From the Centre for Vascular Research, The University of New South Wales and Department of Haematology, The Prince of Wales Hospital, Sydney, New South Wales 2052, Australia
Received for publication, April 25, 2003
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ABSTRACT |
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INTRODUCTION |
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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
(6). 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 (7). Such factors
include Sp1, AP-1, c-Myb, and the highly conserved nuclear phosphoprotein
p300. Two regions of p300 between amino acid residues 328596 and
16782370 independently interact with Ets-1
(8). 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.
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructs0-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 (1577TTCG1574) and 5*2 (1350TTCC1347). 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 (9).
Plasmid and Oligonucleotide TransfectionSmooth muscle cells
were seeded in 100-mm tissue culture plates. At 5060% 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 ActivityTransient 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-1Generation of pGEXEts-1-DBD
has been described previously
(9). Briefly, transformants
were stimulated with 0.1 M
isopropyl-1-thio--D-galactopyranoside for 36 h
following an OD of 0.60.8 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
-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 12 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
-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 AnalysisRecombinant 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 1015 min at 4 °C. Samples were resolved by 6% non-denaturing polyacrylamide gel electrophoresis and visualized by autoradiography.
Western Blot AnalysisSmooth 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') (10) 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 4050% 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 (11, 12) 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 StudiesNuclear extracts of cells were precleared with 30 µl of 1:1 slurry of protein G-SepharoseTM-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 ApoptosisSmooth muscle cells were grown to 5060% 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 x 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.
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RESULTS AND DISCUSSION |
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Existence of Two Putative Ets-1 Elements in the p21WAF1/Cip1 PromoterWe and others have recently determined that p21WAF1/Cip1 plays a mitogenic and anti-apoptotic role in vascular smooth muscle cells2 (10, 13). 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).
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Ets-1 Activates p21WAF1/Cip1 Transcription and Protein ExpressionTo 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.
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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).
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Ets-1 Interacts Selectively with an Ets Element in the p21WAF1/Cip1 PromoterTo 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 (1350GGAA1347) or Oligo-1597/-1558 (1577GGAT1574)) or mutant (mOligo 1370/1334 (1350TTCC1347) or mOligo 1597/1558 (1577TTCG1574)) sequences, respectively. A separate oligonucleotide (32P-labeled Oligo FasL 381/346), bearing the Fas ligand promoter ERE (356GGAA352) (9) bound the Ets-1 protein, as expected (Fig. 5). 32P-Oligo 1370/1334 (1350GGAA1347) also formed a nucleoprotein complex with Ets-1 (Fig. 5), which was abrogated if the probe was substituted with Oligo 1370/1334 (1350TTCC1347) (Fig. 5). A nucleoprotein complex was not detected using 32P-mOligo 1597/1558 (1577GGAT1574) or 32P-mOligo 1597/1558 (1577TTCG1574) (Fig. 5).
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The p21WAF1/Cip1 Promoter Is Differentially Regulated by Ets Motif 1350GGAA1347 and 1577GGAT1574To 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 (1577TTCG1574) in which the 1577GGAT1574 motif had been mutated; however, Ets-1inducibility of the promoter was unchanged (Fig. 6). Mutation of the 1350GGAA1347 element in construct 5*2 (1350TTCC1347) ablated both basal and Ets-1-inducible expression (Fig. 6). Thus, the 1577GGAT1574 motif mediates basal but not Ets-1 activation of the p21WAF1/Cip1 promoter, whereas the 1350GGAA1347 element mediates both basal and Ets-1 inducible p21WAF1/Cip1 promoter-dependent expression.
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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 (14). p300 modulation of p21WAF1/Cip1 transcription is mediated by cooperative interactions with a number of nuclear factors, including Sp1 (15), Sp3 (16), and BRCA1 (17). 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 (1577TTCG1574) bearing the mutant 1577GGAT1574 motif, whereas Ets-1 inducibility of this construct was not changed (Fig. 7A). Unlike wild-type construct 5*2, construct 5*2 (1577TTCG1574) 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 (1350TTCC1347), bearing the mutated 1350GGAA1347 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).
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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
(10,
13). 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
(18). 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.
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The present findings, collectively, demonstrate the existence of a functional ERE (1350GGAA1347) 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 (1577GGAT1574) 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 (19). 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 (20). In contrast, Ets-1 overexpression in human umbilical vein endothelial cells can stimulate apoptosis by modulating the expression of apoptotic genes (21). 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.
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FOOTNOTES |
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Visiting Scholar from the First Clinical College of Harbin Medical
University.
Principal Research Fellow of the National Health and Medical Research
Council of Australia.
¶ To whom correspondence should be addressed. Tel.: 61-2-93852537; Fax: 61-2-9385-1389; E-mail: L.Khachigian{at}unsw.edu.au.
1 The 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.
2 Kavurma, M. M., and Khachigian, L. M. (2003) J. Biol. Chem., in
press.
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ACKNOWLEDGMENTS |
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REFERENCES |
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