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• W2002405735 abstract "The cyclin-dependent kinase inhibitor (CKI) p21WAF1/Cip1 is regulated at the level of transcription by nuclear factors such as the co-activator p300. It is presently unknown whether the Ets family of transcription factors control p21WAF1/Cip1 gene expression. Ets-1 inhibits apoptosis in vascular smooth muscle cells as determined by both fluorescein isothiocyanate-linked annexin V/propidium iodide staining of cells and fluorescence-activated cell sorting analysis and quantitative cytoplasmic histone-associated internucleosomal DNA fragmentation. p21WAF1/Cip1 can play a mitogenic and anti-apoptotic role in smooth muscle cells. Using transient transfection and Western blot analysis, we determined that Ets-1 activates p21WAF1/Cip1 transcription and protein expression. Electrophoretic mobility shift assays revealed that Ets-1 interacts selectively with the –1350GGAA–1347 Ets element in the p21WAF1/Cip1 promoter. Mutation of this element reduced basal and Ets1-inducible p21WAF1/Cip1 promoter-dependent expression. In contrast, the –1577GGAT–1574 motif mediates basal but not Ets-1 activation of the p21WAF1/Cip1 promoter. Co-immunoprecipitation and co-transfection analysis showed that Ets-1 binds p300 and cooperatively activates p21WAF1/Cip1 transcription. The phenotypic importance of Ets-1 regulation of p21WAF1/Cip1 was demonstrated by the capacity of antisense p21WAF1/Cip1 strategies to block Ets-1-inhibition of apoptosis and inhibit Ets-1-induction of proliferation. The cyclin-dependent kinase inhibitor (CKI) p21WAF1/Cip1 is regulated at the level of transcription by nuclear factors such as the co-activator p300. It is presently unknown whether the Ets family of transcription factors control p21WAF1/Cip1 gene expression. Ets-1 inhibits apoptosis in vascular smooth muscle cells as determined by both fluorescein isothiocyanate-linked annexin V/propidium iodide staining of cells and fluorescence-activated cell sorting analysis and quantitative cytoplasmic histone-associated internucleosomal DNA fragmentation. p21WAF1/Cip1 can play a mitogenic and anti-apoptotic role in smooth muscle cells. Using transient transfection and Western blot analysis, we determined that Ets-1 activates p21WAF1/Cip1 transcription and protein expression. Electrophoretic mobility shift assays revealed that Ets-1 interacts selectively with the –1350GGAA–1347 Ets element in the p21WAF1/Cip1 promoter. Mutation of this element reduced basal and Ets1-inducible p21WAF1/Cip1 promoter-dependent expression. In contrast, the –1577GGAT–1574 motif mediates basal but not Ets-1 activation of the p21WAF1/Cip1 promoter. Co-immunoprecipitation and co-transfection analysis showed that Ets-1 binds p300 and cooperatively activates p21WAF1/Cip1 transcription. The phenotypic importance of Ets-1 regulation of p21WAF1/Cip1 was demonstrated by the capacity of antisense p21WAF1/Cip1 strategies to block Ets-1-inhibition of apoptosis and inhibit Ets-1-induction of proliferation. The cyclin-dependent kinase inhibitor p21WAF1/Cip1 was first identified through its interaction with Cdk2 (1Harper J.W. Adami G. Wei N. Keyomarsi K. Elledge S. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5250) Google Scholar) and its regulation of wild-type p53 activity (2El-Deiry W. Tokino T. Velulescu V. Levy D. Parsons R. Trent J. Lin D. Mercer W. Kinzler K. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7957) Google Scholar). The gene is located on human chromosome 6p21.2. An N-terminal domain in p21WAF1/Cip1 inhibits cyclin-dependent kinase activity, whereas a p21WAF1/Cip1 C-terminal domain inhibits proliferating cell nuclear antigen (3Waga S. Hannon G.J. Beach D. Stillman B. Nature. 1994; 369: 574-578Crossref PubMed Scopus (1591) Google Scholar). The transcriptional control of p21WAF1/Cip1 is poorly understood at the present time but is thought to involve the participation of multiple nuclear factors, including p53, Sp family proteins, AP2, E2Fs, signal transducers and activators of transcription (STATs), CCAAT/enhancer-binding protein α, CCAAT/enhancer-binding protein β, Gax, and Runx2 (4Westendorf J.J. Zaidi S.K. Cascino J.E. Kahler R. van Wijnen A.J. Lian J.B. Yoshida M. Stein G.S. Li X. Mol. Cell. Biol. 2002; 22: 7982-7992Crossref PubMed Scopus (282) Google Scholar) (reviewed in Ref. 5Gartel A.L. Tyner A.L. Exp. Cell Res. 1999; 246: 280-289Crossref PubMed Scopus (580) Google Scholar). The ETS family of transcription factors presently consists of ∼30 members and is defined by a conserved DNA-binding ETS domain spanning 85 amino acids that forms a winged helixturn-helix structural motif (6Li R. Pei H. Watson D.K. Oncogene. 2000; 19: 6514-6523Crossref PubMed Scopus (192) Google Scholar). These transcription factors are involved in a diverse array of biological functions, including cellular growth and differentiation. Modulation of gene expression by ETS family members can involve combinatorial interactions with other transcription factors (7Verger A. Duterque-Coquillaud M. Bioessays. 2002; 24: 362-370Crossref PubMed Scopus (119) Google Scholar). Such factors include Sp1, AP-1, c-Myb, and the highly conserved nuclear phosphoprotein p300. Two regions of p300 between amino acid residues 328–596 and 1678–2370 independently interact with Ets-1 (8Jayaraman G. Srinivas R. Duggan C. Ferreira E. Swaminathan S. Somasundaram K. Williams J. Hauser C. Kurkinen M. Dhar R. Weitzman S. Buttice G. Thimmapaya B. J. Biol. Chem. 1999; 274: 17342-17352Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The present study demonstrates the capacity of Ets-1 to regulate p21WAF1/Cip1 gene transcription in a p300-independent and/or -dependent manner. It also demonstrates the phenotypic consequence of Ets-1 regulation of p21WAF1/Cip1 expression on smooth muscle cell proliferation and apoptosis. Cell Culture—WKY-12-22 smooth muscle cells were cultured in Waymouth's MB 752/1 medium (Invitrogen), pH 7.4, supplemented with 10% fetal bovine serum (FBS), 10 μg/ml streptomycin, and 10 units/ml penicillin at 37 °C in a humidified atmosphere of 5% CO2. Cells were passaged every 3 days into 75-cm2 flasks by rinsing twice with phosphate-buffered saline (PBS), 1The abbreviations used are: PBS, phosphate-buffered saline; CMV, cytomegalovirus; Rb, retinoblastoma protein; FasL, Fas ligand; ERE, Ets-1-response element; ELISA, enzyme-linked immunosorbent assay. pH 7.4, and incubation with 0.05% trypsin, 0.02% EDTA in balanced salts solution (ICN-Flow) for 3 min at 37 °C prior to resuspension in growth medium. Plasmid Constructs—0-Luc and 6-Luc (constructs bearing 2.3 and 0.3 kb of the p21WAF1/Cip1 promoter relative to the TATA box, respectively, linked to a luciferase reporter) were generous gifts from Dr. Wafik El-Deiry. In addition, fragments of the p21WAF1/Cip1 promoter were generated using the Erase-a-Base deletion system (Promega). Fragments were then subcloned into pGL3-basic (3*6, 5*2, 7*3, 9*6, 10*3, 11*7, 13*6, and 13*20). Transverse mutations of the Ets-response elements located at –1577/–1574 and –1350/–1347 were generated using the QuikChange site-directed mutagenesis kit (Stratagene) and subsequently named 5*2 (–1577TTCG–1574) and 5*2 (–1350TTCC–1347). pKCR3-Ets-1 was a generous gift from Dr. Ian Cassidy. p300 (CMVbased expression vector) was purchased from Upstate Biotechnology. pGEX-Ets-1-DBD and pKCR3-DN-Ets-1 have been described previously (9Kavurma M.M. Bobryshev Y. Khachigian L.M. J. Biol. Chem. 2002; 277: 36244-36252Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Plasmid and Oligonucleotide Transfection—Smooth muscle cells were seeded in 100-mm tissue culture plates. At ∼50–60% confluence, the cells were transiently transfected with 20 μg of pKCR3-Ets-1 or the internal control plasmid, pKCR3, using FuGENE 6 according to the manufacturer's instructions (Roche Molecular Biochemicals). Assessment of Luciferase Activity—Transient transfections were performed at 60% confluency in 6-well titer plates using FuGENE 6 transfecting agent with indicated constructs. The internal control plasmid, pRL-TK, was also used in transfections. Luciferase activity was determined 24 h following transfection using the Dual-Luciferase assay system (Promega) and normalized to data generated from pRL-TK. Generation of Recombinant Ets-1—Generation of pGEXEts-1-DBD has been described previously (9Kavurma M.M. Bobryshev Y. Khachigian L.M. J. Biol. Chem. 2002; 277: 36244-36252Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Briefly, transformants were stimulated with 0.1 m isopropyl-1-thio-β-d-galactopyranoside for 3–6 h following an OD of 0.6–0.8 at 600 nm. Bacteria were pelleted and sonicated in sonication buffer (50 mm Tris pH 8.5, 50 mm NaCl, 1.43 mm phenylmethylsulfonyl fluoride, 1.44 mm β-mercaptoethanol, and 0.5% Triton X-100). Samples were spun on high speed, and the supernatant was collected into a fresh tube. Glutathione S-transferase-conjugated agarose beads (Sigma) were incubated with the supernatant for 1–2 h at 4 °C on a rotary shaker. Following incubation, beads were washed in 50 mm Tris, pH 8, 100 mm NaCl, 10% glycerol, 1.43 mm phenylmethylsulfonyl fluoride, 1.44 mm β-mercaptoethanol, and 0.5% Nonidet P-40 and eluted with 10 mm reduced glutathione (in 100 mm Tris-HCl, pH 7.5). Recombinant Ets-1 DNA-binding domain (DBD, residues Pro386 to Glu494) was used in electrophoretic mobility shift analysis (EMSA) where indicated. Electrophoretic Mobility Shift Analysis—Recombinant Ets-1-DNA binding domain protein was incubated with indicated 32P-labeled double-stranded oligonucleotides. Reactions proceeded in a total volume of 20 μl (containing 10 mm Tris-HCl, pH 8.0, 50 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, 5% glycerol, 1 μg of salmon sperm DNA, 5% sucrose, 1 μg of poly(dI-dC) and 1 mm phenylmethylsulfonyl fluoride) for 10–15 min at 4 °C. Samples were resolved by 6% non-denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Western Blot Analysis—Smooth muscle cells in 100-mm plates were transfected with 20 μg of pKCR3-Ets-1 or the backbone control plasmid pKCR3. In Western blotting studies detecting phosphorylated Rb, cells were co-transfected with 0.8 μm of either antisense (5′-GAC ATC ACC AGG ATC GGA CAT-3′) (10Weiss R.H. Joo A. Randour C. J. Biol. Chem. 2000; 275: 10285-10290Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) or scrambled (5′-AAG CGT ACT ACG CTA GCA CGA-3′) p21WAF1/Cip1 oligonucleotide. Twenty four hours after transfection, the cells were washed in cold PBS, and total protein was extracted in 150 mm NaCl, 50 mm Tris-HCl, pH 7.5, 1% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100 containing protease inhibitors (10 μg/ml leupeptin, 5 mm EDTA, 1% aprotinin, and 2 mm phenylmethylsulfonyl fluoride). In experiments evaluating the effect of antisense and scrambled p21WAF1/Cip1 on p21WAF1/Cip1 expression, smooth muscle cells were incubated in serum-free medium for 6 h at the confluence of 40–50% prior to transfection with either 0.8 μm antisense or scrambled p21WAF1/Cip1 using FuGENE 6. Eighteen hours after the initial transfection, cells were transfected a second time in the presence of 5% fetal bovine serum. Cells were harvested 24 h following the second transfection. Western blot analysis was performed as described elsewhere (11Taylor L.M. Khachigian L.M. J. Biol. Chem. 2000; 275: 16709-16716Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 12Santiago F.S. Atkins D.A. Khachigian L.M. Am. J. Pathol. 1999; 155: 897-905Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) using rabbit polyclonal antibody recognizing Ets-1 (1:500), Sp1 (1:1000), PU.1 (1:1000), or mouse monoclonal antibodies recognizing p21WAF1/Cip1 (1:300), YY1 (1:1000), or goat polyclonal antibodies targeting phosphorylated Rb (1:500), all of which were purchased from Santa Cruz Biotechnology. Proteins were visualized by chemiluminescence detection (PerkinElmer Life Sciences). Protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad). Immunoprecipitation Studies—Nuclear extracts of cells were precleared with 30 μl of 1:1 slurry of protein G-Sepharose™-radioimmune precipitation assay buffer for 1 h with bi-directional rotation at 4 °C. Ten μl of p300 rabbit polyclonal IgG (Santa Cruz Biotechnology) was incubated with the precleared lysates overnight at 4 °C with gentle shaking. Several washes were performed with centrifugation prior to Western blot analysis. Quantitative Assessment of Apoptosis—Smooth muscle cells were grown to 50–60% confluency in 100-mm plates and transfected with 20 μg of pKCR3-Ets-1 plasmid or pKCR3, with or without 0.8 μm of antisense or scrambled p21WAF1/Cip1. Twenty-four hours after transfection, the cells were trypsinized and seeded into 96-well plates at 10,000 cells per well. Apoptosis was assayed another 24 h later using the Cell Death Detection ELISAPlus kit (Roche Molecular Biochemicals). Results are expressed as total internucleosomal DNA fragmentation as a proportion of the cell population, determined by an automated Coulter Counter. For fluorescein isothiocyanate-linked annexin V/propidium iodide staining, smooth muscle cells (in 100-mm dishes) were transfected with pKCR3-Ets-1 or pKCR3 (20 μg). Cells were washed with ice-cold PBS and resuspended in binding buffer (10 mm HEPES, pH 7.4, 140 mm NaCl, and 2.5 mm CaCl2) at a concentration of 1 × 106 cells/ml. Five μl of annexin V-fluorescein isothiocyanate and 10 μl of propidium iodide (50 μg/ml stock in PBS) was added to 100 μl of the cell suspension. Cells were gently mixed and incubated in the dark for 15 min. This was followed by the addition of 400 μl of binding buffer to each sample prior to analysis by flow cytometry within 1 h. Ets-1 Inhibits Apoptosis in Vascular Smooth Muscle Cells— Previously, we determined that the Fas ligand (FasL) promoter is under the positive regulatory influence of Ets-1 in WKY12-22 vascular smooth muscle cells (9Kavurma M.M. Bobryshev Y. Khachigian L.M. J. Biol. Chem. 2002; 277: 36244-36252Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The effect of Ets-1 on smooth muscle cell apoptosis has not yet been investigated. Given its up-regulation of FasL expression in this cell type, we predicted that Ets-1 would induce apoptosis. WKY12-22 cells were transfected with pKCR3-Ets-1, an SV40-based expression vector encoding c-Ets-1, or its backbone control, and apoptosis was assessed 24 h subsequently by fluorescein isothiocyanatelinked annexin V/propidium iodide staining of cells and fluorescence-activated cell sorting analysis or quantitative cytoplasmic histone-associated internucleosomal DNA fragmentation. Ets-1 inhibited spontaneous levels of both annexin V/propidium iodide staining (Fig. 1A) and DNA fragmentation (Fig. 1B) in this cell type. Western immunoblot analysis on the extracts of cells transfected with pKCR3-Ets-1 or its backbone counterpart demonstrated that Ets-1 was indeed expressed in this system (Fig. 1C), with no change in levels of Sp1, YY1, or the Ets family member PU.1 (Fig. 1C). Existence of Two Putative Ets-1 Elements in the p21WAF1/Cip1 Promoter—We and others have recently determined that p21WAF1/Cip1 plays a mitogenic and anti-apoptotic role in vascular smooth muscle cells 2Kavurma, M. M., and Khachigian, L. M. (2003) J. Biol. Chem., in press. (10Weiss R.H. Joo A. Randour C. J. Biol. Chem. 2000; 275: 10285-10290Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 13Weiss R.H. Randour C.J. Cell. Signal. 2000; 12: 413-418Crossref PubMed Scopus (15) Google Scholar). Using timed 5′-exonuclease digestion, we generated a nested series of Firefly luciferase constructs driven by 5′-deletions of the p21WAF1/Cip1 promoter (Fig. 2A). These constructs were transfected into WKY12-22 cells, and reporter activity was assessed after 24 h by luminometry and normalized for Renilla luciferase activity. Constructs 3*6 and 5*2, bearing 1975 and 1670 bp of the p21WAF1/Cip1 promoter, respectively, generated high basal luciferase activity (Fig. 2B). Fragments of the promoter of 1270-bp length (construct 7*3) or less, however, failed to support the high level of reporter expression observed. The 5′endpoints of p21WAF1/Cip1 constructs 5*2 and 7*3 span two putative cis-acting Ets motifs, 5′-GGAT-3′ and 5′-GGAA-3′, located at positions –1577/–1574 and –1350/–1347 (relative to the transcriptional start site), respectively (Fig. 2C). Ets-1 Activates p21WAF1/Cip1 Transcription and Protein Expression—To determine whether the –1670/–1270 region of the p21WAF1/Cip1 promoter comprised a functional Ets-1response element (ERE), we transfected WKY12-22 cells with increasing amounts of pKCR3-Ets-1 or its backbone. p21WAF1/Cip1 promoter-dependent luciferase activity in cells transfected with construct 5*2 was induced by the Ets-1 expression vector in a dose-dependent manner (Fig. 3A). In contrast, Ets-1 failed to activate reporter expression driven by construct 7*3 (Fig. 3A). Substitution of pKCR3-Ets-1 with pKCR3-DN-Ets-1, which bears the Ets-1 DNA-binding domain but not the activation domain, failed to activate the p21WAF1/Cip1 promoter (Fig. 3B). These findings reveal the existence of a functional ERE in the –1670/–1270 region of the p21WAF1/Cip1 promoter. Western analysis was performed to demonstrate that Ets-1 modulation of p21WAF1/Cip1 transcription is reflected in greater levels of the p21WAF1/Cip1 protein. p21WAF1/Cip1 immunoreactivity was higher in cells transfected with pKCR3-Ets-1 versus pKCR3 alone, indicating that Ets-1 activates endogenous p21WAF1/Cip1 (Fig. 4). In contrast, levels of the zinc finger transcription factor Sp1 were unaltered by Ets-1 overexpression (Fig. 4). Ets-1 Interacts Selectively with an Ets Element in the p21WAF1/Cip1 Promoter—To determine whether Ets-1 interacts with one or both putative EREs in the p21WAF1/Cip1 promoter, we performed electrophoretic mobility shift analysis using 32Plabeled double-stranded oligonucleotides bearing wild type (Oligo –1370/–1334 (–1350GGAA–1347) or Oligo-1597/-1558 (–1577GGAT–1574)) or mutant (mOligo –1370/–1334 (–1350TTCC–1347) or mOligo –1597/–1558 (–1577TTCG–1574)) sequences, respectively. A separate oligonucleotide (32P-labeled Oligo FasL –381/–346), bearing the Fas ligand promoter ERE (–356GGAA–352) (9Kavurma M.M. Bobryshev Y. Khachigian L.M. J. Biol. Chem. 2002; 277: 36244-36252Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) bound the Ets-1 protein, as expected (Fig. 5). 32P-Oligo –1370/–1334 (–1350GGAA–1347) also formed a nucleoprotein complex with Ets-1 (Fig. 5), which was abrogated if the probe was substituted with Oligo –1370/–1334 (–1350TTCC–1347) (Fig. 5). A nucleoprotein complex was not detected using 32P-mOligo –1597/–1558 (–1577GGAT–1574) or 32P-mOligo –1597/–1558 (–1577TTCG–1574) (Fig. 5). The p21WAF1/Cip1 Promoter Is Differentially Regulated by Ets Motif – 1350GGAA – 1347 and – 1577GGAT – 1574—To demonstrate the functional significance of either or both of these Ets motifs, we introduced these same mutations into p21WAF1/Cip1 promoter-reporter constructs and evaluated the effect of co-transfection with pKCR3-Ets-1 or pKCR3 alone. Ets-1 activated luciferase expression driven by 1670 bp of the p21WAF1/Cip1 promoter (Fig. 6). Basal expression was reduced in cells transfected with construct 5*2 (–1577TTCG–1574) in which the –1577GGAT–1574 motif had been mutated; however, Ets-1inducibility of the promoter was unchanged (Fig. 6). Mutation of the –1350GGAA–1347 element in construct 5*2 (–1350TTCC–1347) ablated both basal and Ets-1-inducible expression (Fig. 6). Thus, the –1577GGAT–1574 motif mediates basal but not Ets-1 activation of the p21WAF1/Cip1 promoter, whereas the –1350GGAA–1347 element mediates both basal and Ets-1 inducible p21WAF1/Cip1 promoter-dependent expression. Ets-1 Activation of p21WAF1/Cip1 Promoter Involves Cooperativity with p300 —Previous studies have demonstrated that the activity of the p21WAF1/Cip1 promoter is regulated by the transcriptional co-activator p300 (14Billon N. van Grunsven L.A. Rudkin B.B. Oncogene. 1996; 13: 2047-2054PubMed Google Scholar). p300 modulation of p21WAF1/Cip1 transcription is mediated by cooperative interactions with a number of nuclear factors, including Sp1 (15Owen G.I. Richer J.K. Tung L. Takimoto G. Horwitz K.B. J. Biol. Chem. 1998; 273: 10696-10701Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar), Sp3 (16Xiao H. Hasegawa T. Isobe K. J. Biol. Chem. 2000; 275: 1371-1376Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), and BRCA1 (17Chai Y.L. Cui J. Shao N. Shyam E. Reddy P. Rao V.N. Oncogene. 1999; 18: 263-268Crossref PubMed Scopus (158) Google Scholar). To determine whether Ets-1 modulation of p21WAF1/Cip1 transcription involves interplay with p300, we performed co-transfection experiments using expression vectors for p300 (CMV-based) or Ets-1 together with construct 5*2. p300 and Ets-1 each activated p21WAF1/Cip1 promoter-dependent luciferase expression (Fig. 7A). p21WAF1/Cip1 promoter activity was induced in a synergistic manner when Ets-1 and p300 were co-transfected (Fig. 7A). p300 failed to activate the p21WAF1/Cip1 promoter using construct 5*2 (–1577TTCG–1574) bearing the mutant –1577GGAT–1574 motif, whereas Ets-1 inducibility of this construct was not changed (Fig. 7A). Unlike wild-type construct 5*2, construct 5*2 (–1577TTCG–1574) was not activated by the presence of both p300 and Ets-1 beyond activation in the presence of Ets-1 alone (Fig. 7A). Construct 5*2 (–1350TTCC–1347), bearing the mutated –1350GGAA–1347 element, was refractory to induction by Ets-1 or p300, either alone or in combination (Fig, 7A). Similarly, neither transcription factor activated construct 7*3 (Fig. 7A), whose 5′ end point is 3′ to both of these Ets-1 motifs. Complementary pull-down experiments in which cell extracts were immunoprecipitated with p300 antibodies prior to Western blot analysis for Ets-1 revealed the formation of a physical complex between p300 and Ets-1 (Fig. 7B). Ets-1 Inhibition of Apoptosis Is Mediated by p21WAF1/Cip1— Because the preceding findings show that Ets-1 protects smooth muscle cells from apoptosis and exerts a positive regulatory influence on basal and inducible p21WAF1/Cip1 transcription via sequence-specific interactions with the p21WAF1/Cip1 promoter, we next explored whether Ets-1 inhibition of apoptosis is mediated via p21WAF1/Cip1. We and others have recently determined that p21WAF1/Cip1 can play a mitogenic and anti-apoptotic role in vascular smooth muscle cells2 (10Weiss R.H. Joo A. Randour C. J. Biol. Chem. 2000; 275: 10285-10290Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 13Weiss R.H. Randour C.J. Cell. Signal. 2000; 12: 413-418Crossref PubMed Scopus (15) Google Scholar). We therefore hypothesized that Ets-1 suppression of apoptosis might be rescued by strategies targeting p21WAF1/Cip1. Smooth muscle cell apoptosis, as expected, was reduced upon overexpression of Ets-1 (Fig. 8A). Apoptosis was reversed and, indeed, stimulated by antisense oligonucleotides targeting p21WAF1/Cip1 (Fig. 8A). In contrast, an identical concentration of scrambled oligonucleotide had no effect (Fig. 8A). The capacity of the p21WAF1/Cip1 antisense oligonucleotide, but not the scrambled counterpart, to inhibit endogenous p21WAF1/Cip1 expression is indicated by Western blot analysis in Fig. 8B. To provide additional evidence demonstrating the phenotypic relevance of the Ets-1/p21WAF1/Cip1 system, we quantitated the number of cells in each cohort using a Coulter counter. Cells transfected with pKCR3-Ets-1 grew faster than cells transfected with the backbone alone (Fig. 8C). This induction was abolished by antisense p21WAF1/Cip1, whereas the scrambled oligonucleotide, had no effect (Fig. 8C). Moreover, antisense p21WAF1/Cip1 abrogated phosphorylation of the retinoblastoma protein (Fig. 8D), a marker of G1 → S transition in vascular smooth muscle cells and other cell types (18Kintscher U. Wakino S. Kim S. Jackson S.M. Fleck E. Hsueh W.A. Law R.E. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1216-1224Crossref PubMed Scopus (22) Google Scholar). These studies of apoptosis and proliferation provide complementary evidence that Ets-1 induction of p21WAF1/Cip1 has a profound influence on the smooth muscle cell phenotype. The present findings, collectively, demonstrate the existence of a functional ERE (–1350GGAA–1347) that binds Ets-1 and is required for basal expression of the p21WAF1/Cip1 promoter. A second Ets motif upstream of this site in the promoter (–1577GGAT–1574) failed to bind Ets-1 directly or serve as an ERE but was required for basal activity of the p21WAF1/Cip1 promoter. Ets-1 inhibition of smooth muscle cell apoptosis was rescued by inhibition of p21WAF1/Cip1. Conversely, Ets-1 stimulation of proliferation was reduced by p21WAF1/Cip1 inhibition, thus interconnecting the Ets-1/p21WAF1/Cip1 pathway in the regulation of the smooth muscle cell phenotype. The role of Ets-1 in apoptosis is somewhat controversial. For example, inactivation of Ets-1 increased T-cell apoptosis and terminal B-cell differentiation (19Bories J.C. Willerford D.M. Grevin D. Davidson L. Camus A. Martin P. Stehelin D. Alt F.W. Nature. 1995; 377: 635-638Crossref PubMed Scopus (289) Google Scholar). Similarly, Ets-1(–/–)-RAG-2(–/–) chimeric mice have reduced numbers of mature thymocytes and peripheral T cells. They also display a proliferative defect in response to different activation signals and have increased rates of spontaneous apoptosis (20Muthusamy N. Barton K. Leiden J.M. Nature. 1995; 377: 639-642Crossref PubMed Scopus (286) Google Scholar). In contrast, Ets-1 overexpression in human umbilical vein endothelial cells can stimulate apoptosis by modulating the expression of apoptotic genes (21Teruyama K. Abe M. Nakano T. Iwasaka-Yagi C. Takahashi S. Yamada S. Sato Y. J. Cell. Physiol. 2001; 188: 243-252Crossref PubMed Scopus (66) Google Scholar). By examining both proliferation and apoptosis in smooth muscle cells, the present study demonstrates the anti-apoptotic effect of Ets-1 via the p21WAF1/Cip1 pathway in this cell type. We thank Ms. Marjorie Liu for excellent technical assistance." @default.
• W2002405735 created "2016-06-24" @default.
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• W2002405735 date "2003-07-01" @default.
• W2002405735 modified "2023-10-18" @default.
• W2002405735 title "Ets-1 Protects Vascular Smooth Muscle Cells from Undergoing Apoptosis by Activating p21WAF1/Cip1" @default.
• W2002405735 cites W1482706776 @default.
• W2002405735 cites W1964579258 @default.
• W2002405735 cites W1969499006 @default.
• W2002405735 cites W1994394561 @default.
• W2002405735 cites W1996353071 @default.
• W2002405735 cites W2003282728 @default.
• W2002405735 cites W2015519364 @default.
• W2002405735 cites W2023948035 @default.
• W2002405735 cites W2072088194 @default.
• W2002405735 cites W2077452186 @default.
• W2002405735 cites W2080608291 @default.
• W2002405735 cites W2082451220 @default.
• W2002405735 cites W2083757724 @default.
• W2002405735 cites W2086736649 @default.
• W2002405735 cites W2089218510 @default.
• W2002405735 cites W2089394378 @default.
• W2002405735 cites W2096654038 @default.
• W2002405735 cites W2119824866 @default.
• W2002405735 cites W2143681549 @default.
• W2002405735 cites W2148759751 @default.
• W2002405735 doi "https://doi.org/10.1074/jbc.m304328200" @default.
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