type STAT3, both mutant STAT3 forms are activated by the signal-transducing subunit of the IL-6 receptor, gp130, or by co-transfected IL-3 receptor. Ectopic expression and activation of wild-type STAT3 or STAT3SA in HepG2 hepatoma cells similarly enhance transcription through the IL-6-response element of the HP promoter. This enhancement is specific for STAT3 and cannot be reproduced by STAT1 or STAT5. In contrast, STAT3Δ55C inhibits IL-6-induced transcriptional activation. Interestingly, whereas receptor-activated STAT3 also enhances stimulation of the haptoglobin promoter by dexamethasone through the glucocorticoid receptor, activated STAT3Δ55C reduces the regulation below the level achieved by the glucocorticoid receptor alone. This transdominant action by STAT3Δ55C is dependent on a functional IL-6-responsive element. The data suggest that the carboxyl-terminal domain, but not its serine phosphorylation site of STAT3, is required for transcription as part of the hematopoietin receptor signaling as well as for cooperation with other transcription factors such as the glucocorticoid receptor. Haptoglobin (HP) is one of the major acute phase plasma proteins in the mouse, and its synthesis is additively induced by interleukin (IL)-6 and glucocorticoids. STAT3 serves as the mediator of the IL-6 receptor signal and appears to contribute to the transcriptional induction of acute phase protein genes. The carboxyl-terminal region of STAT3, consisting of an acidic domain and containing a serine phosphorylation site, has been proposed to contribute to the induction process. To assess the role of STAT3 in the transcriptional control of the HP promoter, we applied two mutant forms of STAT3: one with a deletion of the carboxyl-terminal 55 amino acid residues, STAT3Δ55C, and the other with a substitution of serine 727 to alanine, STAT3SA. Like the wild-type STAT3, both mutant STAT3 forms are activated by the signal-transducing subunit of the IL-6 receptor, gp130, or by co-transfected IL-3 receptor. Ectopic expression and activation of wild-type STAT3 or STAT3SA in HepG2 hepatoma cells similarly enhance transcription through the IL-6-response element of the HP promoter. This enhancement is specific for STAT3 and cannot be reproduced by STAT1 or STAT5. In contrast, STAT3Δ55C inhibits IL-6-induced transcriptional activation. Interestingly, whereas receptor-activated STAT3 also enhances stimulation of the haptoglobin promoter by dexamethasone through the glucocorticoid receptor, activated STAT3Δ55C reduces the regulation below the level achieved by the glucocorticoid receptor alone. This transdominant action by STAT3Δ55C is dependent on a functional IL-6-responsive element. The data suggest that the carboxyl-terminal domain, but not its serine phosphorylation site of STAT3, is required for transcription as part of the hematopoietin receptor signaling as well as for cooperation with other transcription factors such as the glucocorticoid receptor. Acute phase response is elicited by the various forms of inflammatory condition, tissue injury, and infection (1Kushner I. Mackiewicz A. Mackiewicz A. Kushner I. Baumann H. Acute Phase Proteins: Molecular Biology, Biochemistry, and Clinical Applications. CRC Press, Boca Raton, FL1993: 4-19Google Scholar). The recruitment of the systemic defense mechanisms is initiated by many cell types at the site of local inflammation and includes macrophages, mast cells, fibroblasts, and endothelial cells and involves the production, release, and distribution of several cytokines. A major component of the systemic acute phase response is the enhanced production of several plasma proteins in the liver that are collectively called acute phase plasma proteins (APPs) 1The abbreviations used are:APPacute phase plasma protein;HPhaptoglobin;ILinterleukin;IL-6RIL-6 receptor;IL-3RIL-3 receptor;bpbase pair;GRglucocorticoid receptor;C/EBPCAAT/enhancer-binding protein;GCSFgranulocyte colony-stimulating factor;GCSFRGCSF receptor;CATchloramphenicol acetyltransferase;EMSAelectrophoretic mobility shift assay;IL-6REinterleukin 6 response element;MAPKmitogen-activated protein kinase;PEA-3polyomavirus enhancer activator-3;SIEsis-inducible element;SIFsis-inducible factor. (1Kushner I. Mackiewicz A. Mackiewicz A. Kushner I. Baumann H. Acute Phase Proteins: Molecular Biology, Biochemistry, and Clinical Applications. CRC Press, Boca Raton, FL1993: 4-19Google Scholar). In most vertebrate species, haptoglobin (HP) is one of the core sets of APPs, and its synthesis is generally increased severalfold. In the mouse, the increase is 30-fold and exceptionally high (2Pajovic S. Jones V.E. Prowse K.R. Berger F.G. Baumann H. J. Biol. Chem. 1994; 269: 2215-2224Google Scholar). HP functions as a hemoglobin-binding protein and is most effective in clearing free hemoglobin from the circulation (3Bowman B.H. Kurosky A. Adv. Hum. Genet. 1982; 12: 189-261Google Scholar). IL-6 is the major inducer of the hepatic production of most APPs, whereas IL-1 and tumor necrosis factor-α act only on a subset of APPs (4Baumann H. Gauldie J. Immunol. Today. 1994; 15: 74-80Google Scholar). Glucocorticoids or dexamethasone enhance the production of many APPs through yet-to-be defined mechanisms. The mouse HP gene is an example of APPs that are strongly induced by both IL-6 and dexamethasone (2Pajovic S. Jones V.E. Prowse K.R. Berger F.G. Baumann H. J. Biol. Chem. 1994; 269: 2215-2224Google Scholar). acute phase plasma protein; haptoglobin; interleukin; IL-6 receptor; IL-3 receptor; base pair; glucocorticoid receptor; CAAT/enhancer-binding protein; granulocyte colony-stimulating factor; GCSF receptor; chloramphenicol acetyltransferase; electrophoretic mobility shift assay; interleukin 6 response element; mitogen-activated protein kinase; polyomavirus enhancer activator-3; sis-inducible element; sis-inducible factor. Signaling by the IL-6 receptor is mediated by the signal-transducing common subunit gp130 through Janus kinases associated with the cytoplasmic domain containing the Box-1 and Box-2 motifs (5Lütticken C. Wegenka U.M. Yuan J. Buschmann J. Schindler C. Ziemiecki A. Harpur A.G. Wilks A.F. Yasukawa K. Taga T. Kishimoto T. Barbieri G. Pellegrini S. Sendtner M. Heinrich P.C. Horn F. Science. 1994; 263: 89-92Google Scholar). Following phosphorylation on the tyrosine residue in the Box-3 motifs of gp130, STAT3 is recruited to the gp130 and is activated by receptor-associated Janus kinases (JAKs) through phosphorylation (6Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell Jr., J.E. Yancopoulous G.D. Science. 1995; 267: 1349-1353Google Scholar). Activated STAT3 dimerizes, translocates to the nucleus, and binds to specific DNA sequences (7Zhong Z. Wen Z. Darnell Jr., J.E. Science. 1994; 264: 95-98Google Scholar). The interaction of STAT3 with specificcis-acting elements has been correlated with enhanced transcription of the responsive target genes including APPs (8Wegenka U.M. Buschmann J. Lutticken C. Heinrich P. Horn F. Mol. Cell. Biol. 1993; 13: 267-288Google Scholar, 9Lai C.-F. Ripperger J. Morella K.K. Wang Y. Gearing D.P. Fey G.H. Baumann H. J. Biol. Chem. 1995; 270: 14847-14850Google Scholar). Recent analyses have suggested that additional modifications of STAT3 protein alter its function as a transcriptional activator. Phosphorylation on serine 727 has been shown to enhance DNA binding affinity and activation of transcription (10Zhang X. Blenis J. Li H.-C. Schindler C. Chen-Kiang S. Science. 1995; 267: 1990-1994Google Scholar, 11Wen Z. Zhong Z. Darnell Jr., J.E. Cell. 1995; 82: 241-250Google Scholar). Deletion of the carboxyl-terminal region, as found in the STAT3β form, rendered the protein inactive as an activator of transcription and as a regulator of differentiation of the myeloid cell line M1 in response to IL-6 (12Shaefer T.S. Sanders L.K. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9097-9101Google Scholar, 13Caldenhoven E. van Dijk T.B. Solari R. Armstrong J. Raaijmakers J.A.M. Lammers J.-W.J. Koenderman L. de Groot R.P. J. Biol. Chem. 1996; 271: 13221-13227Google Scholar, 14Minami M. Inoue M. Wei S. Takeda K. Matsumoto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3963-3966Google Scholar). Although characterization of STAT3 has indicated structural domains relevant for gene induction, the action of critical STAT3 mutant forms, in the context of an IL-6-sensitive gene promoter, has not been described. The promoter of the mouse HP gene has been selected as a model to define the relevance of the carboxyl-terminal peptide and the serine 727 phosphorylation site for regulation by IL-6R and other hematopoietin receptors in hepatic cells. Previously, we have found that the 237-bp HP promoter containscis-acting elements for mediating induction by IL-6 and dexamethasone, and includes two C/EBP binding sites ("A" element at positions −213 to −198 and "C" element at −137 to −123), the IL-6 response element (IL-6RE) ("B" element at positions −165 to −152), and the PEA-3 binding site (at positions −110 to −87) (see Fig. 6 A; Ref. 2Pajovic S. Jones V.E. Prowse K.R. Berger F.G. Baumann H. J. Biol. Chem. 1994; 269: 2215-2224Google Scholar). Although no glucocorticoid receptor (GR) binding site could be identified in the mouse HP promoter, the dexamethasone response appears to be dependent on the presence of the GR and requires the cytokine response region at positions −184 to −106 of the HP promoter (2Pajovic S. Jones V.E. Prowse K.R. Berger F.G. Baumann H. J. Biol. Chem. 1994; 269: 2215-2224Google Scholar). Here we report the specificity with which STAT3 contributes to transcription through the IL-6RE of the HP gene and the relevance of the IL-6RE and STAT3 in mediating induction by IL-6 and dexamethasone. COS-1 and HepG2 cells (15Knowles B.B. Howe C.C. Aden D.P. Science. 1980; 209: 497-499Google Scholar) were maintained in minimal essential medium supplemented with 5 and 10% fetal calf serum, respectively. Mouse hepatoma cells, Hepa-1 (16Bernhard H.P. Darlington G.J. Ruddle F.H. Dev. Biol. 1973; 35: 83-96Google Scholar), were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. Cells were treated with serum-free medium containing 100 ng/ml IL-6, (Genetics Institute), IL-3 (Sandoz), or GCSF (Immunex Corp). Where necessary, 1 μm dexamethasone was used. Expression vector encoding the human GCSFR-gp130 chimeric receptor (GCSFR-gp130(133)) (17Baumann H. Symes A.J. Comeau M.R. Morella K.K. Wang Y. Friend D. Ziegler S.F. Fink J.S. Gearing D.P. Mol. Cell. Biol. 1994; 14: 138-146Google Scholar), IL-3 receptor α (18Hayashida K. Kitamura T. Gorman D.M. Arai K.I. Yokota T. Miyajima A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9655-9659Google Scholar) and β (19Kitamura T. Sato N. Arai K.I. Miyajima A. Cell. 1991; 66: 1165-1174Google Scholar), rat glucocorticoid receptor (pRSVGR) (20Miesfeld R. Rusconi S. Godowski P.J. Maler B.A. Okret S. Wikstrom S.C. Gustafsson J.A. Yamamoto K.R. Cell. 1986; 46: 389-399Google Scholar), and rat STAT3 (9Lai C.-F. Ripperger J. Morella K.K. Wang Y. Gearing D.P. Fey G.H. Baumann H. J. Biol. Chem. 1995; 270: 14847-14850Google Scholar) have been described. Plasmid pmHP(237)-CAT contains the promoter of the Mus domesticus HP gene (from positions −237 to +20 relative to the transcription start site) linked to the CAT gene in pCT (2Pajovic S. Jones V.E. Prowse K.R. Berger F.G. Baumann H. J. Biol. Chem. 1994; 269: 2215-2224Google Scholar). In plasmid pmHP(237)MB-CAT, the IL-6RE sequence, the B element (−163TTACTGGAA−155) was mutated to TAAGTGGCA using the mutagenesis kit (CLONTECH). Plasmid mHP(IL-6RE)-CAT contained four tandem copies of the IL-6RE region of HP promoter (−172 to −151; see Fig. 6 A) in the BglII site of pCAT (Promega). The serine 727 to alanine mutation in STAT3SA was introduced by replacing codon 727, UCC, with GCG. STAT3Δ55C was constructed by adding two stop codons after amino acid residue 715. All of the mutations were verified by DNA sequence analysis, and the cDNA forms were cloned into the human cytomegalovirus-based vector pDC (21Mosley B. Beckmann M.P. March C.J. Idezerda R.L. Gimpel S.S. Vande B.T. Friend D. Alpert A. Anderson D. Jackson J. Wignall J.M. Smith C. Gallis B. Sims J.E. Urdal D. Widmer M.B. Cosman D. Park L.S. Cell. 1989; 59: 335-348Google Scholar). COS-1 cells were transfected by the DEAE-dextran method (22Lopata M.A. Cleveland D.W. Sollner-Webb H. Nucleic Acids Res. 1984; 12: 5707-5717Google Scholar). After a 36-h recovery, the cells were maintained in minimal essential medium without serum for 16 h. After treatment with cytokines for 15 min, whole cell extracts were prepared (23Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Google Scholar). HepG2 cells were transfected by the modified calcium phosphate method (24O'Mahoney J.V. Adams T.E. DNA Cell Biol. 1994; 13: 1227-1232Google Scholar). This method yielded 2.5 ± 5.6% (n = 12) of transfected cells in the culture as judged from in situ staining for the expression of the transfection marker β-galactosidase. The standard plasmid mixtures contained a final concentration of 20–25 μg/ml and included 2 μg of pIE-MUP as an internal marker (25Prowse K.R. Baumann H. Mol. Cell. Biol. 1988; 8: 42-51Google Scholar). After a 24-h recovery, cells were treated with the cytokines for 24 h, and serial dilutions of cell extracts were used to measure CAT activity within the linear range of the assay. Conversion of chloramphenicol to acetylated products was determined. The values were normalized to the expression of the co-transfected major urinary protein (25Prowse K.R. Baumann H. Mol. Cell. Biol. 1988; 8: 42-51Google Scholar) and calculated relative to the untreated control (defined as 1.0). Mean ± S.D. of at least three independently performed experimental series are reproduced in the figures. An aliquot of the whole cell extracts containing 5 μg of protein was used for EMSA. The double-stranded, high affinity sis-inducible element SIEm67 (23Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Google Scholar) served as binding substrate for STAT1 and STAT3, and TB2 served as substrate for STAT5 (26Morella K.K. Lai C. Kumaki S. Kumaki N. Wang Y. Bluman E.M. Witthuhn B.S. Ihle J.N. Giri J. Gearing D.P. Cosman D. Ziegler S.F. Tweardy D.J. Campos S.P. Baumann H. J. Biol. Chem. 1995; 270: 8298-8310Google Scholar). From the same whole cell extracts, aliquots containing 30 μg were also separated on 6 or 7.5% SDS-polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose membrane and were reacted with anti-STAT1 (Transduction Laboratories), anti-STAT3 (C-20, Santa Cruz Biotechnology), monoclonal STAT3 antibody (Transduction Laboratories), STAT5 antibody (Santa Cruz Biotechnology), or phosphotyrosine 705-specific anti-STAT3 (New England Biolabs). Similarly, total cell lysates (30 μg) were analyzed with anti-human GCSFR (Springville Laboratories, RPCI). Immune complexes were visualized by enhanced chemiluminescence reaction (Amersham Corp.). Whole cell extracts from COS-1 cells transiently expressing STAT3 or STAT3SA served as substrates for the MAPK reaction. Twenty μg of whole cell protein in a total of 100 μl of reaction buffer (20 mm Tris-HCl, pH 7.4, 20 mm MgCl2, 20 mm MnCl2, 150 mm NaCl, 8% glycerol, and 50 mm NaF) were incubated with 10 μCi of [γ-32P]ATP and 20 μl of activated MAPK-agarose conjugate (Upstate Biotechnology, Inc.) for 30 min at 37 °C. Reactions were stopped by removing the MAPK beads by brief centrifugation. STAT3 was immunoprecipitated from the supernatant fraction with STAT3 antibody (C-20, Santa Cruz Biotechnology) and then subjected to 7.5% SDS-polyacrylamide gel electrophoresis for autoradiography and Western analysis. To characterize the role of STAT3 in the regulation of mouse HP gene elements, we constructed two key mutant forms of STAT3: STAT3SA with a mutation of the serine 725 to alanine at its carboxyl-terminal kinase substrate site, and STAT3Δ55C with a truncation of the acidic carboxyl-terminal fragment as observed in STAT3β (12Shaefer T.S. Sanders L.K. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9097-9101Google Scholar, 13Caldenhoven E. van Dijk T.B. Solari R. Armstrong J. Raaijmakers J.A.M. Lammers J.-W.J. Koenderman L. de Groot R.P. J. Biol. Chem. 1996; 271: 13221-13227Google Scholar) but lacking the latter's additional 7 residues encoded by the extra exon sequence. We verified the expression of the STAT proteins by transiently transfecting COS-1 cells (Fig.1 A). Western blot analysis (bottom part, mAb) demonstrated that each STAT form was produced in approximately equal amounts, yielding levels that exceeded severalfold that of the endogenous STAT3 (Fig. 1 A,Ctrl). The major portion of the immunodetectable STAT proteins migrated with the expected sizes. However, a minor fraction of STAT3wt and STAT3SA migrated with an approximately 5000 smaller molecular size. These forms probably represent proteolytically modified STAT3 proteins (see similar forms detected in HepG2 cell extracts; Fig. 2 A).Figure 2Effect of STAT proteins on gene induction through the HP promoter. A, HepG2 cells were transfected with a combination of expression vectors for GCSFR-gp130(133) (5 μg/ml), the indicated STAT proteins (10 μg/ml), and empty expression vector to a total of 20 μg/ml. In this experiment, rat STAT1β instead of STAT1α was used for easier detection of the transfected protein in the presence of the relatively abundant endogenous STAT1. After 1 day of recovery, cells were divided in 6-well plates. The cells were treated with IL-6 (Vector Control) or GCSF for 15 min. One part of the collected cells was dissolved directly in SDS sample buffer for Western blot analysis of GCSFR-gp130 (only cytokine-treated cells), and the remaining part was used for preparing whole cell extract. Aliquots containing 5 and 30 μg of protein were used for EMSA and Western analysis, respectively. For EMSA, SIE (all lanesexcept last lane) or TB2 (last lane with STAT5B) served as binding substrate. For STAT protein Western analyses, extracts were electrophoresed in quadruplicate, and proteins on the nitrocellulose membrane were reacted with the indicated antibodies.B, for detecting CAT gene regulation, HepG2 cells were transfected with a similar combination of plasmids as in A, consisting of the reporter mHP(237)-CAT (15 μg/ml) and expression vector for GCSFR-gp130(133) (1 μg/ml) and indicated STAT proteins (5 μg/ml). In this case, rat STAT1α was used. After 1 day of recovery, the cell cultures were divided and treated for 24 h with the indicated cytokine or dexamethasone. The thin layer pattern of one reaction is shown. The -fold stimulation measured in this experimental series is indicated above the autoradiogram. C, HepG2 cells were transfected with a 15 μg/ml of mHP(237)-CAT (upper panel) or mHP(IL-6RE)-CAT (lower panel) and the increasing amounts of expression vector for STAT3, STAT3SA, or STAT3Δ55C as marked. The DNA amount in each transfection was adjusted to a total of 25 μg/ml with pUC13 DNA. Cells were treated with IL-6 for 24 h, and CAT activities were determined. All values are expressed relative to control culture without IL-6 treatment and STAT3 transfection.View Large Image Figure ViewerDownload (PPT) The activation of the transfected STAT proteins by gp130 signals, was demonstrated by the co-transfected chimeric receptor, GCSFR-gp130(133). This receptor consists of the extracellular domain of the human GCSFR fused to the transmembrane and the cytoplasmic domain of 133 residues of human gp130. The chimeric receptor is considered to undergo a GCSF-dependent dimerization that also includes the cytoplasmic gp130 domains. An analogous dimerization process has been predicted for the bona fide ligand-activated IL-6R (27Ward L.D. Howlett G.J. Discolo G. Yasukawa K. Hammacher A. Moritz R.L. Simpson R.J. J. Biol. Chem. 1994; 269: 23286-23289Google Scholar, 28Paonessa G. Graziani R. De Serio A. Savino R. Ciapponi L. Lahm A. Salvati A.L. Toniatti C. Ciliberto G. EMBO J. 1995; 14: 1942-1951Google Scholar). Expression of the chimeric receptor provides the advantage that signaling function of gp130 can be analyzed specifically in transfected cells and independently of the endogenous gp130 (17Baumann H. Symes A.J. Comeau M.R. Morella K.K. Wang Y. Friend D. Ziegler S.F. Fink J.S. Gearing D.P. Mol. Cell. Biol. 1994; 14: 138-146Google Scholar, 29Lai C.-F. Ripperger J. Morella K.K. Wang Y. Gearing D.P. Horseman N.D. Campos S.P. Fey G.H. Baumann H. J. Biol. Chem. 1995; 270: 23254-23257Google Scholar). Extracts from cells treated with or without GCSF indicated that each STAT form was activated by the receptor and yielded comparable SIE-binding activity (Fig. 1 A, upper panel). As shown previously (9Lai C.-F. Ripperger J. Morella K.K. Wang Y. Gearing D.P. Fey G.H. Baumann H. J. Biol. Chem. 1995; 270: 14847-14850Google Scholar) in COS-1 cells not transfected with any STAT expression vectors, GCSFR-gp130 activated detectable SIE-binding activity of endogenous STAT proteins consisting primarily of STAT1. In the presence of overproduced STAT3, however, the predicted SIE binding activities characteristic for STAT3 homodimers (SIF-A complexes, Ref. 23Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Google Scholar) were obtained. Only a minor fraction of heterodimers between STAT3 and endogenous STAT1 (SIF-B complex) appeared as an additional band below the STAT3 homodimer complex. As predicted, the SIE complexes formed with wild-type STAT3 and STAT3SA were recognized by the C-20 anti-STAT3 antibody, yielding a supershifted EMSA pattern (Fig. 1 A,upper panel, lanes 7 and 8). The STAT3Δ55C-containing complex was not recognized by C-20 antibody (lane 9) but showed a characteristic slower mobility probably due to the loss of the acidic C-terminal fragment that bears the epitope for the C-20 antibody (see smaller size on Western blot in Fig. 1 A). Only STAT3Δ55C showed minor SIE-binding activity in untreated cells, which is in agreement with previous data (12Shaefer T.S. Sanders L.K. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9097-9101Google Scholar, 13Caldenhoven E. van Dijk T.B. Solari R. Armstrong J. Raaijmakers J.A.M. Lammers J.-W.J. Koenderman L. de Groot R.P. J. Biol. Chem. 1996; 271: 13221-13227Google Scholar). The reaction with anti-phosphotyrosine STAT3 (Fig. 1 A,lower panel) illustrated that the phosphorylation at tyrosine 705 in each STAT3 form correlated with the DNA binding activity of the protein. To show in our experimental system that overexpressed STAT3Δ55C forms heterodimers with STAT1 or STAT3 (see also Ref. 13Caldenhoven E. van Dijk T.B. Solari R. Armstrong J. Raaijmakers J.A.M. Lammers J.-W.J. Koenderman L. de Groot R.P. J. Biol. Chem. 1996; 271: 13221-13227Google Scholar) we transfected varying amounts of expression vector for STAT3Δ55C, STAT3, and STAT1 into COS-1 cells. DNA binding activity of the STAT proteins was activated through GCSFR-gp130 and determined by EMSA. Dose-dependent formation of heterodimers between STAT3Δ55C and STAT1 (Fig. 1 B) or STAT3 (Fig.1 C) was clearly evident. Moreover, a shift toward predominant STAT3Δ55C homodimers at high STAT3Δ55C expression level was achieved. Analyses of subcellular fractions indicated that activated STAT3Δ55C was translocated to the nucleus, and this translocation was indistinguishable from the wild-type STAT3 or STAT3SA (data not shown). Observing that STAT3SA, contrary to previous predictions (10Zhang X. Blenis J. Li H.-C. Schindler C. Chen-Kiang S. Science. 1995; 267: 1990-1994Google Scholar), was expressed at the same level as wild-type STAT3 and bound to SIE with apparently comparable affinity, we determined whether the point mutation had indeed modified the substrate behavior of STAT3 for MAPK. COS cell extracts containing overexpressed STAT3 or STAT3SA were reacted in vitro with MAPK and [32P]ATP. Immunoprecipitation and gel electrophoresis showed that wild-type STAT3, but not STAT3SA, was prominently labeled (data not shown), indicating that the serine to alanine mutation had effectively removed a principle phosphorylation site in STAT3. Taken together, these results confirmed the expected physicochemical properties of the two mutant STAT3 forms and also indicated that these proteins were essentially indistinguishable in their activation by the gp130 signal, complex formation, and binding to DNA substrates. Hence, these mutant forms fulfilled the preconditions for their application in probing STAT3 function as an inducer of transcription through the HP promoter. To identify the role of STAT proteins in the IL-6 regulation of the mouse HP promoter, we needed to test the IL-6-responsive mHP(237)-CAT construct in hepatic cells, because COS-1 cells failed to reproduce a liver-like regulation of that construct (data not shown). Since no mouse hepatoma cells with IL-6-regulatable endogenous HP gene expression have been identified, we selected HepG2 cells as the experimental system for their prominent IL-6 responsiveness (2Pajovic S. Jones V.E. Prowse K.R. Berger F.G. Baumann H. J. Biol. Chem. 1994; 269: 2215-2224Google Scholar, 9Lai C.-F. Ripperger J. Morella K.K. Wang Y. Gearing D.P. Fey G.H. Baumann H. J. Biol. Chem. 1995; 270: 14847-14850Google Scholar, 17Baumann H. Symes A.J. Comeau M.R. Morella K.K. Wang Y. Friend D. Ziegler S.F. Fink J.S. Gearing D.P. Mol. Cell. Biol. 1994; 14: 138-146Google Scholar). These cells were transfected with pHP(237)-CAT alone or together with expression vectors for the various STAT isoforms to demonstrate the specificity of these proteins to modify signaling by endogenous IL-6R and co-transfected GCSFR-gp130. Although we applied a transient transfection protocol that had been optimized for hepatoma cells (24O'Mahoney J.V. Adams T.E. DNA Cell Biol. 1994; 13: 1227-1232Google Scholar), we could detect only a relative low percentage of transfected HepG2 cells in the culture (see "Experimental Procedures") and lower protein expression than in COS-1 cells (data not shown). Nonetheless, the transfection efficiency was sufficient to verify the presence of the overexpressed STAT proteins by Western blotting and activation of their DNA binding activity through receptor signals by EMSA (Fig.2 A). By comparing the Western blot and EMSA signals for STAT3 in STAT3-transfected cells with that in control or non-STAT3 transfected cells, and by taking into consideration the low transfection efficiency, we estimated that the amount of ectopically expressed STAT3 proteins exceeded the level of endogenous STAT3 by at least 10-fold. Although the low level production of GCSFR-gp130 rendered detection of the receptor protein in the transfected culture difficult, we could determine by Western blot analysis that the expression of GCSFR-gp130 was relatively comparable among separately transfected cell cultures (Fig. 2 A, bottom part). The quantitation of CAT activities (Fig. 2 B) revealed that the expression of the mHP(237)-CAT construct was increased 2–5-fold by the action of GCSFR-gp130 and endogenous IL-6R. The cytokine (e.g. IL-6) response was further enhanced by co-treatment with dexamethasone. Co-expression of STAT1α or STAT5B was ineffective in modulating the regulation. STAT3, as well as STAT3SA, however, enhanced induction in GCSF and IL-6-treated cells by 2–3-fold. In contrast, STAT3Δ55C abrogated induction by the receptors, yet did not significantly affect basal activity of the pHP237-CAT construct. Since overexpressed receptors containing the extracellular domain of GCSFR were noted previously to reduce by unknown mechanisms the signaling by IL-6R in HepG2 cells (17Baumann H. Symes A.J. Comeau M.R. Morella K.K. Wang Y. Friend D. Ziegler S.F. Fink J.S. Gearing D.P. Mol. Cell. Biol. 1994; 14: 138-146Google Scholar), subsequent analyses of IL-6 action were determined only in cells without co-transfected GCSFR-gp130. The influence of the STAT3 forms on mHP-CAT gene regulation through the endogenous IL-6R (Fig. 2 C, upper panel) or GCSFR-gp130 (data not shown) was dependent on the dose of co-transfected STAT3 expression vectors. Generally, a maximal enhancement of the cytokine signal by wild-type STAT3 and STAT3SA was attained with 1–5 μg/ml transfected expression vector. With increasing amounts of STAT3Δ55C expression vector, the cytokine receptor-mediated induction of the reporter gene declined, reaching basal level in some instances. The B element located at positions −165 to −152) of the HP promoter is conserved among HP genes from rodents and primates and has been identified as an IL-6-responsive element (IL-6RE) (33Baumann H. Morella K.K. Jahreis G.P. Marinkovic S. Mol. Cell. Biol. 1990; 10: 5967-5976Google Scholar). Since, for unknown reasons, a binding of the isolated HP IL-6RE sequence to STAT3 could not be detected by the standard EMSA conditions as used in Fig. 1, we verified by functional cell assay that this element was the target of the regulation by STAT3. Four
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