Activation of the STAT Pathway by Angiotensin II in T3CHO/AT1A Cells
1995; Elsevier BV; Volume: 270; Issue: 32 Linguagem: Inglês
10.1074/jbc.270.32.19059
ISSN1083-351X
AutoresG. Jayarama Bhat, Thomas Thekkumkara, Walter G. Thomas, Kathleen Conrad, Kenneth M. Baker,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoWe recently reported that angiotensin II (AII), acting through the STAT (Signal Transducers and Activators of Transcription) pathway, stimulated a delayed SIF (sis-inducing factor)-like DNA binding activity (maximal at 2-3 h) (Bhat, G. J., Thekkumkara, T. J., Thomas, W. G., Conrad, K. M., and Baker, K. M.(1994) J. Biol. Chem. 269, 31443-31449). Using a cell line transfected with the AT1A receptor (T3CHO/AT1A), we further characterized the AII-induced SIF response and explored the possible reasons for the delay in stimulated SIF activity. In cells transfected with a chloramphenicol acetyltransferase reporter plasmid, under the control of a SIE (sis-inducing element), AII markedly stimulated chloramphenicol acetyltransferase activity. The delayed SIF activation by AII was not due to a requirement for the release of other SIF inducing factors into the medium and contrasts with the rapid (5 min) induction elicited by the cytokine, interleukin-6 (IL-6). Interestingly, both agents stimulated tyrosine phosphorylation of Stat92 and predominantly the formation of SIF complex A. We tested the hypothesis that AII initially activated an inhibitory pathway, which was responsible for delaying the maximal SIF stimulation until 2 h. Pretreatment of cells for 15 min with AII resulted in significant inhibition of the IL-6-induced nuclear SIF response (10 min) and Stat92 tyrosine phosphorylation, which was blocked by EXP3174, an AT1 receptor antagonist. This inhibition was transient with return of the IL-6-induced SIF response at 2 h, suggesting that the delayed maximal activation of SIF by AII occurs following an initial transient inhibitory phase. Pretreatment of cells with phorbol 12-myristate 13-acetate for 15 min, to activate protein kinase C, resulted in inhibition of the IL-6-induced SIF response (10 min). However, down-regulation of protein kinase C activity prevented phorbol 12-myristate 13-acetate, but not AII mediated inhibition of the IL-6-induced SIF response. Although the mechanism is not clear, the results presented in this paper raise the interesting possibility that the activation of SIF/Stat92 by AII is characterized by an initial inhibitory phase, followed by the induction process. The observation that AII and IL-6 utilize similar components of the STAT pathway and that AII can cross-talk with IL-6 signaling through inhibition of IL-6-induced SIF/Stat92, implies a modulatory role for AII in cellular responses to cytokines. We recently reported that angiotensin II (AII), acting through the STAT (Signal Transducers and Activators of Transcription) pathway, stimulated a delayed SIF (sis-inducing factor)-like DNA binding activity (maximal at 2-3 h) (Bhat, G. J., Thekkumkara, T. J., Thomas, W. G., Conrad, K. M., and Baker, K. M.(1994) J. Biol. Chem. 269, 31443-31449). Using a cell line transfected with the AT1A receptor (T3CHO/AT1A), we further characterized the AII-induced SIF response and explored the possible reasons for the delay in stimulated SIF activity. In cells transfected with a chloramphenicol acetyltransferase reporter plasmid, under the control of a SIE (sis-inducing element), AII markedly stimulated chloramphenicol acetyltransferase activity. The delayed SIF activation by AII was not due to a requirement for the release of other SIF inducing factors into the medium and contrasts with the rapid (5 min) induction elicited by the cytokine, interleukin-6 (IL-6). Interestingly, both agents stimulated tyrosine phosphorylation of Stat92 and predominantly the formation of SIF complex A. We tested the hypothesis that AII initially activated an inhibitory pathway, which was responsible for delaying the maximal SIF stimulation until 2 h. Pretreatment of cells for 15 min with AII resulted in significant inhibition of the IL-6-induced nuclear SIF response (10 min) and Stat92 tyrosine phosphorylation, which was blocked by EXP3174, an AT1 receptor antagonist. This inhibition was transient with return of the IL-6-induced SIF response at 2 h, suggesting that the delayed maximal activation of SIF by AII occurs following an initial transient inhibitory phase. Pretreatment of cells with phorbol 12-myristate 13-acetate for 15 min, to activate protein kinase C, resulted in inhibition of the IL-6-induced SIF response (10 min). However, down-regulation of protein kinase C activity prevented phorbol 12-myristate 13-acetate, but not AII mediated inhibition of the IL-6-induced SIF response. Although the mechanism is not clear, the results presented in this paper raise the interesting possibility that the activation of SIF/Stat92 by AII is characterized by an initial inhibitory phase, followed by the induction process. The observation that AII and IL-6 utilize similar components of the STAT pathway and that AII can cross-talk with IL-6 signaling through inhibition of IL-6-induced SIF/Stat92, implies a modulatory role for AII in cellular responses to cytokines. Angiotensin II (AII) 1The abbreviations used are: AIIangiotensin IIG-proteinguanyl nucleotide-binding proteinAT1Aangiotensin II receptor subtype 1ACHO-K1Chinese hamster ovary cellsPDGFplatelet-derived growth factorIFN-γinterferon-γIL-6interleukin-6SIEsis-inducing elementSIFsis-inducing factorJAKJanus kinaseTGF-βtransforming growth factor-βSTATsignal transducers and activators of transcriptionPMAphorbol 12-myristate 13-acetatePKCprotein kinase CEXPEXP3174CATchloramphenicol acetyltransferase. 1The abbreviations used are: AIIangiotensin IIG-proteinguanyl nucleotide-binding proteinAT1Aangiotensin II receptor subtype 1ACHO-K1Chinese hamster ovary cellsPDGFplatelet-derived growth factorIFN-γinterferon-γIL-6interleukin-6SIEsis-inducing elementSIFsis-inducing factorJAKJanus kinaseTGF-βtransforming growth factor-βSTATsignal transducers and activators of transcriptionPMAphorbol 12-myristate 13-acetatePKCprotein kinase CEXPEXP3174CATchloramphenicol acetyltransferase. stimulates a variety of physiological responses related to the regulation of blood pressure, salt, and fluid homeostasis(1Raizada M.K. Phillips M.I. Summers C. Cellular and Molecular Biology of Renin-Angiotensin System. CRC Press, Inc., Boca Raton, FL1993Google Scholar, 2Lindpainter K. Ganten D. The Cardiac Renin-Angiotensin System. Fatura Publishing Co., Inc., Armonk, NY1994Google Scholar). In addition, AII promotes growth responses in many cells, including cardiomyocytes, cardiac fibroblasts, and vascular smooth muscle cells(3Aceto J.F. Baker K.M. Am. J. Physiol. 1990; 258: H806-H813PubMed Google Scholar, 4Sadoshima J. Izumo S. Circ. Res. 1993; 73: 413-423Crossref PubMed Scopus (1285) Google Scholar, 5Schorb W. Booz G.W. Dostal D.E. Conrad K.M. Chang K.C. Baker K.M. Circ. Res. 1993; 72: 1245-1254Crossref PubMed Scopus (363) Google Scholar, 6Berk B.C. Vekshtein V. Gordon H.M. Tsuda T. Hypertension. 1989; 13: 305-314Crossref PubMed Scopus (516) Google Scholar, 7Weber H. Taylor D.S. Molloy C.J. J. Clin. Invest. 1994; 93: 788-798Crossref PubMed Scopus (145) Google Scholar, 8Dostal D.E. Baker K.M. Trends Cardiovasc. Med. 1993; 3: 67-74Crossref PubMed Scopus (70) Google Scholar). Angiotensin II exerts effects through specific G-protein coupled receptors, predominantly the AT1 receptor subtype. AT1 receptors couple to intracellular calcium mobilization, activation of tyrosine kinases such as p125FAK, p46SHC, and p54SHC, and induction of serine/threonine kinases, including protein kinase C (PKC) and mitogen-activated protein kinases (2Lindpainter K. Ganten D. The Cardiac Renin-Angiotensin System. Fatura Publishing Co., Inc., Armonk, NY1994Google Scholar, 9Sadoshima J. Izumo S. Cir. Res. 1993; 73: 424-438Crossref PubMed Scopus (366) Google Scholar, 10Marrero M.B. Paxton W.G. Duff J.L. Berk B.C. Bernstein K.E. J. Biol. Chem. 1994; 269: 10935-10939Abstract Full Text PDF PubMed Google Scholar, 11Booz G.W. Dostal D.E. Singer H.A. Baker K.M. Am. J. Physiol. 1994; 267: C1308-C1318Crossref PubMed Google Scholar, 12Schorb W. Peeler T.C. Madigan N.N. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 19626-19632Abstract Full Text PDF PubMed Google Scholar, 13Molloy C. Taylor D.S. Weber H. J. Biol. Chem. 1993; 268: 7338-7345Abstract Full Text PDF PubMed Google Scholar, 14Schorb W. Conrad K.M. Singer H.A. Dostal D.E. Baker K.M. J. Mol. Cell. Cardiol. 1995; 27: 1151-1160Abstract Full Text PDF PubMed Scopus (72) Google Scholar). Angiotensin II may act directly through these signaling pathways or indirectly via the release of growth factors such as PDGF and TGF-β, as demonstrated for rat vascular smooth muscle cells(4Sadoshima J. Izumo S. Circ. Res. 1993; 73: 413-423Crossref PubMed Scopus (1285) Google Scholar, 7Weber H. Taylor D.S. Molloy C.J. J. Clin. Invest. 1994; 93: 788-798Crossref PubMed Scopus (145) Google Scholar). Like other growth factors, AII induces a rapid increase of the growth associated nuclear proto-oncogenes c-myc, c-fos, and c-jun and several cellular genes including tenascin, fibronectin, and collagen(4Sadoshima J. Izumo S. Circ. Res. 1993; 73: 413-423Crossref PubMed Scopus (1285) Google Scholar, 15Naftilan A.J. Gilliland G.K. Eldridge C.S. Kraft A.S. Mol. Cell. Biol. 1990; 10: 5536-5540Crossref PubMed Scopus (136) Google Scholar, 16Taubman M.B. Berk B.C. Izumo S. Tsuda T. Alexander R.W. Nadal-Ginard B. J. Biol. Chem. 1989; 264: 526-530Abstract Full Text PDF PubMed Google Scholar, 17Crabos M. Roth M. Hahn A.W.A. Earne P. J. Clin. Invest. 1994; 93: 2372-2378Crossref PubMed Scopus (238) Google Scholar, 18Crawford D.C. Chobanian A.V. Brecher P. Circ. Res. 1993; 74: 727-739Crossref Scopus (210) Google Scholar, 19Sharifi B.G. Lafleur D.W. Pirola C.J. Forrester J.S. Fagin J.A. J. Biol. Chem. 1992; 267: 23910-23915Abstract Full Text PDF PubMed Google Scholar). These studies indicate that AII can induce rapid changes in gene expression and function, that may ultimately lead to increased cell growth(20Dostal D.E. Baker K.M. Peach M.J. Maggi M. Greenen V. Horizons in Endocrinology. Raven Press, New York1991: 265-272Google Scholar). angiotensin II guanyl nucleotide-binding protein angiotensin II receptor subtype 1A Chinese hamster ovary cells platelet-derived growth factor interferon-γ interleukin-6 sis-inducing element sis-inducing factor Janus kinase transforming growth factor-β signal transducers and activators of transcription phorbol 12-myristate 13-acetate protein kinase C EXP3174 chloramphenicol acetyltransferase. angiotensin II guanyl nucleotide-binding protein angiotensin II receptor subtype 1A Chinese hamster ovary cells platelet-derived growth factor interferon-γ interleukin-6 sis-inducing element sis-inducing factor Janus kinase transforming growth factor-β signal transducers and activators of transcription phorbol 12-myristate 13-acetate protein kinase C EXP3174 chloramphenicol acetyltransferase. Growth factors and cytokines transduce signaling through a common pathway (STAT pathway) from receptor to the nucleus(21Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4907) Google Scholar). The c-fos regulatory element SIE (sis-inducing element) has been used extensively to study the activation of STAT pathways by many ligands, including PDGF, epidermal growth factor, IFN-γ, and IL-6(22Ruff-Jamison S. Chen K. Cohen S. Science. 1993; 261: 1733-1736Crossref PubMed Scopus (240) Google Scholar, 23Silvennoinen O. Schindler C. Schlessinger J. Levy D.E. Science. 1993; 261: 1736-1739Crossref PubMed Scopus (295) Google Scholar, 24Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Crossref PubMed Scopus (633) Google Scholar, 25Fu X-Y. Zhang J-J. Cell. 1993; 74: 1135-1145Abstract Full Text PDF PubMed Scopus (271) Google Scholar, 26Shuai K. Stark G.R. Kerr I.M. Darnell Jr., J.E. Science. 1993; 261: 1744-1746Crossref PubMed Scopus (678) Google Scholar, 27Ruff-Jamison S. Zhong Z. Wen Z. Chen K. Darnell Jr., J.E. Cohen S. J. Biol. Chem. 1994; 269: 21933-21935Abstract Full Text PDF PubMed Google Scholar, 28Shuai K. Horvath C.M. Huang L.H.T. Qureshi S.A. Cowburn D. Darnell Jr., J.E. Cell. 1994; 76: 821-828Abstract Full Text PDF PubMed Scopus (676) Google Scholar). Binding of the ligand to the receptor activates tyrosine kinases, which phosphorylate monomeric STAT proteins in the cytoplasm, leading to dimerization and formation of complexes referred to as SIF (sis-inducing factor)(29Wagner B.J. Hays T.E. Hoban C.J. Cochran B.H. EMBO J. 1990; 9: 4477-4484Crossref PubMed Scopus (550) Google Scholar). SIF subsequently translocates to the nucleus and interacts with SIE, or SIE-like elements, in the promoter of genes to induce expression. Depending upon the ligand, SIF appears in three different forms: complex A, B, and C(22Ruff-Jamison S. Chen K. Cohen S. Science. 1993; 261: 1733-1736Crossref PubMed Scopus (240) Google Scholar, 24Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Crossref PubMed Scopus (633) Google Scholar). PDGF and epidermal growth factor induce all three complexes; IL-6 induces mainly complex A and IFN-γ mainly complex C. We have recently shown (30Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 31443-31449Abstract Full Text PDF PubMed Google Scholar) that AII stimulates the STAT pathway and induces predominantly SIF complex A in both neonatal rat cardiac fibroblasts and CHO-K1 cells expressing AT1A receptors (T3CHO/AT1A). Like growth factors, activation of SIF by AII was post-translational and required the actions of tyrosine kinases. Angiotensin II-induced SIF complexes contained tyrosine phosphorylated Stat91, with activation occurring in the cytoplasm followed by translocation to the nucleus. However, with respect to the time course of SIF activation, the AII-induced response significantly differed from that of growth factor/cytokine responses. Unlike the rapid induction of SIF by cytokines and growth factors (maximal in less than 30 min), AII-induced activity although detectable at 30 min, was maximal at 2-3 h(30Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 31443-31449Abstract Full Text PDF PubMed Google Scholar). In the present study, using T3CHO/AT1A cells, we explored the possible reasons for the delayed maximal SIF activation by AII. Our data indicate that AII directly activates SIF activity and that the delayed maximal activation of SIF is not due to the secondary release of other SIF inducing factors into the medium. We also provide evidence that AII evokes a PKC independent, transient inhibitory effect on the rapid SIF induction by IL-6. We propose that similar transient inhibitory mechanisms may be responsible for delaying the maximal SIF activation by AII (2 h). Cell culture media, fetal bovine serum, antibiotics, Geneticin, tissue culture flasks, IL-6, and TGF-β, were purchased from Life Technologies, Inc.; AII was purchased from U. S. Biochemical Corp; Nitrocellulose membranes were purchased from Amersham Corp; [γ-32P]ATP was from DuPont NEN; polyclonal antibodies to Stat92 and protein A/G-agarose were purchased from Santa Cruz Biotechnology; monoclonal antibodies to phosphotyrosine and PDGF were purchased from Upstate Biotechnology; goat anti-rabbit IgG and rabbit anti-mouse IgG were purchased from Bio-Rad; and other chemicals were purchased from Sigma. T3CHO/AT1A cells (31Thekkumkara T.J. Du J. Dostal D.E. Motel T.J. Thomas W.G. Baker K.M. Mol. Cell. Biochem. 1995; 146: 79-89Crossref PubMed Scopus (41) Google Scholar) were grown in α-minimum essential medium containing 10% fetal bovine serum and 200 μg/ml Geneticin antibiotic for 12-24 h, serum starved for 12 h, and treated with one or more growth factors or cytokines at the following concentrations: AII, 100 nM; PDGF, 10 ng/ml; TGF-β, 5 ng/ml; IL-6, 10 ng/ml. Where indicated, the non-peptide AT1 receptor antagonist EXP3174 was added to a final concentration of 100 μM. For PKC down-regulation, cells were exposed to PMA (250 nM) for 24 h, prior to stimulation with different agonists. At the time of experiments, cultures were subconfluent. Nuclear extracts were prepared and gel mobility shift assays were performed as described previously(30Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 31443-31449Abstract Full Text PDF PubMed Google Scholar), using 32P-labeled, double stranded oligonucleotides representing the SIE-DNA (top strand: 5′-CAGTTCCCGTCAATC-3′). Immunoprecipitations were performed according to previously described methods(30Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 31443-31449Abstract Full Text PDF PubMed Google Scholar), on protein extracts obtained from the nuclei of cells untreated or treated with AII or IL-6. One hundred μg of protein from each sample was diluted with an equal volume of immunoprecipitation buffer (1 × = 10 mM Tris (pH 7.4), 150 mM NaCl, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate) and immunoprecipitated with 1 μg of anti-Stat92 antibody and protein A/G-agarose. Immunocomplexes were harvested by centrifugation, washed 3 times with immunoprecipitation buffer, proteins resolved by 8% polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane (Amersham), and incubated with antiphosphotyrosine antibody(30Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 31443-31449Abstract Full Text PDF PubMed Google Scholar). Immunoreactive bands were visualized using a chemiluminescence Western blotting system (Amersham) according to the manufacturer's instructions. The blots were stripped (Amersham) and reprobed with polyclonal anti-Stat92 antibody as described previously(30Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 31443-31449Abstract Full Text PDF PubMed Google Scholar). The parental plasmid pBLCAT2 was obtained from B. Luckow and G. Schutz(32Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar). This plasmid has a bacterial chloramphenicol acetyltransferase gene under the control of the thymidine kinase (tk) promoter. DNA representing three tandem repeats of SIE (see above) and mutant SIE (5′-CAGCCACCGTCAATC-3′) were inserted at the HindIII-BamHI site of the pBLCAT2 vector to generate SIE/pBLCAT2 and m.SIE/pBLCAT2, respectively. T3CHO/AT1A cells were grown in 60-mm tissue culture plates to 50-60% confluence until the day of transfection. Cells were transfected with 2 μg of pBLCAT2 (vector), SIE/pBLCAT2, or m.SIE/pBLCAT2 using Lipofectin according to the manufacturer's (Life Technologies, Inc.) instructions. To measure the transfection efficiency, cells were co-transfected with pSV-β-galactosidase plasmid (Promega). Twenty-four hours after transfections, the cells were serum starved for 3 h and AII was added for 24 h. Cells were collected and extracts prepared by three cycles of freezing and thawing. Extracts were heated to 60°C for 10 min, sedimented at 14,000 rpm in a microcentrifuge, and protein concentrations determined (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211715) Google Scholar). CAT assay was performed as described previously with minor modifications(34Crabb D.W. Minth C.D. Dixon J.E. Methods Enzymol. 1989; 168: 690-701Crossref PubMed Scopus (12) Google Scholar). The reaction mixture contained 140 mM Tris-HCl (pH 7.8), 0.2 μCi of [14C]chloramphenicol, 4 mM acetyl-coenzyme A, and 40 μg of cell extract in a final volume of 150 μl. The mixture was incubated at 37°C, for 1 h and then extracted with ethyl acetate. The organic phase was transferred, dried, and the pellet redissolved in 15 μl of ethyl acetate. The labeled chloramphenicol and acetylated derivatives were separated by ascending thin layer chromatography using chloroform/methanol (95:5, v/v) and the chromatograms subjected to autoradiography at −70°C. We reported previously (30Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 31443-31449Abstract Full Text PDF PubMed Google Scholar) that AII induces maximal SIF activity between 2 and 3 h in T3CHO/AT1A cells. This delay in maximal response to AII, compared to the rapid activation (30 min) by cytokines and growth factors, is not due to a requirement for new protein synthesis. Given the observation that, in rat aortic smooth muscle cells, AII causes an increase in the expression/secretion of growth factors such as PDGF and TGF-β(4Sadoshima J. Izumo S. Circ. Res. 1993; 73: 413-423Crossref PubMed Scopus (1285) Google Scholar, 7Weber H. Taylor D.S. Molloy C.J. J. Clin. Invest. 1994; 93: 788-798Crossref PubMed Scopus (145) Google Scholar), we first considered the possibility that the delayed SIF induction resulted from secondary release of growth factors. We tested the ability of different agents (PDGF, TGF-β, and IL-6) to stimulate SIF activity in T3CHO/AT1A cells. Nuclear extracts were made from treated (PDGF, TGF-β, IL-6, 30 min; AII, 2 h) and untreated cells and analyzed in an gel mobility shift assay. As shown in Fig. 1, PDGF and TGF-β failed to induce SIF activity (lanes 3 and 5), while induction was observed with AII (positive control; lane 2) and IL-6 (lane 4). These results suggest that AII induced SIF activity is not due to the release of PDGF and TGF-β in these cells. To investigate the possibility that AII stimulation causes the release of SIF inducing factors, we collected conditioned medium from T3CHO/AT1A cells treated with AII for 2 h, the time of maximal stimulation. This conditioned media was tested for the ability to induce SIF activity at early (30 min) and delayed (2 h) time points. As shown in Fig. 2, conditioned medium from cells exposed to AII induced significant levels of SIF, only at 2 h (lane 6), but not at 30 min (lane 4), as would be expected if the response was mediated by a released factor. However, the activity at 2 h was completely blocked by pretreatment with the AT1 receptor antagonist, EXP3174 (lane 7). These results indicate that AII acts directly through the AT1A receptor to induce SIF activity and that the delayed activity is not secondary to the release of other SIF inducing factors. In order to determine whether continuous receptor occupancy is required for AII-mediated SIF induction, we first treated the cells with AII (100 nM) for varying periods (5, 15, and 35 min and 1 h), and then with 100-fold excess of the AT1 receptor antagonist EXP3174 until 2 h. Nuclear extracts were made from these cells and analyzed in a gel mobility shift assay. As shown in Fig. 3A, cells exposed to AII for 5 min and 15 min before the addition of EXP3174 showed no or very little SIF induction (lanes 3 and 4). Cells exposed to AII for 35 min and 1 h before the addition of EXP3174 showed significant SIF induction (lanes 5 and 6). These results indicate that a 15-35 min continuous exposure to AII is required for SIF induction. To determine whether T3CHO/AT1A cells respond appropriately to a known rapid SIF inducer (IL-6)(24Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Crossref PubMed Scopus (633) Google Scholar), we compared the time course of SIF activation by AII, to that of IL-6. For these experiments, we treated serum-starved T3CHO/AT1A cells with AII and IL-6 for varying periods of time, up to 6 h. Nuclear extracts were prepared and analyzed in a gel mobility shift assay. As we have reported previously(30Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 31443-31449Abstract Full Text PDF PubMed Google Scholar), in this cell line and in neonatal cardiac fibroblasts, AII stimulates predominantly a delayed SIF-A, with maximal activity at 2 h. In contrast, IL-6 stimulated high levels of SIF activity (complex A) as early as 5 min (Fig. 3B), consistent with the findings of others using different cell types(24Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Crossref PubMed Scopus (633) Google Scholar). These results suggest that the delayed SIF response is not an aberration of this cell line, but rather a characteristic of AII stimulation. Since IL-6 induced SIF (complex A) has been demonstrated to contain Stat92 (Stat3, also referred to as acute phase response factor)(35Akira S. Nishio Y. Inoue M. Wang X-J. Wei S. Matsusaka T. Yoshida K. Sudo T. Naruto M. Kishimoto T. Cell. 1994; 77: 63-71Abstract Full Text PDF PubMed Scopus (853) Google Scholar, 36Wegenka U.M. Lutticken C. Buschmann J. Yuan J. Lottspeich F. Muller-Estrel W. Schindler C. Roeb E. Heinrich P.C. Horn F. Mol. Cell. Biol. 1994; 14: 3186-3196Crossref PubMed Scopus (231) Google Scholar, 37Raz R. Durbin J.E. Levy D.E. J. Biol. Chem. 1994; 269: 24391-24395Abstract Full Text PDF PubMed Google Scholar), we tested the relatedness of the AII and IL-6-induced complexes using anti-Stat92 antibody in a gel mobility supershift assay. Nuclear extracts were prepared from AII and IL-6-stimulated T3CHO/AT1A cells and incubated with Stat92 antibody. As shown in Fig. 4, the anti-Stat92 antibodies recognized SIF complex A in both AII and IL-6-treated nuclear extracts, as demonstrated by the complete disappearance of SIF complex A and the formation of supershift complexes (lanes 3 and 7). When AII and IL-6-induced nuclear extracts were incubated with IgG from a nonimmunized rabbit, supershifted complexes were not observed demonstrating the specificity of protein-antibody interactions (lanes 5 and 9). We conclude from these results that Stat92 or an antigenically related protein is likely to be part of AII-induced SIF complex A. Our previous report on AII (30Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 31443-31449Abstract Full Text PDF PubMed Google Scholar) and the results obtained in Fig. 3B on IL-6, demonstrate that AII and IL-6 induce maximal nuclear SIF activation at different time points with AII at 2-3 h and IL-6 at 5-30 min. The longer time required for maximal SIF activation by AII is different from the rapid activation by most cytokines and growth factors, where the maximal response is detected in less than 30 min(22Ruff-Jamison S. Chen K. Cohen S. Science. 1993; 261: 1733-1736Crossref PubMed Scopus (240) Google Scholar, 23Silvennoinen O. Schindler C. Schlessinger J. Levy D.E. Science. 1993; 261: 1736-1739Crossref PubMed Scopus (295) Google Scholar, 24Sadowski H.B. Shuai K. Darnell Jr., J.E. Gilman M.Z. Science. 1993; 261: 1739-1744Crossref PubMed Scopus (633) Google Scholar). We speculated as to whether AII could activate an inhibitory pathway, in the initial phase following AII exposure, which was responsible for delaying the maximal SIF stimulation until 2 h. Since both AII and IL-6 appear to utilize Stat92-related protein for SIF-A formation (see Fig. 4), if AII promotes an inhibitory pathway, pretreatment of T3CHO/AT1A cells with AII would be expected to affect the rapid induction of SIF by IL-6. To test this hypothesis, T3CHO/AT1A cells were pretreated with AII for 15 min, following which IL-6 was added for an additional 10 min. As a positive control for stimulation of SIF activity, cells were treated with AII (25 min and 2 h) or IL-6 alone (10 min). Nuclear extracts were prepared and subjected to electrophoretic mobility shift assays and immunoprecipitations. As shown in Fig. 5, pretreatment of T3CHO/AT1A cells with AII for 15 min resulted in significant inhibition of the IL-6-induced SIF response at 10 min (lane 5). Pre-exposure of cells to EXP3174, an AT1 receptor antagonist, prevented the inhibitory action of AII (lane 6), demonstrating that this phenomena is mediated through the transfected AT1A receptor. Since both AII and IL-6-induced SIF complexes contain Stat92/or a related protein (see Fig. 4), we determined whether the inhibition of the IL-6-induced SIF response by AII was reflected in the degree of tyrosine phosphorylation of Stat92. The nuclear extracts corresponding to the samples described in Fig. 5 were immunoprecipitated with anti-Stat92 antibody, separated by SDS-polyacrylamide gel electrophoresis, blotted, and probed with antiphosphotyrosine antibodies. As shown in Fig. 6A, AII caused the tyrosine phosphorylation of a single 92-kDa protein at 2 h (lane 3). In contrast, IL-6 induced the tyrosine phosphorylation of two proteins, of molecular mass 92 and 89 kDa (lane 4), consistent with the finding of others(36Wegenka U.M. Lutticken C. Buschmann J. Yuan J. Lottspeich F. Muller-Estrel W. Schindler C. Roeb E. Heinrich P.C. Horn F. Mol. Cell. Biol. 1994; 14: 3186-3196Crossref PubMed Scopus (231) Google Scholar). A recent report suggests that the two forms of Stat92 results from differential phosphorylation(38Lutticken C. Coffer P. Yuan J. Schwartz C. Caldenhoven E. Schindler C. Kruijer W. Heinrich P.C. Horn F. FEBS Lett. 1995; 360: 137-143Crossref PubMed Scopus (93) Google Scholar). In untreated nuclei, both forms of tyrosine-phosphorylated proteins were not observed (lane 1). Interestingly, pretreatment with AII markedly reduced the IL-6-induced tyrosine phosphorylation of both forms of Stat92 (lane 5). The AT1 receptor antagonist EXP3174 prevented the inhibitory action of AII on IL-6-induced tyrosine phosphorylation (lane 6). To determine whether Stat92 related proteins were present in the unphosphorylated form in the nucleus, the blot in Fig. 6A was stripped and reprobed with anti-Stat92 antibody. Fig. 6B shows that Stat92-related proteins are present mainly in the nucleus of AII (2 h) and IL-6-treated cells (lanes 3 and 4), consistent with the pattern observed by others, in which only tyrosine-phosphorylated STAT proteins translocate to the nucleus (21Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4907) Google Scholar, 22Ruff-Jamison S. Chen K. Cohen S. Science. 1993; 261: 1733-1736Cr
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