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• W2101781012 abstract "The proteoglycan versican is pro-atherogenic and central to vascular injury and repair events. We identified the signaling pathways and promoter elements involved in regulation of versican expression in vascular smooth muscle cells. Phosphatidylinositol 3-kinase inhibitor, LY294002, significantly decreased versican-luciferase (Luc) promoter activity and endogenous mRNA levels. We further examined the roles of protein kinase B and glycogen synthase kinase (GSK)-3β, downstream effectors of phosphatidylinositol 3-kinase, in the regulation of versican transcription. Co-transfection of dominant negative and constitutively active protein kinase B constructs with a versican-Luc construct decreased and increased promoter activity, respectively. Inhibition of GSK-3β activity by LiCl augmented accumulation of β-catenin and caused induction of versican-Luc activity as well as versican mRNA levels. β-Catenin has no DNA binding domain, therefore it cannot directly induce transcription of the versican promoter. Software analysis of the versican promoter revealed two potential binding sites for T-cell factors (TCFs), proteins that confer transcriptional activation of β-catenin. Electrophoretic mobility shift and supershift assays revealed specific binding of human TCF-4 and β-catenin to oligonucleotides corresponding to a potential TCF binding site in the versican promoter. In addition to binding assays, we directly assessed the dependence of versican promoter activity on TCF binding sites. Site-directed mutagenesis of the TCF site located -492 bp relative to the transcription start site markedly diminished versican-Luc activity. Co-transfection of TCF-4 with versican-Luc did not increase promoter activity, but addition of β-catenin and TCF-4 significantly stimulated basal versican promoter activity. Our findings suggest that versican transcription is predominantly mediated by the GSK-3β pathway via the β-catenin-TCF transcription factor complex in smooth muscle cells, wherein such regulation contributes to the normal or aberrant formation of provisional matrix in vascular injury and repair events. The proteoglycan versican is pro-atherogenic and central to vascular injury and repair events. We identified the signaling pathways and promoter elements involved in regulation of versican expression in vascular smooth muscle cells. Phosphatidylinositol 3-kinase inhibitor, LY294002, significantly decreased versican-luciferase (Luc) promoter activity and endogenous mRNA levels. We further examined the roles of protein kinase B and glycogen synthase kinase (GSK)-3β, downstream effectors of phosphatidylinositol 3-kinase, in the regulation of versican transcription. Co-transfection of dominant negative and constitutively active protein kinase B constructs with a versican-Luc construct decreased and increased promoter activity, respectively. Inhibition of GSK-3β activity by LiCl augmented accumulation of β-catenin and caused induction of versican-Luc activity as well as versican mRNA levels. β-Catenin has no DNA binding domain, therefore it cannot directly induce transcription of the versican promoter. Software analysis of the versican promoter revealed two potential binding sites for T-cell factors (TCFs), proteins that confer transcriptional activation of β-catenin. Electrophoretic mobility shift and supershift assays revealed specific binding of human TCF-4 and β-catenin to oligonucleotides corresponding to a potential TCF binding site in the versican promoter. In addition to binding assays, we directly assessed the dependence of versican promoter activity on TCF binding sites. Site-directed mutagenesis of the TCF site located -492 bp relative to the transcription start site markedly diminished versican-Luc activity. Co-transfection of TCF-4 with versican-Luc did not increase promoter activity, but addition of β-catenin and TCF-4 significantly stimulated basal versican promoter activity. Our findings suggest that versican transcription is predominantly mediated by the GSK-3β pathway via the β-catenin-TCF transcription factor complex in smooth muscle cells, wherein such regulation contributes to the normal or aberrant formation of provisional matrix in vascular injury and repair events. Findings from our laboratory and others indicate that the proteoglycan (PG) 1The abbreviations used are: PG, proteoglycan; ECM, extracellular matrix; GSK, glycogen synthase kinase; Luc, luciferase; MCDB, molecular and cellular developmental biology-131; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; SMC, smooth muscle cell; TCF, T-cell factor; LEF, lymphoid enhancer factor; PDGF, platelet-derived growth factor; DN-PKB, dominant negative mutant of PKB1 construct; CA-PKB, constitutively activated mutant of PKB1; CtBP, C-terminal binding protein; wt-PTEN, wild-type PTEN; mut-PTEN, mutant PTEN; EMSA, electrophoretic mobility shift assay.1The abbreviations used are: PG, proteoglycan; ECM, extracellular matrix; GSK, glycogen synthase kinase; Luc, luciferase; MCDB, molecular and cellular developmental biology-131; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; SMC, smooth muscle cell; TCF, T-cell factor; LEF, lymphoid enhancer factor; PDGF, platelet-derived growth factor; DN-PKB, dominant negative mutant of PKB1 construct; CA-PKB, constitutively activated mutant of PKB1; CtBP, C-terminal binding protein; wt-PTEN, wild-type PTEN; mut-PTEN, mutant PTEN; EMSA, electrophoretic mobility shift assay. versican is one of several extracellular matrix (ECM) molecules that accumulates in vascular lesions (1.Farb A. Kolodgie F.D. Hwang J.Y. Burke A.P. Tefera K. Weber D.K. Wight T.N. Virmani R. Circulation. 2004; 110: 940-947Crossref PubMed Scopus (186) Google Scholar, 2.Lin H. Wilson J.E. Roberts C.R. Horley K.J. Winters G.L. Costanzo M.R. McManus B.M. J. Heart Lung Transplant. 1996; 15: 1233-1247PubMed Google Scholar, 3.Lin H. Kanda T. Hoshino Y. Takase S. Kobayashi I. Nagai R. McManus B.M. Cardiovasc. Pathol. 1998; 7: 31-37Crossref PubMed Scopus (9) Google Scholar). Versican is generally considered to be pro-atherogenic because of its ability to trap cholesterol-rich lipoproteins (4.Williams K.J. Tabas I. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 551-561Crossref PubMed Google Scholar, 5.Williams K.J. Curr. Opin. Lipidol. 2001; 12: 477-487Crossref PubMed Scopus (71) Google Scholar, 6.Rahmani M. McDonald P.C. Wong B.W. McManus B.M. Can. J. Cardiol. 2004; 20: 58B-65BPubMed Google Scholar) in addition to its crucial role in regulation of cell adhesion, survival, proliferation, and migration and ECM assembly, all fundamental processes involved in vascular disease (4.Williams K.J. Tabas I. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 551-561Crossref PubMed Google Scholar, 5.Williams K.J. Curr. Opin. Lipidol. 2001; 12: 477-487Crossref PubMed Scopus (71) Google Scholar, 6.Rahmani M. McDonald P.C. Wong B.W. McManus B.M. Can. J. Cardiol. 2004; 20: 58B-65BPubMed Google Scholar, 7.Wight T.N. Merrilees M.J. Circ. Res. 2004; 94: 1158-1167Crossref PubMed Scopus (290) Google Scholar). The complete versican gene structure has been elucidated in humans (8.Iozzo R.V. Naso M.F. Cannizzaro L.A. Wasmuth J.J. McPherson J.D. Genomics. 1992; 14: 845-851Crossref PubMed Scopus (52) Google Scholar, 9.Naso M.F. Zimmermann D.R. Iozzo R.V. J. Biol. Chem. 1994; 269: 32999-33008Abstract Full Text PDF PubMed Google Scholar) and the mouse (10.Naso M.F. Morgan J.L. Buchberg A.M. Siracusa L.D. Iozzo R.V. Genomics. 1995; 29: 297-300Crossref PubMed Scopus (29) Google Scholar). The human and murine genes prove to be remarkably conserved in genomic organization. Both versican genes extend for ∼90 kb and contain 15 exons that align in an identical manner with the protein sub-domains. Naso et al. (9.Naso M.F. Zimmermann D.R. Iozzo R.V. J. Biol. Chem. 1994; 269: 32999-33008Abstract Full Text PDF PubMed Google Scholar) report that the human versican gene has one transcription start site. Meanwhile, sequence analysis reveals potential binding sites for several transcription factors in addition to the TATA box. Transient expression assays of reporter constructs driven by an 876-bp (-632/+240 relative to the transcriptional start site) piece of the versican promoter in HeLa cells and IMR-90 embryonic lung fibroblasts have shown significant expression. These results indicate that the human versican 5′-flanking sequence contains promoter and enhancer elements able to drive reporter gene expression in cells derived from epithelial or mesenchymal tissues (9.Naso M.F. Zimmermann D.R. Iozzo R.V. J. Biol. Chem. 1994; 269: 32999-33008Abstract Full Text PDF PubMed Google Scholar). Various growth factors and mediators influence the expression of versican. Studies using arterial smooth muscle cells (SMCs) have demonstrated that transforming growth factor β1 and platelet-derived growth factor (PDGF)-AB increase versican mRNA levels, core protein synthesis, and glycosaminoglycan chain length (11.Schonherr E. Jarvelainen H.T. Sandell L.J. Wight T.N. J. Biol. Chem. 1991; 266: 17640-17647Abstract Full Text PDF PubMed Google Scholar). Similarly, normal human gingival fibroblasts respond to treatment with either transforming growth factor β or PDGF-BB by increasing versican mRNA levels (12.Kahari V.M. Larjava H. Uitto J. J. Biol. Chem. 1991; 266: 10608-10615Abstract Full Text PDF PubMed Google Scholar, 13.Haase H.R. Clarkson R.W. Waters M.J. Bartold P.M. J. Cell. Physiol. 1998; 174: 353-361Crossref PubMed Scopus (53) Google Scholar). In contrast, the pro-inflammatory cytokine interleukin-1β appears to decrease the synthesis of this PG in human gingival fibroblasts and arterial SMCs (14.Qwarnstrom E.E. Jarvelainen H.T. Kinsella M.G. Ostberg C.O. Sandell L.J. Page R.C. Wight T.N. Biochem. J. 1993; 294: 613-620Crossref PubMed Scopus (29) Google Scholar). Data from recent investigations suggest that versican synthesis by mesanchymal cells can be regulated by physical stimuli, including cell density and mechanical strain (15.Lee R.T. Yamamoto C. Feng Y. Potter-Perigo S. Briggs W.H. Landschulz K.T. Turi T.G. Thompson J.F. Libby P. Wight T.N. J. Biol. Chem. 2001; 276: 13847-13851Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). In vitro experiments using monkey arterial SMCs have shown that PDGF-BB stimulates versican core protein expression; this signaling apparently occurs through a receptor tyrosine kinase-dependent, protein kinase C-independent pathway (16.Schonherr E. Kinsella M.G. Wight T.N. Arch. Biochem. Biophys. 1997; 339: 353-361Crossref PubMed Scopus (77) Google Scholar). Angiotensin II-mediated stimulation of SMC versican expression is regulated by epidermal growth factor receptor-dependent tyrosine kinase pathways (17.Shimizu-Hirota R. Sasamura H. Mifune M. Nakaya H. Kuroda M. Hayashi M. Saruta T. J. Am. Soc. Nephrol. 2001; 12: 2609-2615PubMed Google Scholar). The canonical Wnt-wingless signaling pathway regulates various biologic processes including early embryogenesis and neoplasia by increasing the stability and transcriptional activity of a key mediator, β-catenin (18.Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar, 19.Taipale J. Beachy P.A. Nature. 2001; 411: 349-354Crossref PubMed Scopus (1172) Google Scholar, 20.Korswagen H.C. Clevers H.C. Cold Spring Harbor Symp. Quant. Biol. 1999; 64: 141-147Crossref PubMed Scopus (22) Google Scholar). In the absence of Wnt ligand, GSK-3 promotes the phosphorylation of β-catenin at key serine/threonine residues, targeting it for degradation through the ubiquitin-ligase pathway (21.Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2143) Google Scholar). In response to Wnt, the GSK-3-binding protein inhibits GSK-3 activity (22.Ding V.W. Chen R.H. McCormick F. J. Biol. Chem. 2000; 275: 32475-32481Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Some growth factors can regulate GSK-3 activity by mediating its phosphorylation at serine 9 independent of Wnt ligand. Although serine 9 phosphorylation of GSK-3 is associated with its inactivation, Wnt ligand does not necessarily regulate this phosphorylation (23.Doble B.W. Woodgett J.R. J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1748) Google Scholar). GSK-3 inactivation leads to β-catenin stabilization and translocation into the nucleus, where it binds to T-cell factor(TCF)/lymphoid enhancer factor (LEF) family proteins to form a transcription factor complex that activates target genes such as the matrix metalloproteinase-7, fibronectin, vascular endothelial growth factor, cyclin D1, and c-myc (24.Goodwin A.M. D'Amore P.A. Angiogenesis. 2002; 5: 1-9Crossref PubMed Scopus (152) Google Scholar). A variety of mitogenic stimuli including Wnts, insulin, epidermal growth factor, and PDGF result in catalytic inactivation of GSK-3. The catalytic inactivation of GSK-3 induced by most polypeptide mitogens is reversible by treatment with serine/threonine-specific phosphates (25.Cross D.A. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (420) Google Scholar). The inactivation event has been demonstrated to be due to phosphorylation of serine 21 and serine 9 of GSK-3α and GSK-3β, respectively (26.Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (751) Google Scholar, 27.Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (504) Google Scholar, 28.Shaw M. Cohen P. Alessi D.R. FEBS Lett. 1997; 416: 307-311Crossref PubMed Scopus (214) Google Scholar). These residues are specific targets for several protein-serine kinases, including PKB, pp90rsk, and cyclic AMP-dependent protein kinase A (29.Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4337) Google Scholar, 30.Fang X. Yu S.X. Lu Y. Bast Jr., R.C. Woodgett J.R. Mills G.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11960-11965Crossref PubMed Scopus (634) Google Scholar). The inactivating biochemical consequence of phosphorylation by all three enzymes is identical; what differs is the initiating signal. Thus, activation of the phosphatidylinositol 3-kinase (PI3K) pathway (usually via receptor tyrosine kinase activation) results in stimulation of PKB. Inactivation of GSK-3 in response to many mitogens can be inhibited by antagonists of PI3K such as LY294002 inhibitor (25.Cross D.A. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (420) Google Scholar). These mechanisms are all independent of Wnt-induced regulation of GSK-3. Most protein kinases are induced by cellular stimuli, whereas GSK-3 is shut down. In addition, the enzyme has a broad variety of target proteins, most of which are inactivated by phosphorylation of GSK-3. Thus, inhibition of this one enzyme will tend to induce the functions of a diverse array of targets including transcription factors and other regulatory molecules (31.Hardt S.E. Sadoshima J. Circ. Res. 2002; 90: 1055-1063Crossref PubMed Scopus (332) Google Scholar). Despite the importance of versican in vascular pathophysiology, the function and regulation of expression of this versatile molecule in vitro and in vivo are unknown. Our results suggest that a signaling molecule activated by 3-phosphoinositides, namely PKB, plays a critical role in serum-stimulated versican transcription. Furthermore, we provide evidence that phosphorylation and inhibition of GSK-3β by PKB and subsequent activation of the β-catenin-TCF complex are essential for transcription from the versican promoter. Isolation and Primary Culture of Rat Aortic SMCs—A rat aortic SMC culture was established by a modification of the enzymatic dispersion technique (32.Thyberg J. Int. Rev. Cytol. 1996; 169: 183-265Crossref PubMed Google Scholar, 33.Campbell J.H. Campbell G.R. Clin. Sci. (Lond.). 1993; 85: 501-513Crossref PubMed Scopus (136) Google Scholar). Briefly, four adult male Fisher rats (275–350 g) were euthanized in accordance with ethical guidelines set by the University of British Columbia Animal Care Committee. The thoracic aorta was removed and immediately washed in MCDB-131 medium (Sigma-Aldrich). Enzyme I (0.5 mg/ml collagenase II; Worthington Biochemical Corp., Freehold, NJ) was applied to the exposed media, and the tissue was incubated for 20 min at 37 °C to loosen the media from the underlying adventitia. Medial strips were removed with sterile forceps, taking care not to reach the adventitial layer, transferred to a 35-mm tissue culture dish containing 500 μl of Enzyme II (0.5 mg/ml collagenase II, 0.2 mg/ml elastase; Worthington Biochemical Corp.), and minced. Medial tissue from the aortas of all four rats was pooled, additional Enzyme II solution was added to the dish, and the tissue was incubated at 37 °C for 2.5 h with pipetting at regular intervals to disperse cells. Liberated cells were subsequently pelleted at 1000 rpm for 5 min, resuspended in 1 ml of MCDB-131 containing 20% newborn calf serum, and seeded into a 35-mm tissue culture dish. Cells were grown to confluence, released by trypsinization, and subcultured at a density of 1.0 × 104 cells/cm2 in MCDB-131 supplemented with 5% newborn calf serum. Cell Culture—Rat vascular SMCs were maintained in MCDB-131 plus 5% newborn calf serum as described previously in a controlled atmosphere of air-CO2 (5%) at 37 °C until confluence (6–7 days). Confluent SMCs at passages 4–8 were used for experiments. Human prostate cancer PC3 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 5% fetal bovine serum (Invitrogen) at 37 °C in 5% CO2. A stable HeLa cell line expressing dominant negative mutant of PKB1 construct (DN-PKB), constitutively activated mutant of PKB1 (CA-PKB), and corresponding empty plasmids was generated and selected as described previously (34.Esfandiarei M. Luo H. Yanagawa B. Suarez A. Dabiri D. Zhang J. McManus B.M. J. Virol. 2004; 78: 4289-4298Crossref PubMed Scopus (104) Google Scholar). Generation of Promoter Reporter, Mutant, and Deletion Constructs—A 752-bp versican promoter (-634/+118) and a shorter fragment (-438/+118) corresponding to the versican promoter sequences (9.Naso M.F. Zimmermann D.R. Iozzo R.V. J. Biol. Chem. 1994; 269: 32999-33008Abstract Full Text PDF PubMed Google Scholar) were generated by PCR from human genomic DNA with the appropriate sets of primers (available upon request). These inserts were cloned into a pGL3 basic vector (Promega) by standard molecular biology techniques and called wt-versican-Luc and -438del-versican-Luc as depicted in Fig. 5A. The putative TCF binding sites (TCCCTTTGATGG and TTCTTTGCTGAT at positions -546 and -492 bp, respectively) contained in the wt-versican-Luc were mutated as denoted in Fig. 5A by site-directed mutagenesis using a QuikChange mutagenesis kit from Stratagene as described previously (35.Yeung L.H. Read J.T. Sorenson P. Nelson C.C. Gleave M. Jia W. Rennie P.S. Biochem. J. 2003; 371: 843-855Crossref PubMed Google Scholar). The mutated inserts were generated by PCR and then inserted into the promoterless luciferase vector pGL3-Basic. All constructs were verified by sequencing. Plasmid Constructs—The DN-PKB (Upstate Biotechnology), CA-PKB (Upstate Biotechnology), and empty vector control were used in transient and stable transfection of SMCs and HeLa cells, respectively. The cDNAs for wild-type PTEN (wt-PTEN) and its mutant (mut-PTEN) were kindly provided by J. Dixon (Department of Pharmacology, University of California, San Diego, CA) and subcloned into pXJ41-neo expression vector kindly provided by C. Pallen (Department of Pediatrics, University of British Columbia, Vancouver, British Columbia, Canada). A dominant stable β-catenin construct was a kind gift of B. Gumbiner (Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY). The expression vectors TCF-1E and TCF-4E harboring the wild-type human TCF-4 gene and chimeric TCF-1 construct lacking C-terminal binding protein (CtBP) structural domain, respectively, were a gift from A. Hecht (Max-Planck-Institute of Immunobiology, Freiburg, Germany) (36.Hecht A. Stemmler M.P. J. Biol. Chem. 2003; 278: 3776-3785Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The myc-tagged dominant negative TCF-4 (ΔNTCF-4) was kindly provided by H. Clevers (Department of Immunology, University Medical Center Utrecht, The Netherlands) (37.Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2918) Google Scholar). Wild-type GSK-3β was a kind gift from G. Cooper (Department of Pathology, Harvard Medical School, Boston, MA) (38.Pap M. Cooper G.M. J. Biol. Chem. 1998; 273: 19929-19932Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar). Transfection and Luciferase Activity Assays—Starved SMCs were transiently transfected in 6-well plates using up to 2 μg of plasmid DNA and FuGENE 6 reagent (Roche Applied Science) according to the procedure recommended by the manufacturer. In brief, a 3:1 ratio of FuGENE 6 reagent (in microliters) to plasmid (in micrograms) was incubated for 30 min at room temperature in incomplete medium before addition to 70–80% subconfluent cells in medium containing the mediator of interest or in complete medium for the period of time indicated in the figure legends. After the indicated incubation period, cells were lysed, and luciferase activities were measured with a kit from Promega according to the manufacturer's protocol. Protein concentrations were measured with a Bradford protein assay kit from Bio-Rad, and luciferase values were normalized to the obtained protein concentrations. In some transfection experiments, normalization was done by LacZ reporter (Promega), and β-galactosidase assays were performed according to the manufacturer's protocol. Immunoblotting—SMCs were grown in 6-well plates. Cells were lysed with 200 μl of lysis buffer. For PKB, lysis buffer contained 50 mm HEPES (pH 7.6), 1 mm EDTA, 5 mm EGTA, 10 mm MgCl2, 50 mm β-glycerophosphate, 1 mm Na3VO4, 10 mm NaF, 30 mm sodium pyrophosphate, 2 mm dithiothreitol, and 1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride. For GSK-3, lysis buffer contained 20 mm Tris (pH 7.5), 25 mm β-glycerophosphate, 100 mm NaCl, 1 mm Na3VO4, 2 mm EGTA, 2 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride. Samples were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with anti-phospho-PKB (Ser-473; Cell Signaling Technology) or anti-phospho-GSK-3 (Ser-9; Oncogene Research Product) antibodies. Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies were used as secondary antibodies. The levels of wt-PTEN and mut-PTEN were determined 48 h post-transfection of PC3 cells by Western blot analysis using antibody against PTEN (Santa Cruz Biotechnology). The bound secondary antibody was detected by enhanced chemiluminescence (Amersham Biosciences). RNA Extraction and cDNA Synthesis—Total RNA was isolated from treated and untreated SMCs using the RNeasy Mini Kit according to the manufacturer's protocol (Qiagen). All preparations were treated with RNase-free DNase (Qiagen) to remove genomic DNA. RNA (0.5–1 μg) was reverse transcribed in a total volume of 20 μl in the presence of 200 units of SuperScript RNase H-Reverse Transcriptase (Invitrogen), 40 units of RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen), and 0.5 μg of Oligo(dT) Primer (Invitrogen) according to the manufacturer's instructions. SYBR Green Quantitative Real-time Reverse Transcription-PCR— Quantitative two-step real-time reverse transcription-PCR was performed using a LightCycler (Roche Applied Science) in order to assess versican mRNA expression in SMCs. β-Actin was used as a housekeeping gene. Primer pairs were designed to flank an intron-containing sequence. PCR conditions used included 3 mm Mg2+, 0.3 μm forward and reverse primers, and 2 μl of LightCycler FastStart DNA Master SYBR Green I Mix (Roche Applied Science) in a final volume of 20 μl. The samples were loaded in the LightCycler glass capillaries, closed, centrifuged, and placed in the LightCycler rotor. The cycling program consisted of 10 min of initial denaturation at 95 °C, 45 cycles of 95 °C for 5 s, 55 °C for 5 s, and 72 °C for 20 s, and single detection for 1 s with a single fluorescence acquisition (ramp rates, 20 °C/s). The analytical melting program was 95 °C for 0 s and 65 °C for 15 s, increasing to 95 °C at a ramp rate of 0.2 °C/s, with continuous fluorescence acquisition. Each sample was run in triplicate. A standard curve was included in each run. Standards were prepared by cloning the target sequence into plasmid DNA. The data were analyzed by using the second-derivative maximum of each amplification reaction and relating it to its respective standard curve. The results from the quantitative PCR were expressed as the ratio of the mean target gene measurements to the mean housekeeping gene value for a given sample (target:reference). Electrophoretic Mobility Shift Assay—Individual oligonucleotides were 5′-end [γ-32P]deoxyadenine triphosphate-labeled with T4 polynucleotide kinase. Labeled oligonucleotides (100 ng) were annealed to equimolar amounts of their complementary strands (unlabeled) by heating to 95 °C for 5 min in Tris-EDTA supplemented with 50 mm NaCl and slowly cooling to room temperature. Double-stranded oligonucleotide probes were purified on a 5% (w/v) polyacrylamide, 0.5× Tris-borate EDTA non-denaturing gel; eluted in 500 μl of elution buffer (0.6 m NH4OAC, 0.1% (w/v) SDS, and 1 mm EDTA); and ethanol precipitated prior to use in electrophoretic mobility shift assays (EMSAs). Nuclear extracts used in EMSAs were isolated from SW480 human colorectal carcinoma cells, using a modified version of the method of Dignam et al. (39.Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9150) Google Scholar). Final nuclear protein preparations were collected in buffer C (400 mm NaCl, 20 mm HEPES (pH 7.4), 25% glycerol, 1.5 mm MgCl2, 0.2 mm EDTA, and 0.5 mm dithiothreitol). For band shift experiments, 20,000 cpm of labeled oligoduplex probes were added to 5 μl of nuclear extracts in 40 μl of DNA binding buffer (10% glycerol, 1 mm EDTA, 1 mm dithiothreitol, and 60 mm KCl). To prevent nonspecific binding of nuclear proteins, poly(dI-dC) was added to a concentration of 100 ng/μl, and the binding reaction was incubated at room temperature for 10 min prior to electrophoresis. For supershift experiments, 1 μg of goat anti-TCF-4 polyclonal antibody (N-20; 0.4 μg/μl; Santa Cruz Biotechnology) was added to the binding reaction and incubated for 10 min at room temperature prior to electrophoresis. For EMSA interference experiments, 0.2 and 0.4 μg of goat anti-β-catenin polyclonal antibody was used (H-102; 0.4 μg/μl; Santa Cruz Biotechnology). For both supershift and interference experiments, antibody control reactions contained an equivalent mass of normal mouse IgG, and antibody negative reactions were supplemented with an equal volume of phosphate-buffered saline. Protein-DNA complexes were separated from unbound DNA using a 5% (w/v) polyacrylamide, 0.5× Tris-borate EDTA non-denaturing gel run at a constant voltage of 400 V for 1 h. The oligonucleotides corresponding to the TCF/LEF site from versican promoter used for EMSAs were as follows: 5′-ACTTCCCTTTGATGGGACAG-3′ and 5′-CTGTCCCATCAAAGGGAAGT-3′. The 3-Phosphoinositide-dependent Signaling Mediates Versican Transcription in Vascular SMCs—We examined the role of the PI3K-PKB pathway in induction of versican transcription as a result of serum stimulation in vascular SMCs. First, the levels of activated PKB were determined by Western blot analysis using phosphospecific antibody, after a 30-min pre-incubation of quiescent SMCs with different concentrations of LY294002, a pharmacological inhibitor of PI3K, followed by serum stimulation. LY294002 inhibited activation of downstream PKB at all concentrations tested (Fig. 1A). Next we examined whether activation of PI3K and subsequent generation of 3-phosphoinositides is necessary for the stimulation of versican transcription by serum. We transfected SMCs with wild-type versican-Luc reporter construct, and 24 h after transfection, serum-starved, growth-arrested cells were pretreated with LY294002 for 30 min prior to stimulation with 5% newborn calf serum for 24 h. Versican promoter activation was significantly inhibited (Fig. 1B). We next determined the role of the PI3K pathway in the induction of expression of versican mRNA by real-time reverse transcription-PCR analysis. SMCs were growth-arrested for 24 h, pretreated with LY294002 for 30 min, and stimulated with 5% serum for 24 h. The induction of versican mRNA expression by serum was significantly suppressed by LY294002 pretreatment (Fig. 1C). These results suggest that reduced production of 3-phosphoinositides inhibits versican transcription (Fig. 1, A and B). To examine further the role of 3-phosphoinositides, we investigated the role of PTEN (phosphatase and tensin homolog deleted on chromosome 10) in control of versican expression. PTEN is a phosphatase that selectively dephosphorylates the 3 position of both phosphatidylinositol-3,4,5-trisphosphate and phosphatidylinositol-3,4-bisphosphate (40.Maehama T. Dixon J.E. Trends Cell Biol. 1999; 9: 125-128Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar), antagonizing the diverse downstream signaling effector pathways activated by PI3K-derived phospholipids. To investigate the effects of this protein on versican expression levels, we overexpressed it in a cell line normally lacking PTEN, the PC3 prostate" @default.
• W2101781012 created "2016-06-24" @default.
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• W2101781012 title "Regulation of the Versican Promoter by the β-Catenin-T-cell Factor Complex in Vascular Smooth Muscle Cells" @default.
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