Platelet-Derived Growth Factor-BB Mediates Cell Migration through Induction of Activating Transcription Factor 4 and Tenascin-C
2012; Elsevier BV; Volume: 180; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2012.02.009
ISSN1525-2191
AutoresKristine P. Malabanan, Anjali V. Sheahan, Levon M. Khachigian,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoThe acute response to vascular cell injury, which underpins vasculo-occlusive pathologies such as atherogenesis and restenosis after percutaneous coronary intervention, involves a complex series of molecular events that alter patterns of gene expression and favor a synthetic phenotype. One transcription factor that has been implicated in this process is the evolutionarily conserved mammalian stress response pathway regulator activating transcription factor 4 (ATF-4). Here, we show for the first time that both mRNA and protein levels of ATF-4 are induced in smooth muscle cells (SMCs) by the potent migratory factor PDGF-BB through PDGFR-β. PDGF-BB also stimulates the expression of tenascin-C (TN-C), an extracellular matrix glycoprotein that regulates the activity of focal adhesion complexes, facilitating the SMC migration that underlies negative vascular remodeling in response to injury. Overexpression of ATF-4 increased transcript levels of the four TN-C isoforms in rat vascular SMCs, and ATF-4 knockdown inhibited PDGF-BB-inducible TN-C expression in vitro and injury-inducible TN-C protein expression in the balloon-injured rat artery wall. Furthermore, we show that ATF-4 is required for PDGF-BB-inducible SMC migration in response to injury. PDGF-BB-induced migration was also compromised in ATF-4 null mEFs, and this effect was rescued by the addition of TN-C. Our findings thus demonstrate the role of ATF-4 in both injury- and PDGF-BB-inducible TN-C expression and cell migration. The acute response to vascular cell injury, which underpins vasculo-occlusive pathologies such as atherogenesis and restenosis after percutaneous coronary intervention, involves a complex series of molecular events that alter patterns of gene expression and favor a synthetic phenotype. One transcription factor that has been implicated in this process is the evolutionarily conserved mammalian stress response pathway regulator activating transcription factor 4 (ATF-4). Here, we show for the first time that both mRNA and protein levels of ATF-4 are induced in smooth muscle cells (SMCs) by the potent migratory factor PDGF-BB through PDGFR-β. PDGF-BB also stimulates the expression of tenascin-C (TN-C), an extracellular matrix glycoprotein that regulates the activity of focal adhesion complexes, facilitating the SMC migration that underlies negative vascular remodeling in response to injury. Overexpression of ATF-4 increased transcript levels of the four TN-C isoforms in rat vascular SMCs, and ATF-4 knockdown inhibited PDGF-BB-inducible TN-C expression in vitro and injury-inducible TN-C protein expression in the balloon-injured rat artery wall. Furthermore, we show that ATF-4 is required for PDGF-BB-inducible SMC migration in response to injury. PDGF-BB-induced migration was also compromised in ATF-4 null mEFs, and this effect was rescued by the addition of TN-C. Our findings thus demonstrate the role of ATF-4 in both injury- and PDGF-BB-inducible TN-C expression and cell migration. The response to vascular cell injury is a complex process of transcription and vascular remodeling exacerbated by the penetration of inflammatory cells into the damaged vessel, which in turn creates the activated environment that stimulates the migration of smooth muscle cells (SMC) from the media into the intima.1Libby P. Ridker P.M. Maseri A. Inflammation and atherosclerosis.Circulation. 2002; 105: 1135-1143Crossref PubMed Scopus (5900) Google Scholar, 2Libby P. Schwartz S.M. Brogi E. Tanaka H. Clinton S. A cascade model for restenosis.Circ Res. 1992; 86: III47-III52Google Scholar SMC hyperplasia contributes to the formation of the lesion, with loss of lumen diameter and vascular contractility, processes that underpin pathologies such as atherogenesis and restenosis after percutaneous coronary intervention. SMC migration and hyperplasia are preceded by the modulation of SMC phenotype from a contractile state to a synthetic state, characterized by a decrease in the number of myofilaments and contractile proteins and the development of a more epithelioid shape, resembling its embryonic dedifferentiated counterparts.3Willis A.I. Pierre-Paul D. Sumpio B.E. Gahtan V. Vascular smooth muscle cell migration: current research and clinical implications.Vasc Endovascular Surg. 2004; 38: 11-23Crossref PubMed Scopus (90) Google Scholar, 4Campbell G.R. Campbell J.H. Smooth muscle phenotypic changes in arterial wall homeostasis: implications for the pathogenesis of atherosclerosis.Exp Mol Pathol. 1985; 42: 139-162Crossref PubMed Scopus (319) Google Scholar, 5Campbell J.H. Campbell G.R. The role of smooth muscle cells in atherosclerosis.Curr Opin Lipidol. 1994; 5: 323-330Crossref PubMed Scopus (76) Google Scholar Platelet-derived growth factor subunit B homodimer (PDGF-BB) has been shown to be a potent and selective factor promoting phenotypic switching of SMC into the synthetic state, facilitating its proliferation and migration.6Owens G.K. Kumar M.S. Wamhoff B.R. Molecular regulation of vascular smooth muscle cell differentiation in development and disease.Physiol Rev. 2004; 84: 767-801Crossref PubMed Scopus (2582) Google Scholar, 7Yoshida T. Gan Q. Shang Y. Owens G.K. Platelet-derived growth factor-BB represses smooth muscle cell marker genes via changes in binding of MKL factors and histone deacetylases to their promoters.Am J Physiol Cell Physiol. 2007; 292: C886-C895Crossref PubMed Scopus (88) Google Scholar Indeed, PDGF-BB expression by macrophages in models of atherogenesis has been shown to mediate SMC migration into the tunica intima.8Newby A.C. Zaltsman A.B. Fibrous cap formation or destruction–the critical importance of vascular smooth muscle cell proliferation, migration and matrix formation.Cardiovasc Res. 1999; 41: 345-360Crossref PubMed Scopus (275) Google Scholar At the transcriptional level, PDGF-BB effects phenotypic changes by regulating the expression of a number of pathophysiologically relevant genes, one of which is tenascin-C (TN-C; also known as cytotactin),9LaFleur D.W. Fagin J.A. Forrester J.S. Rubin S.A. Sharifi B.G. Cloning and characterization of alternatively spliced isoforms of rat tenascin Platelet-derived growth factor-BB markedly stimulates expression of spliced variants of tenascin mRNA in arterial smooth muscle cells.J Biol Chem. 1994; 269: 20757-20763Abstract Full Text PDF PubMed Google Scholar, 10Wallner K. Shah P.K. Sharifi B.G. Balloon catheterization induces arterial expression of new tenascin-C isoform.Atherosclerosis. 2002; 161: 75-83Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar a large extracellular matrix glycoprotein that modulates cell adhesion. TN-C is composed of six polypeptides, each containing epidermal growth factor-like (EGFL) repeats, fibronectin type III (FN III) domains (some conserved and some alternatively spliced), and a C-terminal fibrinogen globe. The N-terminal tenascin assembly domain allows the individual polypeptides to form a hexabrachion structure.11Jones P.L. Jones F.S. Tenascin-C in development and disease: gene regulation and cell function.Matrix Biol. 2000; 19: 581-596Crossref PubMed Scopus (260) Google Scholar TN-C promotes SMC migration either by sterically blocking fibronectin-integrin interactions12Majesky M.W. Neointima formation after acute vascular injury Role of counteradhesive extracellular matrix proteins.Tex Heart Inst J. 1994; 21: 78-85PubMed Google Scholar or activating components of adhesion complexes, such as PDGFR-β, αvβ3 integrin, focal adhesion kinase (FAK), and proto-oncogene tyrosine-protein kinase Src (SRC).13Ishigaki T. Imanaka-Yoshida K. Shimojo N. Matsushima S. Taki W. Yoshida T. Tenascin-C enhances crosstalk signaling of integrin alphavbeta3/PDGFR-beta complex by SRC recruitment promoting PDGF-induced proliferation and migration in smooth muscle cells.J Cell Physiol. 2011; 22l6: 2617-2624Crossref Scopus (64) Google Scholar The C isoform of tenascin (TN-C) is induced by mechanical injury14Feng Y. Yang J.H. Huang H. Kennedy S.P. Turi T.G. Thompson J.F. Libby P. Lee R.T. Transcriptional profile of mechanically induced genes in human vascular smooth muscle cells.Circ Res. 1999; 85: 1118-1123Crossref PubMed Scopus (128) Google Scholar and has been found to contribute to intimal hyperplasia in a mouse model of graft artery stenosis.15Sawada Y. Onoda K. Imanaka-Yoshida K. Maruyama J. Yamamoto K. Yoshida T. Shimpo H. Tenascin-C synthesized in both donor grafts and recipients accelerates artery graft stenosis.Cardiovasc Res. 2007; 74: 366-376Crossref PubMed Scopus (25) Google Scholar In addition, TN-C protein is highly expressed in both murine and human atherosclerotic plaques.16von Lukowicz T. Silacci M. Wyss M.T. Trachsel E. Lohmann C. Buck A. Luscher T.F. Neri D. Matter C.M. Human antibody against C domain of tenascin-C visualizes murine atherosclerotic plaques ex vivo.J Nucl Med. 2007; 48: 582-587Crossref PubMed Scopus (27) Google Scholar, 17Pedretti M. Rancic Z. Soltermann A. Herzog B.A. Schliemann C. Lachat M. Neri D. Kaufmann P.A. Comparative immunohistochemical staining of atherosclerotic plaques using F16, F8 and L19: three clinical-grade fully human antibodies.Atherosclerosis. 2010; 208: 382-389Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Several factors have been shown to transcriptionally regulate TN-C expression in fibroblasts.18McKean D.M. Sisbarro L. Ilic D. Kaplan-Alburquerque N. Nemenoff R. Weiser-Evans M. Kern M.J. Jones P.L. FAK induces expression of Prx1 to promote tenascin-C-dependent fibroblast migration.J Cell Biol. 2003; 161: 393-402Crossref PubMed Scopus (69) Google Scholar, 19Jinnin M. Ihn H. Asano Y. Yamane K. Trojanowska M. Tamaki K. Tenascin-C upregulation by transforming growth factor-beta in human dermal fibroblasts involves Smad3, Sp1, and Ets1.Oncogene. 2004; 23: 1656-1667Crossref PubMed Scopus (108) Google Scholar Interspecies alignment of the TN-C proximal promoter reveals some conserved transactivation sites that lend themselves to acute up-regulation, including a strain-responsive element, several Ets binding sites, and a homeodomain binding site close to the TATA box.11Jones P.L. Jones F.S. Tenascin-C in development and disease: gene regulation and cell function.Matrix Biol. 2000; 19: 581-596Crossref PubMed Scopus (260) Google Scholar In human dermal fibroblasts, PDGF-AA activates the TN-C promoter through Sp1, Ets-1, and Ets-2.20Jinnin M. Ihn H. Asano Y. Yamane K. Trojanowska M. Tamaki K. Platelet derived growth factor induced tenascin-C transcription is phosphoinositide 3-kinase/Akt-dependent and mediated by Ets family transcription factors.J Cell Physiol. 2006; 206: 718-727Crossref PubMed Scopus (34) Google Scholar However, transcription factors controlling TN-C expression in SMC in the context of vascular injury have not previously been investigated, nor the involvement of the mammalian stress response pathway regulator activating transcription factor 4 (ATF-4) in this critical process. In earlier work, we showed that ATF-4 is induced after SMC injury in vitro and in balloon-injured rat arteries.21Malabanan K.P. Khachigian L.M. Activating transcription factor-4 and the acute vascular response to injury.J Mol Med. 2010; 88: 545-552Crossref PubMed Scopus (15) Google Scholar, 22Malabanan K. Kanellakis P. Bobik A. Khachigian L.M. ATF-4 induced by FGF-2 regulates VEGF-A transcription in vascular SMCs and mediates intimal thickening in rat arteries following balloon injury.Circ Res. 2008; 103: 378-387Crossref PubMed Scopus (46) Google Scholar ATF-4 (also known as CREB-2, TaxREB67, and C/ATF) is a ubiquitous basic/leucine zipper domain (bZIP) transcription factor and a member of the cAMP-responsive element-binding (CREB) protein family.23Lee K.A. Hai T.Y. SivaRaman L. Thimmappaya B. Hurst H.C. Jones N.C. Green M.R. A cellular protein, activating transcription factor, activates transcription of multiple E1A-inducible adenovirus early promoters.Proc Natl Acad Sci USA. 1987; 84: 8355-8359Crossref PubMed Scopus (147) Google Scholar Here, using knockdown, deficiency, and overexpression strategies, we demonstrate the requirement of ATF-4 in injury- and PDGF-BB-inducible TN-C expression and SMC wound repair. Primary rat aortic smooth muscle cells (RASMCs) were obtained from Cell Applications (San Diego, CA) and cultured in Waymouth's medium, pH 7.4, containing 10% fetal bovine serum (FBS) and antibiotics as described previously.24Khachigian L.M. Owensby D.A. Chesterman C.N. A tyrosinated peptide representing the alternatively spliced exon of the platelet-derived growth factor A-chain binds specifically to cultured cells and interferes with binding of several growth factors [Erratum appeared in J Biol Chem 1992, 267:6449].J Biol Chem. 1992; 267: 1660-1666Abstract Full Text PDF PubMed Google Scholar Cells were rendered quiescent by incubation in Waymouth's medium without FBS for 24 hours. Cells were not used beyond passage 8 in experiments. Atf4−/− and Atf4+/+ mouse embryonic fibroblasts (SV40-transformed mEFs) were obtained from Dr. David Ron (Skirball Institute of Biomolecular Medicine, New York University School of Medicine) and grown in complete medium consisting of advanced Dulbecco's modified Eagle's medium (DMEM) with nonessential amino acids (Invitrogen-Life Technologies, Carlsbad, CA) and 10% FBS, 10 units/mL penicillin, 10 μg/mL streptomycin, 2 mmol/L l-glutamine, and 55 μmol/L β-mercaptoethanol. The ATF-4-pcDNA3 used has been described previously.22Malabanan K. Kanellakis P. Bobik A. Khachigian L.M. ATF-4 induced by FGF-2 regulates VEGF-A transcription in vascular SMCs and mediates intimal thickening in rat arteries following balloon injury.Circ Res. 2008; 103: 378-387Crossref PubMed Scopus (46) Google Scholar For transfection, cells were allowed to grow to 60% to 70% confluence, then were transfected with indicated constructs using FUGENE 6 transfection agent (Roche Applied Science-Roche Molecular Biochemicals, Mannheim, Germany) or RNAiMAX transfection reagent (Invitrogen-Life Technologies). For PDGF receptor inhibition, growth-quiescent RASMCs were pretreated with inhibitors to PDGFR-β (AG1295, 10 μmol/L; Calbiochem) or PDGFR-α (ab35765; 1 μg/mL; Abcam, Cambridge, MA) for 1 hour, before growth factor exposure. Total RNA was prepared from cells that were exposed to PDGF-BB (Sigma-Aldrich, St. Louis, MO) with TRIzol reagent in accordance with the manufacturer's instructions (Invitrogen-Life Technologies). RNA was reverse-transcribed to cDNA using oligo(dT) primers and SuperScript II Reverse Transcriptase (Invitrogen-Life Technologies). PCR was performed in a 20 μL reaction containing 2.5 mmol/L MgCl2, 0.1 mmol/L dNTP, 0.1 μmol/L primers, 1 μL cDNA, and 1 U of Platinum Taq polymerase (Invitrogen-Life Technologies) using a GeneAmp PCR system 2400 thermal cycler (Applied Biosystems-Life Technologies, Foster City, CA). ATF-4 and GAPDH PCR cycling conditions were as described previously.22Malabanan K. Kanellakis P. Bobik A. Khachigian L.M. ATF-4 induced by FGF-2 regulates VEGF-A transcription in vascular SMCs and mediates intimal thickening in rat arteries following balloon injury.Circ Res. 2008; 103: 378-387Crossref PubMed Scopus (46) Google Scholar TN-C primers and cycling conditions used were as described previously.9LaFleur D.W. Fagin J.A. Forrester J.S. Rubin S.A. Sharifi B.G. Cloning and characterization of alternatively spliced isoforms of rat tenascin Platelet-derived growth factor-BB markedly stimulates expression of spliced variants of tenascin mRNA in arterial smooth muscle cells.J Biol Chem. 1994; 269: 20757-20763Abstract Full Text PDF PubMed Google Scholar Quantitative real-time RT-PCR (RT-qPCR) was performed using SYBR Green PCR master mix (Applied Biosystems-Life Technologies). Briefly, total RNA was isolated and reverse-transcribed as described above. PCR was performed in a 10 μL reaction, consisting of 5 μL SYBR Green PCR master mix and 0.3 μmol/L of forward and reverse primers. PCR amplification was performed using a Corbett Rotor-Gene RG-6000 system (Qiagen, Valencia, CA). The PCR conditions consisted of an initial hold step of 50°C for 2 minutes and then activation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds, 60°C for 15 seconds, and 72°C for 10 seconds. The CT value of ATF-4, Egr-1, JunB, or TN-C amplification was normalized to that of β-actin control. PCR products were visualized on ethidium bromide-stained agarose gels to ensure product of right size and no primer dimerization. All initial runs were performed with simultaneous generation of efficiency curves. Data were generated from at least triplicate samples. To amplify the different TN-C isoforms, the following RT-qPCR primers were used: for the Large isoform, Tnc_Large_Fwd: 5′-CCAGGAGGACTCAGGTCAGTGGACCTA-3′ and Tnc_Large_Rev: 5′-AGG GATCTCCTCTGTCAAGACCTCAAC-3′; for the A1A2 isoform, Tnc_Large_Fwd and A1A2_Rev: 5′-CTTGGGAGAGCCCATGGCTGTCAAGACCTCAAC-3′; for the D-isoform, Tnc_Inter_Fwd: 5′-AAGGCATCCACAGAAGCTGAA-3′ and Tnc_Inter_Rev: 5′-GAGGGTTATTTCTTGTGGTTCAGAGTT-3′; for the Small isoform, Tnc_Small_Fwd: 5′-GAAGGCATCCACAGCCATGG-3′ and Tnc_Small_Rev: 5′-TCACCAGTCGGGTCTCAGTATC-3′; for Egr-1, Egr-1_Fwd: 5′-GCCTTT TGCCTGTGACATTT-3′ and Egr-1 Rev: 5′-AGCCCGGAGAGGAGTAAGAG-3′; for Jun B, JunB Fwd: 5′-CACGACGACTCATACGCAGC-3′ and JunB Rev: 5′-GACCCTTGAGACCCCGATAA-3′; and for β-actin, β-actin_Fwd: 5′-AGCCATGTACGTAGCCATCC-3′ and β-actin_Rev: 5′-CTCTCAGCTGTGGTGGTGAA-3′. Primers used to amplify ATF-4 were as described previously.22Malabanan K. Kanellakis P. Bobik A. Khachigian L.M. ATF-4 induced by FGF-2 regulates VEGF-A transcription in vascular SMCs and mediates intimal thickening in rat arteries following balloon injury.Circ Res. 2008; 103: 378-387Crossref PubMed Scopus (46) Google Scholar RASMCs were cultured in 100-mm tissue culture plates and treated with PDGF-BB for various lengths of time. For collection of cell lysates, cell monolayers were washed twice in 1× PBS; total protein was extracted in 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.5), 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 5 mmol/L EDTA, 10 mg/mL leupeptin, 1% aprotinin, and 2 mmol/L phenylmethylsulfonyl fluoride. For secreted TN-C in the media, 5 mL of the media was concentrated (Vivaspin concentrator, 30 kDa molecular weight cutoff; GE Healthcare, Little Chalfont, UK) to a final volume of 300 μL. Protein sample (10 to 20 μg of lysate or 50 μL concentrated tunica media) was loaded onto a 6% to 10% SDS–polyacrylamide gel and electroblotted onto a polyvinylidene difluoride nylon membrane (EMD Millipore, Bedford, MA). Membranes were blocked in 0.05% Tween 20 (v/v) PBS containing 5% skim milk, then incubated with the following primary antibodies: ATF-4 (sc-22800) and TN-C (sc-20932) from Santa Cruz Biotechnology (SCBT; Santa Cruz, CA) and β-actin (A5316) from Sigma-Aldrich. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Dako, Carpinteria, CA). Proteins were visualized by chemiluminescence detection (NEN Life Sciences, Boston, MA). RASMCs were seeded in Petri dishes and growth was arrested with serum-free medium for 24 hours before transfection with 0.4 μmol/L small interfering RNA (siRNA; Qiagen) targeting endogenous rat ATF-4.22Malabanan K. Kanellakis P. Bobik A. Khachigian L.M. ATF-4 induced by FGF-2 regulates VEGF-A transcription in vascular SMCs and mediates intimal thickening in rat arteries following balloon injury.Circ Res. 2008; 103: 378-387Crossref PubMed Scopus (46) Google Scholar siRNA technology provides an invaluable tool for exploring target gene function.25Bhindi R. Fahmy R.G. Lowe H.C. Chesterman C.N. Dass C.R. Cairns M.J. Saravolac E.G. Sun L.Q. Khachigian L.M. Brothers in arms: DNA enzymes, short interfering RNA, and the emerging wave of small-molecule nucleic acid-based gene-silencing strategies.Am J Pathol. 2007; 171: 1079-1088Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar At 24 hours after serum arrest, ATF-4 or TN-C isoforms were inducibly expressed by the addition of PDGF-BB for the appropriate length of time, and total RNA was extracted and analyzed as described above. For wounding assays, the RASMC or mEF cell monolayer was scratched using a sterile toothpick; the growth medium was changed, and PDGF-BB was added. Wound closure was measured by counting the number of cells in the wound area at 24 hours after the scratch. Samples were prepared in triplicate, and cell counts were taken as the average of three fields per sample under ×100 magnification. Atf4+/+ and Atf4−/− mEFs were grown in minimal-serum medium for 24 hours, before seeding at a density of 5 × 104 cells in 0.1% FBS DMEM in the top chamber of a Millicell cell culture insert (EMD Millipore) with an 8 μm pore size for 24-well dual-chamber migration assays. Inserts were coated with TN-C or bovine serum albumin (2 μg/insert). This was achieved by the addition of TN-C or bovine serum albumin in PBS solution to the apical chamber, incubation for 2 hours at 37°C, and then removal of excess liquid and air-drying in the hood for 30 minutes. Inserts were equilibrated with 0.1% FBS DMEM before cell seeding. The bottom chamber contained either 0.1% FBS DMEM or 0.1% FBS DMEM with 50 ng/mL PDGF-BB. Three inserts were prepared for each treatment group. The migration assay was allowed to proceed at 37°C and 5% CO2 for 24 hours, at which point the cells were washed three times with 1× PBS to dislodge nonmigratory cells and then were fixed in Quick DIP I solution (Thermo Fisher Scientific, Australia) for 15 minutes. The inserts were washed three times with 1× PBS, stained with Fronine Quick DIP II solution for 15 minutes, and then were washed three more times with 1× PBS. The inserts were mounted on glass slides using ProLong Gold anti-fade reagent with DAPI (Invitrogen-Life Technologies) and then were visualized under a fluorescent microscope. The number of cells that had migrated through each membrane was determined by counting the number of cells in three randomly selected microscopic fields at ×100 magnification. Balloon catheter injury to the carotid artery of adult Sprague Dawley rats (450 to 550 g) was performed as described previously.22Malabanan K. Kanellakis P. Bobik A. Khachigian L.M. ATF-4 induced by FGF-2 regulates VEGF-A transcription in vascular SMCs and mediates intimal thickening in rat arteries following balloon injury.Circ Res. 2008; 103: 378-387Crossref PubMed Scopus (46) Google Scholar After injury, 100 μL of PBS containing 10 μL FUGENE 6 and 50 μg siRNA or the vehicle itself was infused into the ligated segment for 20 minutes at 100 mm Hg. The rats were sacrificed 14 days later. Tissue processing and immunohistochemistry using TN-C antibodies were performed essentially as described previously.22Malabanan K. Kanellakis P. Bobik A. Khachigian L.M. ATF-4 induced by FGF-2 regulates VEGF-A transcription in vascular SMCs and mediates intimal thickening in rat arteries following balloon injury.Circ Res. 2008; 103: 378-387Crossref PubMed Scopus (46) Google Scholar, 26Santiago F.S. Ishii H. Shafi S. Khurana R. Kanellakis P. Bhindi R. Ramirez M. Bobik A. Martin J. Chesterman C.N. Zachary I. Khachigian L. Yin Yang-1 inhibits vascular smooth muscle cell growth and intimal thickening by repressing p21WAF1/Cip1 transcription and p21WAF1/Cip1-Cdk4-cyclin D1 assembly.Circ Res. 2007; 101: 146-155Crossref PubMed Scopus (60) Google Scholar Digital images of sections were captured using a DP-70 Olympus microscope. Relative staining in the neointima was determined using ImageJ version 1.44 (NIH, Bethesda, MD). The mean integrated density values (measured in pixels) were determined on three representative sections of equal area, using two fields of view from four rats per group. For data presented in histograms, results are expressed as means ± SE performed in triplicate or quadruplicate. Data are representative of three to five independent determinations. Data were analyzed for statistical significance (P < 0.05) using two-tailed Student's t-test or analysis of variance between groups. Building on our previous demonstration that both in vitro cell injury and FGF-2 induce ATF-4 mRNA expression,22Malabanan K. Kanellakis P. Bobik A. Khachigian L.M. ATF-4 induced by FGF-2 regulates VEGF-A transcription in vascular SMCs and mediates intimal thickening in rat arteries following balloon injury.Circ Res. 2008; 103: 378-387Crossref PubMed Scopus (46) Google Scholar we explored the possibility that PDGF-BB, a potent SMC migratory factor,27Jawien A. Bowen-Pope D.F. Lindner V. Schwartz S.M. Clowes A.W. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty.J Clin Invest. 1992; 89: 507-511Crossref PubMed Scopus (593) Google Scholar could regulate ATF-4 expression in SMCs. RT-qPCR analysis of PDGF-BB-treated RASMCs revealed an increase in ATF-4 mRNA levels within 15 minutes after exposure to the growth factor (50 ng/mL), which peaked at 1 hour (Figure 1A). This induction was dose-responsive, with ATF-4 transcript levels induced at PDGF-BB levels as low as 5 ng/mL and highest transcript levels induced at 25 to 50 ng/mL (Figure 1B). Western blot analysis of PDGF-BB-treated RASMCs revealed an increase in ATF-4 protein expression within 30 minutes, peaking at 2 to 4 hours (Figure 1C). Altered expression of this stress response regulator by PDGF-BB has hitherto not been reported in any cell type. PDGF-BB has effects on ATF-4 in non-SMCs. For example, we have observed a profound induction of ATF-4 mRNA levels by PDGF-BB in NIH-3T3 fibroblasts (unpublished data). From previous microarray analysis comparing differential gene expression in response to injury compromised by the absence of ATF-4, we identified TN-C as a target for investigation. TN-C is expressed in a variety of isoforms through alternative splicing of certain FN III repeats. Primers were designed to detect the four TN-C isoforms expressed in SMC.10Wallner K. Shah P.K. Sharifi B.G. Balloon catheterization induces arterial expression of new tenascin-C isoform.Atherosclerosis. 2002; 161: 75-83Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar RT-qPCR analysis showed that PDGF-BB treatment (50 ng/mL) of RASMCs increased expression of the TN-C Large isoform within 2 hours, reaching nearly 20-fold after 6 to 8 hours (Figure 1D). A similar trend was observed for the three other isoforms: the A1A2, D-isoform, and Small isoforms (Figure 1D), with transcript levels of all four TN-C isoforms returning to basal levels by 24 hours (data not shown). Two PDGF-BB inducible TN-C protein variants, 240 and 280 kDa, have been shown to contribute to SMC migration.10Wallner K. Shah P.K. Sharifi B.G. Balloon catheterization induces arterial expression of new tenascin-C isoform.Atherosclerosis. 2002; 161: 75-83Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Consistently, Western blot analysis revealed an increase in the two TN-C protein isoforms in the media by RASMCs treated with PDGF-BB, peaking at 6 hours of growth factor exposure (Figure 1E). To investigate the regulatory role of ATF-4 in mediating TN-C transcription, we transfected a CMV-driven expression vector ATF-4-pcDNA3 (20 μg) into RASMCs and used its backbone (pcDNA3) as a control. RT-PCR analysis with primers used to characterize TN-C in SMCs9LaFleur D.W. Fagin J.A. Forrester J.S. Rubin S.A. Sharifi B.G. Cloning and characterization of alternatively spliced isoforms of rat tenascin Platelet-derived growth factor-BB markedly stimulates expression of spliced variants of tenascin mRNA in arterial smooth muscle cells.J Biol Chem. 1994; 269: 20757-20763Abstract Full Text PDF PubMed Google Scholar showed that ATF-4 overexpression induced mRNA levels of all four TN-C isoforms detected to date10Wallner K. Shah P.K. Sharifi B.G. Balloon catheterization induces arterial expression of new tenascin-C isoform.Atherosclerosis. 2002; 161: 75-83Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar: Large (1700 bp), A1A2 (1000 bp), D-isoform (650 bp), and Small (360 bp), with GAPDH levels showing even loading (Figure 2A). RT-qPCR analysis of the TN-C Large, A1A2, D-isoform, and Small isoforms after 24 hours also revealed a significant induction with ATF-4 overexpression, compared with the backbone alone (Figure 2B). Likewise, Western blot analysis revealed increased TN-C protein levels in the media when ATF-4 was overexpressed (Figure 2C). To our knowledge, this is the first demonstration of ATF-4 regulation of TN-C expression. To determine which PDGF receptor mediates PDGF-BB-inducible ATF-4 and TN-C expression, we pretreated growth-quiescent RASMCs with inhibitors of PDGFR-β (AG1295, 10 μmol/L) or PDGFR-α (ab35765, 1 μg/mL) for 1 hour, before stimulation with PDGF-BB (50 ng/mL) for 1 and 6 hours. RT-qPCR analysis showed that inhibition of PDGFR-β significantly inhibited ATF-4 mRNA levels after 1 hour (Figure 3A) and TN-C Large mRNA levels after 6 hours (Figure 3C), whereas PDGFR-α inhibition had no such effect. A similar trend was observed for the other TN-C isoforms (Figure 3C). Efficacy of inhibition of PDGFR-β at this concentration of AG1295 was demonstrated by RT-qPCR analysis of a known downstream gene, Egr1 (Figure 3B). Prior studies have also shown incomplete inhibition with the same concentration of AG1295 in the presence of PDGF-BB in the same cell type.28Mochizuki S. Brassart B. Hinek A. Signaling pathways transduced through the elastin receptor facilitate proliferation of arterial smooth muscle cells.J Biol Chem. 2002; 277: 44854-44863Crossref PubMed Scopus (198) Google Scholar To examine the effect of silencing ATF-4 on PDGF-BB-inducible TN-C expression, we transfected RASMCs with siRNA targeting endogenous rat ATF-4 (0.4 μmol/L siRNA). The scrambled counterpart (ATF-4 scr) served as a control. The siRNA blocked PDGF-BB (50 n
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