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• W2015584527 abstract "Previously, we have demonstrated that STAT-5B plays a role in thrombin-induced vascular smooth muscle cell (VSMC) growth and motility. To learn more about the role of STAT-5B in vessel wall remodeling, we examined its involvement in platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMC growth and motility and balloon injury-induced neointima formation. PDGF-BB activated STAT-5B as measured by its tyrosine phosphorylation, DNA binding, and reporter gene activity. PDGF-BB induced cyclin D1 expression, CDK4 activity, and Rb protein phosphorylation, leading to VSMC growth and motility, and these responses were suppressed by the blockade of STAT-5B. Increased cyclin D1 levels, CDK4 activity, and Rb protein phosphorylation were observed in 1-week balloon-injured arteries compared with uninjured arteries, and these responses were also suppressed by adenovirus-mediated expression of dnSTAT-5B. In addition, adenovirus-mediated expression of dnSTAT-5B attenuated balloon injury-induced smooth muscle cell migration from media to intima and their proliferation in intima, resulting in reduced neointima formation. These observations indicate that STAT-5B plays an important role in PDGF-BB-induced VSMC growth and motility in vitro and balloon injury-induced neointima formation in vivo. Previously, we have demonstrated that STAT-5B plays a role in thrombin-induced vascular smooth muscle cell (VSMC) growth and motility. To learn more about the role of STAT-5B in vessel wall remodeling, we examined its involvement in platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMC growth and motility and balloon injury-induced neointima formation. PDGF-BB activated STAT-5B as measured by its tyrosine phosphorylation, DNA binding, and reporter gene activity. PDGF-BB induced cyclin D1 expression, CDK4 activity, and Rb protein phosphorylation, leading to VSMC growth and motility, and these responses were suppressed by the blockade of STAT-5B. Increased cyclin D1 levels, CDK4 activity, and Rb protein phosphorylation were observed in 1-week balloon-injured arteries compared with uninjured arteries, and these responses were also suppressed by adenovirus-mediated expression of dnSTAT-5B. In addition, adenovirus-mediated expression of dnSTAT-5B attenuated balloon injury-induced smooth muscle cell migration from media to intima and their proliferation in intima, resulting in reduced neointima formation. These observations indicate that STAT-5B plays an important role in PDGF-BB-induced VSMC growth and motility in vitro and balloon injury-induced neointima formation in vivo. Increased vascular smooth muscle cell (VSMC) mass is a major contributing factor in atherosclerosis and neointima formation after percutaneous transluminal angioplasty.1Ross R Atherosclerosis—an inflammatory disease.N Engl J Med. 1999; 340: 115-126Crossref PubMed Scopus (19370) Google Scholar, 2Hansson GK Libby P Schonbeck U Yan ZQ Innate and adaptive immunity in the pathogenesis of atherosclerosis.Circ Res. 2002; 91: 281-291Crossref PubMed Scopus (862) Google Scholar, 3Schwartz SM deBlois D O'Brien ER The intima. Soil for atherosclerosis and restenosis.Circ Res. 1995; 77: 445-465Crossref PubMed Scopus (901) Google Scholar A broad spectrum of molecules including cytokines, growth factors, hormones, and oxidants that are produced by a variety of cell types at the sites of vascular injury have been reported to influence the growth of VSMCs in vitro.4Itoh H Mukoyama M Pratt RE Gibbons GH Dzau VJ Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II.J Clin Invest. 1993; 91: 2268-2274Crossref PubMed Scopus (463) Google Scholar, 5Nugent MA Karnovsky MJ Edelman ER Vascular cell-derived heparan sulfate shows coupled inhibition of basic fibroblast growth factor binding and mitogenesis in vascular smooth muscle cells.Circ Res. 1993; 73: 1051-1060Crossref PubMed Scopus (86) Google Scholar, 6Rauch BH Millette E Kenagy RD Daum G Fischer JW Clowes AW Syndecan-4 is required for thrombin-induced migration and proliferation in human vascular smooth muscle cells.J Biol Chem. 2005; 280: 17507-17511Crossref PubMed Scopus (60) Google Scholar, 7Abid MR Yano K Guo S Patel VI Shrikhande G Spokes KC Ferran C Aird WC Forkhead transcription factors inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia.J Biol Chem. 2005; 280: 29864-29873Crossref PubMed Scopus (101) Google Scholar, 8Spinetti G Wang M Monticone R Zhang J Zhao D Lakatta EG Rat aortic MCP-1 and its receptor CCR2 increase with age and alter vascular smooth muscle cell function.Arterioscler Thromb Vasc Biol. 2004; 24: 1397-1402Crossref PubMed Scopus (155) Google Scholar, 9Liu Z Dronadula N Rao GN A novel role for nuclear factor of activated T cells in receptor tyrosine kinase and G protein-coupled receptor agonist-induced vascular smooth muscle cell motility.J Biol Chem. 2004; 279: 41218-41226Crossref PubMed Scopus (54) Google Scholar, 10Rao GN Berk BC Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression.Circ Res. 1992; 70: 593-599Crossref PubMed Google Scholar Because a large number of molecules are apparently involved in these vessel wall diseases, understanding the biochemical mechanisms that are common to the mitogenic effects of many of these substances may lead to the development of better therapeutic agents against the progression of these vascular lesions. Indeed, approaches that target the inhibition of VSMC proliferation have been found to be more promising in arresting the development of neointima.11Ahn JD Morishita R Kaneda Y Lee SJ Kwon KY Choi SY Lee KU Park JY Moon IJ Park JG Yoshizumi M Ouchi Y Lee IK Inhibitory effects of novel AP-1 decoy oligonucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo.Circ Res. 2002; 90: 1325-1332Crossref PubMed Scopus (115) Google Scholar, 12Johnson TW Wu YX Herdeg C Baumbach A Newby AC Karsch KR Oberhoff M Stent-based delivery of tissue inhibitor of metalloproteinase-3 adenovirus inhibits neointimal formation in porcine coronary arteries.Arterioscler Thromb Vasc Biol. 2005; 25: 754-759Crossref PubMed Scopus (55) Google Scholar, 13Morishita R Gibbons GH Horiuchi M Ellison KE Nakama M Zhang L Kaneda Y Ogihara T Dzau VJ A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo.Proc Natl Acad Sci USA. 1995; 92: 5855-5859Crossref PubMed Scopus (487) Google Scholar, 14Tanner FC Boehm M Akyurek LM San H Yang ZY Tashiro J Nabel GJ Nabel EG Differential effects of the cyclin-dependent kinase inhibitors p27kip1, p21cip1 and p16ink4 on vascular smooth muscle cell proliferation.Circulation. 2000; 101: 2022-2025Crossref PubMed Scopus (215) Google Scholar Toward elucidation of signal transduction mechanisms that are ubiquitously involved in the mitogenic actions of various VSMC agonists, we have shown previously that both receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) agonists such as platelet-derived growth factor-BB (PDGF-BB) and thrombin, respectively, activate nuclear factor of activated T cells (NFATs) and signal transducer and activator of transcription-3 (STAT-3) in stimulating VSMC growth and/or motility.9Liu Z Dronadula N Rao GN A novel role for nuclear factor of activated T cells in receptor tyrosine kinase and G protein-coupled receptor agonist-induced vascular smooth muscle cell motility.J Biol Chem. 2004; 279: 41218-41226Crossref PubMed Scopus (54) Google Scholar, 15Liu Z Zhang C Dronadula N Li Q Rao GN Blockade of nuclear factor of activated T cells activation signaling suppresses balloon injury-induced neointima formation in a rat carotid artery model.J Biol Chem. 2005; 280: 14700-14708Crossref PubMed Scopus (83) Google Scholar, 16Yellaturu CR Rao GN Cytosolic phospholipase A2 is an effector of janus-activated kinase/signal transducers and activators of transcription signaling and is involved in platelet-derived growth factor BB-induced growth in vascular smooth muscle cells.J Biol Chem. 2003; 278: 9986-9992Crossref PubMed Scopus (38) Google Scholar, 17Neeli I Liu Z Dronadula N Ma ZA Rao GN An essential role of Jak-2/STAT-3/cPLA2 axis in platelet-derived growth factor BB-induced vascular smooth muscle cell motility.J Biol Chem. 2004; 279: 46122-46128Crossref PubMed Scopus (46) Google Scholar, 18Dronadula N Liu Z Wang C Cao H Rao GN STAT-3-dependent expression of cPLA2 is required for thrombin-induced vascular smooth muscle cell motility.J Biol Chem. 2005; 280: 3112-3120Crossref PubMed Scopus (35) Google Scholar Early studies from various laboratories have shown that STATs play an important role in the regulation of cytokine-induced gene expression.19Darnell Jr, JE Kerr IM Stark GR Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5062) Google Scholar, 20Horvath CM Wen Z Darnell Jr, JE A STAT protein domain that determines DNA sequence recognition suggests a novel DNA-binding domain.Genes Dev. 1995; 9: 984-994Crossref PubMed Scopus (452) Google Scholar, 21Teglund S McKay C Schuetz E vanDeursen JM Stravopodis D Wang D Brown M Bodner S Grosveld G Ihle JN Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses.Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1080) Google Scholar However, subsequent studies have revealed their involvement in a variety of cellular responses including cell differentiation, proliferation, and apoptosis.22Wu YY Bradshaw RA Activation of the Stat3 signaling pathway is required for differentiation by interleukin-6 in PC12–E2 cells.J Biol Chem. 2000; 275: 2147-2156Crossref PubMed Scopus (56) Google Scholar, 23Iavnilovitch E Groner B Barash I Overexpression and forced activation of stat5 in mammary gland of transgenic mice promotes cellular proliferation, enhances differentiation, and delays postlactational apoptosis.Mol Cancer Res. 2002; 1: 32-47PubMed Google Scholar, 24Bromberg JF Wrzeszczynska MH Devgan G Zhao Y Pestell RG Albanese C Darnell Jr, JE Stat3 as an oncogene.Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2517) Google Scholar, 25Cui Y Riedlinger G Miyoshi K Tang W Li C Deng CX Robinson GW Hennighausen L Inactivation of Stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation.Mol Cell Biol. 2004; 24: 8037-8047Crossref PubMed Scopus (405) Google Scholar, 26Udy GB Towers RP Snell RG Wilkins RJ Park SH Ram PA Waxman DJ Davey HW Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression.Proc Natl Acad Sci USA. 1997; 94: 7239-7244Crossref PubMed Scopus (838) Google Scholar, 27Stephanou A Brar BK Scarabelli TM Jonassen AK Yellon DM Marber MS Knight RA Latchman DS Ischemia-induced STAT-1 expression and activation play a critical role in cardiomyocyte apoptosis.J Biol Chem. 2000; 275: 10002-10008Crossref PubMed Scopus (184) Google Scholar, 28Huang YQ Li JJ Karpatkin S Thrombin inhibits tumor cell growth in association with upregulation of p21waf/cip1 and caspases via a p53-independent STAT-1-dependent pathway.J Biol Chem. 2000; 275: 6462-6468Crossref PubMed Scopus (89) Google Scholar In addition, selectivity among various STATs in mediating these cellular effects has been observed.22Wu YY Bradshaw RA Activation of the Stat3 signaling pathway is required for differentiation by interleukin-6 in PC12–E2 cells.J Biol Chem. 2000; 275: 2147-2156Crossref PubMed Scopus (56) Google Scholar, 23Iavnilovitch E Groner B Barash I Overexpression and forced activation of stat5 in mammary gland of transgenic mice promotes cellular proliferation, enhances differentiation, and delays postlactational apoptosis.Mol Cancer Res. 2002; 1: 32-47PubMed Google Scholar, 24Bromberg JF Wrzeszczynska MH Devgan G Zhao Y Pestell RG Albanese C Darnell Jr, JE Stat3 as an oncogene.Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2517) Google Scholar, 25Cui Y Riedlinger G Miyoshi K Tang W Li C Deng CX Robinson GW Hennighausen L Inactivation of Stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation.Mol Cell Biol. 2004; 24: 8037-8047Crossref PubMed Scopus (405) Google Scholar, 26Udy GB Towers RP Snell RG Wilkins RJ Park SH Ram PA Waxman DJ Davey HW Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression.Proc Natl Acad Sci USA. 1997; 94: 7239-7244Crossref PubMed Scopus (838) Google Scholar, 27Stephanou A Brar BK Scarabelli TM Jonassen AK Yellon DM Marber MS Knight RA Latchman DS Ischemia-induced STAT-1 expression and activation play a critical role in cardiomyocyte apoptosis.J Biol Chem. 2000; 275: 10002-10008Crossref PubMed Scopus (184) Google Scholar, 28Huang YQ Li JJ Karpatkin S Thrombin inhibits tumor cell growth in association with upregulation of p21waf/cip1 and caspases via a p53-independent STAT-1-dependent pathway.J Biol Chem. 2000; 275: 6462-6468Crossref PubMed Scopus (89) Google Scholar Specifically, whereas STAT-1 has been found to be involved in the modulation of apoptosis,27Stephanou A Brar BK Scarabelli TM Jonassen AK Yellon DM Marber MS Knight RA Latchman DS Ischemia-induced STAT-1 expression and activation play a critical role in cardiomyocyte apoptosis.J Biol Chem. 2000; 275: 10002-10008Crossref PubMed Scopus (184) Google Scholar, 28Huang YQ Li JJ Karpatkin S Thrombin inhibits tumor cell growth in association with upregulation of p21waf/cip1 and caspases via a p53-independent STAT-1-dependent pathway.J Biol Chem. 2000; 275: 6462-6468Crossref PubMed Scopus (89) Google Scholar STAT-3 and STAT-5 were reported to play a role in the regulation of cell growth and differentiation.22Wu YY Bradshaw RA Activation of the Stat3 signaling pathway is required for differentiation by interleukin-6 in PC12–E2 cells.J Biol Chem. 2000; 275: 2147-2156Crossref PubMed Scopus (56) Google Scholar, 23Iavnilovitch E Groner B Barash I Overexpression and forced activation of stat5 in mammary gland of transgenic mice promotes cellular proliferation, enhances differentiation, and delays postlactational apoptosis.Mol Cancer Res. 2002; 1: 32-47PubMed Google Scholar, 24Bromberg JF Wrzeszczynska MH Devgan G Zhao Y Pestell RG Albanese C Darnell Jr, JE Stat3 as an oncogene.Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2517) Google Scholar Recently, we have observed a role for STAT-5B in GPCR agonist thrombin-induced VSMC growth and motility.29Cao H Dronadula N Rizvi F Li Q Gerthoffer WT Rao GN A novel role for STAT-5B in the regulation of Hsp27-FGF-2 axis facilitating thrombin-induced vascular smooth muscle cell DNA synthesis and motility.Circ Res. 2006; 98: 913-922Crossref PubMed Scopus (30) Google Scholar PDGF-BB is the most potent VSMC mitogen and chemoattractant.1Ross R Atherosclerosis—an inflammatory disease.N Engl J Med. 1999; 340: 115-126Crossref PubMed Scopus (19370) Google Scholar, 3Schwartz SM deBlois D O'Brien ER The intima. Soil for atherosclerosis and restenosis.Circ Res. 1995; 77: 445-465Crossref PubMed Scopus (901) Google Scholar, 16Yellaturu CR Rao GN Cytosolic phospholipase A2 is an effector of janus-activated kinase/signal transducers and activators of transcription signaling and is involved in platelet-derived growth factor BB-induced growth in vascular smooth muscle cells.J Biol Chem. 2003; 278: 9986-9992Crossref PubMed Scopus (38) Google Scholar, 17Neeli I Liu Z Dronadula N Ma ZA Rao GN An essential role of Jak-2/STAT-3/cPLA2 axis in platelet-derived growth factor BB-induced vascular smooth muscle cell motility.J Biol Chem. 2004; 279: 46122-46128Crossref PubMed Scopus (46) Google Scholar Therefore, to understand the role of STAT-5B in vascular wall remodeling further, in the present investigation we have studied its involvement in VSMC growth and motility in response to PDGF-BB in vitro and balloon injury in vivo. Here, we report for the first time that PDGF-BB stimulates STAT-5B activation leading to induction of cyclin D1 expression, CDK4 activity, and Rb protein phosphorylation in VSMCs and thereby the growth and motility of these cells. In addition, increases in cyclin D1 levels, CDK4 activity, Rb protein phosphorylation, smooth muscle cell (SMC) migration from media to intima, and their proliferation in intima were observed in arteries after balloon injury as compared with uninjured arteries, and these responses were suppressed by adenovirus-mediated expression of dnSTAT-5B resulting in reduced neointima formation. Together, these results point to a potential role for STAT-5B in vessel wall remodeling in response to injury. Aprotinin, phenylmethyl sulfonyl fluoride, sodium orthovanadate, leupeptin, HEPES, β-glycerophosphate, and sodium fluoride were purchased from Sigma Chemical Company (St. Louis, MO). Recombinant human PDGF-BB (220-BB) was from R&D Systems Inc. (Minneapolis, MN). Anti-STAT-3 antibodies (SC-482), anti-STAT-5 antibodies (SC-835, SC-836), anti-STAT-5B antibodies (SC-1656), anti-CDK4 antibodies (SC-260), and STAT-5 consensus-binding oligonucleotides (5′-AGATTTCTAGGAATTCAATCC-3′) (SC-2565) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho STAT-3 antibodies (9145), anti-phospho STAT-5 antibodies (9351), anti-PY20 antibodies (9411), and anti-phospho pRb antibodies (9307) were supplied by Cell Signaling Technology (Beverly, MA). Anti-cyclin D1 antibodies (RB-010-P), anti-STAT-5B antibodies (RB-096-P1ABX), and anti-proliferating cell nuclear antigen (PCNA) antibodies (MS-106-P) were from NeoMarkers (Fremont, CA). Anti-STAT-5B antibodies (06-969) were also obtained from Upstate (Lake Placid, NY). Monoclonal anti-histone antibodies (MAB 1276) were from Chemicon (Temecula, CA). The in situ cell death detection kit, TMR Red, was purchased from Roche Diagnostics (Indianapolis, IN). Truncated Rb protein (3109) was obtained from QED Bioscience, San Diego, CA. Rat cyclin D1 short interfering RNA (siRNA) duplexes (siRNA3: sense, 5′-GCGCGUACCCUGACACCAAUU-3′; antisense, 5′-UUGGUGUCAGGGUACGCGCUU-3′; siRNA4: sense, 5′-CCGAGAAGUUGUGCAUCUAUU-3′; antisense, 5′-UAGAUGCACAACUUCUCGGUU-3′); scrambled control siRNA (5′-UAGCGACUAAACACAUCAA-3′) were made by Dharmacon (Lafayette, CO). T4 polynucleotide kinase was obtained from Promega (Madison, WI). [γ32P]ATP (3000 Ci/mmol), [14C]chloramphenicol (59 mCi/mmol), and protein-A-Sepharose (CL-4B) were from Amersham Biosciences (Piscataway, NJ). Thymidine [methyl-3H] (20 Ci/mmol) was obtained from Perkin-Elmer (Boston, MA). All of the primers and the putative STAT-binding oligonucleotides from rat cyclin D1 promoter were made by IDT (Coralville, IA). Rat VSMCs were isolated and subcultured as described previously.29Cao H Dronadula N Rizvi F Li Q Gerthoffer WT Rao GN A novel role for STAT-5B in the regulation of Hsp27-FGF-2 axis facilitating thrombin-induced vascular smooth muscle cell DNA synthesis and motility.Circ Res. 2006; 98: 913-922Crossref PubMed Scopus (30) Google Scholar VSMCs were used between 4 and 12 passages. Human aortic smooth muscle cells (HASMCs) were obtained from Cascade Biologics, Inc. (Portland, OR) and subcultured in Medium 231 containing smooth muscle growth supplements, 10 μg/ml gentamicin, and 0.25 μg/ml amphotericin B. Cultures were maintained at 37°C in a humidified 95% air and 5% CO2 atmosphere. HASMCs were growth-arrested by incubating in Medium 231 without smooth muscle growth supplements for 48 hours and used to perform the experiments unless otherwise stated. HASMCs were used between 6 and 10 passages. VSMC DNA synthesis was measured by pulse-labeling cells with 1 μCi/ml [3H]thymidine for the last 12 hours of the 24-hour incubation period as described previously.29Cao H Dronadula N Rizvi F Li Q Gerthoffer WT Rao GN A novel role for STAT-5B in the regulation of Hsp27-FGF-2 axis facilitating thrombin-induced vascular smooth muscle cell DNA synthesis and motility.Circ Res. 2006; 98: 913-922Crossref PubMed Scopus (30) Google Scholar VSMCs at 72 hours of appropriate treatments were trypsinized, rinsed with and suspended in 1 ml of phosphate-buffered saline (PBS), and counted using hemocytometer. VSMC motility was measured using a wounding assay as described previously.29Cao H Dronadula N Rizvi F Li Q Gerthoffer WT Rao GN A novel role for STAT-5B in the regulation of Hsp27-FGF-2 axis facilitating thrombin-induced vascular smooth muscle cell DNA synthesis and motility.Circ Res. 2006; 98: 913-922Crossref PubMed Scopus (30) Google Scholar Although migration is difficult to quantify in vivo, the accumulation of cells in the intima early (∼4 days) after injury is considered to be mostly attributable to the migration of SMCs from the injured media.30Jackson CL Raines EW Ross R Reidy MA Role of endogenous platelet-derived growth factor in arterial smooth muscle cell migration after balloon catheter injury.Arterioscler Thromb. 1993; 13: 1218-1226Crossref PubMed Scopus (229) Google Scholar Therefore, the in vivo SMC migration was determined as described by Bendeck and colleagues.31Bendeck MP Zempo N Clowes AW Galardy RE Reidy MA Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat.Circ Res. 1994; 75: 539-545Crossref PubMed Scopus (542) Google Scholar In brief, 4 days after balloon injury, the carotid arteries were fixed in vivo with 10% buffered formalin at physiological pressure. The middle 1 cm of the denuded (injured) and uninjured common carotid arteries were cut and fixed in ice-cold acetone for 10 minutes. The arteries were then opened longitudinally and pinned down onto an agar plate with the luminal surface facing upward. The arteries were rinsed in PBS and then placed in 3% H2O2 to block endogenous peroxidase activity. Nonspecific protein binding was blocked by incubating the arteries in 5% normal goat serum in PBS for 30 minutes. The arteries were incubated with anti-histone monoclonal antibody diluted 1:100 in PBS for 1 hour, followed by incubation in biotinylated goat anti-mouse IgG for 30 minutes. Peroxidase labeling was performed by using the ABC kit (Vector Laboratories, Burlingame, CA), and the signals were visualized by using the DAB kit (Vector Laboratories). After each step, the slides were rinsed three times for 5 minutes each in PBS. Finally, the opened arteries were placed intimal side up on glass slides with coverslips. As a negative control, samples of the same specimens without the primary antibody were used. The intimal surface of the vessel was examined under a light microscope at ×200, and the total number of intimal cell nuclei per square millimeter of surface area were counted. After appropriate treatments, VSMC nuclear extracts were prepared and analyzed for STAT-5 DNA binding activity as described previously.29Cao H Dronadula N Rizvi F Li Q Gerthoffer WT Rao GN A novel role for STAT-5B in the regulation of Hsp27-FGF-2 axis facilitating thrombin-induced vascular smooth muscle cell DNA synthesis and motility.Circ Res. 2006; 98: 913-922Crossref PubMed Scopus (30) Google Scholar VSMCs were transfected with 1× pSp1GLECAT plasmid in serum- and antibiotic-free Dulbecco's modified Eagle's medium using FuGENE 6 reagent (Invitrogen, Carlsbad, CA). The 1× pSp1GLECAT contains STAT-5-responsive sequence of the human Sp2.1 promoter and thereby drives the CAT reporter gene expression.32Benitah SA Valeron PF Rui H Lacal JC STAT5a activation mediates the epithelial to mesenchymal transition induced by oncogenic RhoA.Mol Biol Cell. 2003; 14: 40-53Crossref PubMed Scopus (38) Google Scholar Cells were then quiesced and treated with and without PDGF-BB (20 ng/ml) for the indicated times, and cell extracts were prepared. In the case of testing the role of STAT-5B, cells were transduced first with the control Ad-GFP or Ad-dnSTAT-5B virus followed by transfection with pSp1GLECAT plasmid DNA. Cell extracts were normalized for protein and assayed for CAT activity using [14C]chloramphenicol and acetyl coenzyme A as substrates as described previously.18Dronadula N Liu Z Wang C Cao H Rao GN STAT-3-dependent expression of cPLA2 is required for thrombin-induced vascular smooth muscle cell motility.J Biol Chem. 2005; 280: 3112-3120Crossref PubMed Scopus (35) Google Scholar ChIP assay was performed on VSMCs by using the ChIP assay kit following the supplier's protocol (Upstate Biotechnology). STAT-5B-DNA complexes were immunoprecipitated using anti-STAT-5B antibodies (catalog no. SC-1656 from Santa Cruz Biotechnology or catalog no. 06-969 from Upstate Biotechnology). Preimmune serum (SC-2338) was used as a negative control. Precipitated DNA was extracted using phenol-chloroform, and the DNA fragments from the aqueous phase were purified using QIAquick columns (catalog no. 28104; Qiagen, Valencia, CA). The purified DNA was used as template for polymerase chain reaction (PCR) amplification using primers (forward, 5′-CAACGAAGCCAATCGGGAAGCTTC-3′; reverse, 5′-CACCCTATACTTAAGCGGAGAGAA-3′) flanking the putative STAT-binding site located at −978 in rat cyclin D1 promoter region.33Kitazawa S Kitazawa R Maeda S Transcriptional regulation of rat cyclin D1 gene by CpG methylation status in promoter region.J Biol Chem. 1999; 274: 28787-28793Crossref PubMed Scopus (77) Google Scholar The PCR products were resolved on 2% agarose gel and stained with ethidium bromide. After appropriate treatments, protein extracts from cells or tissues were prepared using lysis buffer (20 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 1% Nonidet P-40, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 50 mmol/L glycerophosphate, 10 mmol/L NaF, and 1 mmol/L sodium orthovanadate). An equal quantity of total protein (200 μg) from each sample was immunoprecipitated with anti-CDK4 antibodies (2 μg) overnight at 4°C followed by incubation with protein-A-Sepharose beads for 1 hour at room temperature. CDK4 activity in the immunocomplexes was assayed at 30°C in a kinase reaction mix that contained 25 mmol/L HEPES, pH 7.4, 10 mmol/L MgCl2, 1 mmol/L EGTA, 200 μg/ml truncated Rb protein, 1 μCi of [γ32P]ATP, and 50 μmol/L ATP. After 30 minutes of incubation, the kinase reaction was stopped by the addition of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer and subsequent boiling for 5 minutes. After heat denaturation, the reaction products were separated by electrophoresis on 0.1% sodium dodecyl sulfate and 10% polyacrylamide gels. The 32P-labeled Rb protein was visualized by autoradiography, and the band intensities were quantitated using NIH Image J (National Institutes of Health, Bethesda, MD). DnSTAT-5A and dnSTAT-5B expression plasmids were constructed by deletion of their C-terminal regions starting from amino acids 713 to 793 in the case of the former one and from amino acids 718 to 793 in the case of the latter one.34Wang D Stravopodis D Teglund S Kitazawa J Ihle JN Naturally occurring dominant negative variants of Stat5.Mol Cell Biol. 1996; 16: 6141-6148Crossref PubMed Scopus (226) Google Scholar Construction of Ad-GFP, Ad-dnSTAT-5A, and Ad-dnSTAT-5B were described previously.15Liu Z Zhang C Dronadula N Li Q Rao GN Blockade of nuclear factor of activated T cells activation signaling suppresses balloon injury-induced neointima formation in a rat carotid artery model.J Biol Chem. 2005; 280: 14700-14708Crossref PubMed Scopus (83) Google Scholar, 29Cao H Dronadula N Rizvi F Li Q Gerthoffer WT Rao GN A novel role for STAT-5B in the regulation of Hsp27-FGF-2 axis facilitating thrombin-induced vascular smooth muscle cell DNA synthesis and motility.Circ Res. 2006; 98: 913-922Crossref PubMed Scopus (30) Google Scholar After balloon injury, solutions (100 μl) of Ad-GFP (1010 pfu/ml) or Ad-dnSTAT-5B (1010 pfu/ml) were infused into the ligated segment of the common carotid artery for 30 minutes as described previously.15Liu Z Zhang C Dronadula N Li Q Rao GN Blockade of nuclear factor of activated T cells activation signaling suppresses balloon injury-induced neointima formation in a rat carotid artery model.J Biol Chem. 2005; 280: 14700-14708Crossref PubMed Scopus (83) Google Scholar Viral transductions were evaluated at 1 week after balloon injury by isolation of arteries followed either by cryostat cross-sectioning and examining for green fluorescence protein expression or by preparing tissue extracts and Western blot analysis of GFP levels using its specific antibodies. All of the animal protocols were performed in accordance with the relevant guidelines and regulations approved by the Internal Animal Care and Use Committee of the University of Tennessee Health Science Center. Balloon injury was performed essentially as described previously by us.15Liu Z Zhang C Dronadula N Li Q Rao GN Blockade of nuclear factor of activated T cells activation signaling suppresses balloon injury-induced neointima formation in a rat carotid artery model.J Biol Chem. 2005; 280: 14700-14708Crossref PubMed Scopus (83) Google Scholar In brief, rats weighing 250 to 300 g were anesthetized by injecting (intraperitoneally) ketamine (60 mg/kg) and xylazine (5 mg/kg). Under a stereomicroscope, the right common, external and internal, carotid arteries were exposed by a longitudinal midline cervical incision, and blood flow was temporarily interrupted by ligation of the common and internal carotid arteries using vessel clips. External carotid artery was ligated permanently. A 2F Fogarty arterial embolectomy catheter was introduced through an arteriotomy in the external carotid artery just below the ligature and advanced to the common carotid artery. To produce carotid artery injury, the balloon was inflated with saline and passed six times with rotation from just under the proximal edge of the o" @default.
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• W2015584527 date "2007-10-01" @default.
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• W2015584527 title "Suppression of Activation of Signal Transducer and Activator of Transcription-5B Signaling in the Vessel Wall Reduces Balloon Injury-Induced Neointima Formation" @default.
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