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J. Biol. Chem., Vol. 277, Issue 39, 36244-36252, September 27, 2002

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Ets-1 Positively Regulates Fas Ligand Transcription via Cooperative Interactions with Sp1^*

Mary M. Kavurma, Yuri Bobryshev^§, and Levon M. Khachigian^¶

From the  Centre for Thrombosis and Vascular Research and ^§ Surgical Professional Unit, St. Vincents Hospital, The University of New South Wales, Sydney 2052, Australia

Received for publication, January 16, 2002, and in revised form, April 16, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The FasL/Fas system has been implicated in smooth muscle cell apoptosis and atherosclerotic plaque instability, a process that can lead to plaque rupture, precipitating myocardial infarction and sudden death. The transcriptional mechanisms regulating FasL gene expression in vascular smooth muscle cells are poorly understood. We recently described a novel mechanism mediating inducible FasL gene expression in smooth muscle cells involving the zinc finger transcription factor Sp1 (Kavurma, M. M., Santiago, F. S., Bofocco, E., and Khachigian, L. M. (2001) J. Biol. Chem. 276, 4964-4971). We now show that FasL gene expression is governed by cooperative activation between Sp1 and the Ets family of transcription factors. The overexpression of Ets-1 was sufficient to induce FasL promoter-dependent expression and protein synthesis. Ets-1 activation of the promoter was abrogated either by deletion or mutation of the Sp1 binding site. The overexpression of Ets-1 together with Sp1 produced cooperative activation of the FasL promoter. Sp1 induction of the FasL promoter was abrogated by an Ets-1 mutant lacking the activation domain. Conversely, Ets-1 activation of the promoter was blocked by an Sp1 mutant bearing the DNA-binding domain. The mutation of the ³⁶⁵GGAA³⁶² element in the FasL promoter abolished Ets-1 activation and attenuated Sp1-inducible gene expression. Immunoprecipitation and supershift experiments revealed that endogenous Ets-1 and Sp1 physically interact and co-occupy this site. Thus, FasL gene expression in vascular smooth muscle cells is mediated by cooperativity between Ets-1 and Sp1.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Fas Ligand (FasL)¹ is a cytotoxic type II transmembrane protein and member of the tumor necrosis factor family (1). The engagement of FasL with its receptor Fas initiates trimerization of Fas, resulting in the recruitment of the death-inducing signaling complex (1, 2) whose principal components include accessory molecules Fas-associated death domain and pro-caspase 8 (2). Immediately after recruitment, pro-caspase 8 is proteolytically processed and in turn activates the execution phases of apoptosis (3-5).

FasL has been implicated in atherosclerotic plaque instability (6, 7). Smooth muscle cells are the principal cells in the plaque capable of producing collagen fibers required to maintain tensile strength (8, 9). FasL-dependent smooth muscle cell apoptosis within vulnerable regions may lead to plaque instability and rupture, processes that can precipitate myocardial infarction and sudden death. The molecular mechanisms regulating FasL gene expression in vascular smooth muscle cells are poorly understood. However, studies in other cell types have revealed the existence of binding sites in the FasL promoter for NFAT (10), Egr-1 (10), Egr-2 (10), Egr-3 (11), NFB (12), and AP-1 (12). We recently reported that FasL gene expression in smooth muscle cells is controlled by Sp1, which is in turn regulated by the atypical protein kinase  (PKC) (13).

Ets proteins have been implicated in the regulation of genes involved in diverse cellular processes, such as proliferation, differentiation, development, transformation, and apoptosis (14-16). Ets factors typically bear a conserved winged helix-turn-helix DNA-binding domain (17) that recognize a core motif ^5'GGA(A/T)^3' whose flanking nucleotides determine specificity (14, 17). Members of this family include Ets-1, Ets-2, PU.1, Fli-1, GABP, Elf-1, Sap-1, PEA-3, Elk-1, Elk-2, erg and ergB. A key characteristic that Ets transcription factors display is their ability to regulate transcription through interactions with other nuclear factors. For example, Ets proteins functionally interact with NFB (18), AP-1 (19), Pax (20), Tax (21, 22), and Sp1 (21, 23). Here we demonstrate that FasL transcription in smooth muscle cells is regulated by Ets-1, cooperative interactions with Sp1, and a distinct recognition element in the FasL promoter.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Transfections and Luciferase Assays-- WKY12-22/WKY3M-22 smooth muscle cells were maintained in Waymouth's medium (Invitrogen), pH 7.4, supplemented with 1 mM L-glutamine (Invitrogen), 10 units/ml penicillin, 10 µg/ml streptomycin, and 10% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO₂. Transient transfections were performed at 60% confluency together with the indicated constructs and 1 µg of the internal control plasmid pRL-TK using FuGENE 6 transfection agent (Roche Molecular Biochemicals). Luciferase activity was quantified 24 h after transfection using the dual luciferase assay system (Promega), and luciferase activity was normalized with the data generated from pRL-TK.

Plasmid Constructs-- The fragments of the FasL promoter was amplified by PCR from FasL-hsLuc (a gift of Dr. Shailaja Kasibhatla, La Jolla Institute of Cellular Immunology, San Diego, CA) and blunt end-cloned into pGL3-basic (756FasL-hsLuc, 395FasL-hsLuc, 322FasL-hsLuc, and 271FasL-hsLuc). mSp1-FasL-hsLuc has been described previously (13). 756mGGAAFasL-hsLuc was generated using the QuikChange site-directed mutagenesis kit (Stratagene). The following primers were used to generate 756mGGAAFasL-hsLuc, 5'-GCTCTGCAGGATCCCATTCCGGTGAGCATAGCCTAC-3' and 5'-GTAGGCTATGCTCACCGGAATGGGATCCTGCAGACG-3'. Ets-1-pKCR3 was obtained from Ian Cassidy (Department of Biochemistry and Molecular Biology, University of Queensland, Queensland, Australia), and the backbone pKCR3 was generated by restriction enzyme digest EcoR1 separating the chicken Ets-1 cDNA. pEBGNLS and pEBGSp1 were obtained from Gerald Thiel (Institute for Genetics, University of Cologne, Cologne, France), CMV-Sp1 was obtained from Robert Tjian (Howard Hughes Medical Institute, University of California, Los Angeles, CA), Fli-1-pcDNA3 was obtained from Melissa Holmes (Center for Thrombosis and Vascular Research, University of New South Wales, New South Wales, Australia), and PU.1-pcDNA3 was obtained from Robert Passey (Department of Pathology, University of New South Wales, New South Wales, Australia). DN-Ets-1 and pGEXEts-1DBD (Ets-DNA-binding domain) were generated by the amplification of the DNA-binding domain by PCR and blunt end-cloned into pKCR3 or pGEX4T-2 (Amersham Biosciences), respectively. The primer sequences are as follows: forward Ets-1 domain, 5'-CCTGTCATTCCTGCCGC-3' and, reverse Ets-1 domain, 5'-CCTCTGGTGTGTAGCCC-3'.

Western Blot for FasL-- WKY3M-22 smooth muscle cells were transfected with the indicated constructs for 24 h. The medium was then replaced with serum-free medium for an additional 12 h. The supernatant was collected and concentrated using Centricon-10 columns (Millipore), and proteins from the supernatant were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon-P transfer membranes (Millipore). The membrane was blocked overnight in phosphate-buffered saline containing 5% skim milk and 0.05% Tween 20. FasL was detected with FasL polyclonal antibodies (1:100, Santa Cruz Biotechnology) and chemiluminescence.

Immunohistochemistry-- Immunohistochemical analysis on human carotid endarterectomy specimens was performed essentially as described previously (24).

Immunoprecipitation Studies-- Immunoprecipitation experiments were performed in accordance with the manufacturer's guidelines (Dynal). 3 µg of Sp1 rabbit polyclonal IgG (Santa Cruz Biotechnology) and 15 µl of magnetic Dynabeads (M-20 Sheep anti-Rabbit IgG, Dynal) were incubated overnight at 4 °C with gentle shaking. Smooth muscle cell nuclear extracts (20 µl) were incubated with the washed coated beads for 60 min using bidirectional rotation at 4 °C. Several washes were performed prior to Western blot analysis.

Generation of Recombinant Glutathione S-Transferase Ets-1 Protein-- BL21 transformants of pGEXEts-1DBD (residues Pro-386 to Glu-494) were grown to A_0.6-0.8 at 600 nm and stimulated with 0.1 M isopropyl-1-thio--D-galactopyranoside for 3-6 h at 30 °C with shaking. Bacteria were pelleted and sonicated in 50 mM Tris, pH 8.5, 50 mM NaCl, 1.43 mM phenylmethylsulfonyl fluoride, 1.44 mM -mercaptoethanol, and 0.5% Triton X-100. Glutathione S-transferase-conjugated agarose beads were added to the supernatant and incubated on a rotary shaker for 1 h at 4 °C. The beads were washed several times in wash buffer (50 mM, Tris pH 8.0, 100 mM NaCl, 10% glycerol, 1.43 mM phenylmethylsulfonyl fluoride, 1.44 mM -mercaptoethanol, and 0.5% Nonidet P-40) and eluted at 22 °C for 10 min in 100 mM Tris-HCl, pH 7.5 and 10 mM of reduced glutathione.

DNase I Footprint Analysis-- 756FasL-hsLuc was digested with EcoRI and HindIII to generate a 475-bp fragment of the FasL promoter prior to 5' end labeling with [-³²P]dATP and purification using MicroBio-Spin Columns (Bio-Rad). This labeled double-stranded fragment was further digested with BglII and subsequently purified. DNase I footprint analysis was performed using 70,000 cpm of probe, 1 mg/ml poly(dI·dC), 10 mg/ml BSA, 10× footprint buffer (50% glycerol, 50 mM MgCl₂, 500 mM NaCl, and 25 mM HEPES, pH 8.0) and increasing amounts of recombinant Ets-1. Reactions proceeded on ice for 1 h followed by digestion with 2 units of DNase I in 25 mM NaCl, 10 mM HEPES, pH 5.0, 5 mM MgCl₂, and 1 mM CaCl₂ for 5 min. The digestions were stopped by the addition of phenol-chloroform, and the DNA was precipitated with ethanol and resuspended in 5 µl of formamide-loading buffer prior to boiling for 2 min. The reactions were resolved on a 10% sequencing gel and visualized by PhosphorImager.

Electrophoretic Mobility Shift Assay (EMSA)-- WKY12-22 nuclear extracts or recombinant Ets-1 protein were incubated with indicated ³²P-labeled double-stranded oligonucleotides 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. The mixture was incubated for 10-15 min at 4 °C. In competition assays, nuclear extracts were incubated with unlabeled double-stranded oligonucleotide or Ets-1, Sp1, and pCNA antibody (Santa Cruz Biotechnology) prior to the addition of the probe. The samples were resolved by 6% non-denaturing polyacrylamide gel electrophoresis and visualized by autoradiography.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Ets-1 Is Expressed in Human Atherosclerotic Lesions-- We recently determined that FasL gene expression in vascular smooth muscle cells is under the transcriptional control of the zinc finger transcription factor Sp1, which in turn is regulated by atypical PKC (13). This pathway has important implications to smooth muscle cell apoptosis and the potentially fatal consequences of atherosclerotic plaque instability but has not yet been localized to a pathologic setting. An immunohistochemical analysis of human atherosclerotic plaques revealed constitutive Sp1 expression throughout the diseased lesion as well as normal media (Fig. 1). In contrast, the activated form of PKC (phosphorylated on Thr⁴¹⁰) was exclusively detected in atherosclerotic lesions particularly within shoulders (Fig. 1), which are prone to later rupture (25). FasL immunopositivity was also confined to the lesion (Fig. 1). Both PKC-Thr⁴¹⁰ and FasL expression was localized to -SM actin (Ac)⁺ smooth muscle cells undergoing apoptosis based on in situ TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) (Fig. 1) and CPP32 (caspase 3) staining (data not shown). Because Sp1 has been reported to physically associate with the prototypical helix-turn-helix Ets family member Ets-1 in non-vascular cell types (23, 26), we hypothesized that Ets-1 may be involved in FasL gene regulation, possibly in concert with Sp1. To date, Ets-1 has not been localized in human atherosclerotic arteries, nor has it been linked to the FasL transcription in any cell type. Immunohistochemical analysis revealed that Ets-1 is expressed by -actin⁺ smooth muscle cells in human atherosclerotic lesions, coincident with Sp1 and FasL immunoreactivity (Fig. 1).

View larger version (125K): [in this window] [in a new window]   Fig. 1.   Ets-1, Sp1, and FasL immunoreactivity in smooth muscle cells of human carotid atherosclerotic plaques. Smooth muscle cells are identified by -SM-actin+ staining. TUNEL+ and PKC-Thr410 immunopositivity is indicated in the figure. The figure is representative of staining observed in three carotid endarterectomy specimens. I, intima; M, media; C, core.

Ets-1 Activates FasL Gene Expression-- Inspection of the FasL promoter revealed the existence of several putative Ets binding sites (EBS). To determine the capacity of Ets-1 to modulate endogenous FasL protein expression in vascular smooth muscle cells, we performed Western immunoblot analysis with antibodies targeting FasL. The overexpression of Ets-1 compared with the backbone control vector pKCR3 increased FasL immunoreactivity (Fig. 2A). Sp1 overexpression in this system also increased FasL protein levels (Fig. 2A), indicating that Ets-1 like Sp1 is sufficient to confer FasL gene expression. To determine whether FasL is under the transcriptional control of Ets-1, transient transfection analysis was performed with the Firefly luciferase construct FasL-hsLuc under the influence of 1.3 kilobases of the FasL promoter, and the reporter activity was determined. FasL promoter activity was induced by Ets-1. In contrast, expression vectors generating Fli-1 and PU.1 had no effect on FasL promoter-dependent expression (Fig. 2B). A cotransfection of FasL-hsLuc with increasing concentrations of Ets-1-pKCR3 revealed dose-dependent Ets-1 activation of the FasL promoter (Fig. 3A). These data demonstrate selective activation of FasL by this prototypical Ets family factor.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Ets-1 activates FasL gene expression in smooth muscle cells. A, Western blot analysis using samples from WKY3M-22 cells overexpressing Ets-1 and Sp1. 40 µg of Ets-1-pKCR3 and 15 µg of CMV-Sp1 (together with respective backbone controls) were used. Coomassie Blue-stained gel indicates unbiased protein loading. B, Ets-1 selectively activates the FasL promoter. Transient transfection analysis in WKY12-22 cells overexpressing Ets-1 with FasL-hsLuc and Ets family members is shown. 3 µg of indicated expression constructs were used, 3 µg of backbones pKCR3 and pCDNA3 were used, and 15 µg of FasL promoter reporter construct were used throughout. Firefly luciferase activity was normalized to Renilla activity. Error bars represent the mean ± S.E. The data are representative of two independent determinations.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Ets-1 activates the FasL promoter. A, Ets-1 activates FasL promoter activity in a dose-dependent manner. Transient transfections with WKY12-22 cells expressing Ets-1 with FasL-hsLuc or mSp1FasL-hsLuc are shown. B, Ets-1 and Sp1 activate the FasL promoter in a cooperative manner, which is abrogated by the Sp1 mutation in the FasL promoter. C, cooperativity between Ets-1 and Sp1 is demonstrated at the level of FasL protein. 40 and 60 µg of Ets-1-pKCR3 and 15 µg of CMV-Sp1 (together with respective backbone control vectors) were transfected into WKY3M-22 smooth muscle cells for FasL Western analysis. Coomassie Blue-stained gel indicates unbiased protein loading. Unless indicated otherwise, cotransfection with 15 µg of FasL-hsLuc or mSp1FasL-hsLuc was used throughout. Luciferase activity was normalized to Renilla activity. Error bars represent the mean ± S.E. The data are representative of two independent determinations.

Ets-1 Activation of FasL Expression Is Sp1-dependent-- To determine whether Sp1 is involved in Ets-1 induction of the FasL promoter, we performed a transient co-transfection analysis using Ets-1-pKCR3 together with mSp1FasL-hsLuc, a FasL promoter construct bearing a mutation in the Sp1 binding element (⁸²GGGCGG²⁷⁷ to ²⁸²TTTCTT²⁷⁷). This mutant reporter construct was unable to mediate Ets-1 activation of the FasL promoter over a 20-fold concentration range of the Ets-1 expression vector (Fig. 3A), indicating that the Sp1 binding element is essential for Ets-1 activation of the FasL promoter. We next expressed Ets-1 and Sp1 with the FasL promoter in a transient co-transfection setting. Ets-1 and Sp1 each independently induced wild-type FasL promoter-dependent expression (Fig. 3B). Co-expression of both factors was more effective than the expression of either factor alone, producing cooperative activation of the FasL promoter (Fig. 3B). However, mSp1FasL-hsLuc failed to support Sp1, Ets-1, or Sp1/Ets-1 activation (Fig. 3B), demonstrating that the cooperativity between Ets-1 and Sp1 is mediated via the ²⁸²GGGCGG²⁷⁷ element in the FasL promoter. To demonstrate Ets-1/Sp1 cooperativity at the level of endogenous FasL, we evaluated the effect of Ets-1 and Sp1 on FasL protein expression by Western blot analysis. FasL expression induced by a fixed amount of Ets-1 and Sp1 was augmented when the stoichiometry between these factors was increased in favor of Ets-1 (Fig. 3C). In contrast, FasL immunoreactivity was not detected when identical amounts of the respective backbones were used (Fig. 3C).

Mutants of Ets-1 or Sp1 Ablate Cooperative Activation of FasL Promoter-- To provide further support for positive collaboration between Ets-1 and Sp1 at the FasL promoter in smooth muscle cells, we generated a dominant-negative (DN) mutant of Ets-1 bearing only the DNA-binding domain. Sp1 activation of the FasL promoter (Fig. 4, column 3) was abrogated in the presence of DN-Ets-1 mutant (Fig. 4, column 4). Conversely, the Ets-1 activation of the promoter (Fig. 4, column 7) was blocked by DN-Sp1 (pEBGSp1) containing the DNA-binding domain of Sp1 (Fig. 4, column 8). These results demonstrate the reliance of intact Sp1 for Ets-1 activation of the FasL promoter and intact Ets-1 for Sp1 activation. Moreover, these data suggest that the Ets-1/Sp1 cooperativity observed may involve the physical interaction between these factors in these cells.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Ets-1 and Sp1 cooperate to activate the FasL promoter. Cooperativity between Ets-1 and Sp1 is established using DN-mutant constructs. Co-transfection analysis in WKY12-22 smooth muscle cell is shown. Unless indicated, 15 µg of FasL-hsLuc were used throughout. Luciferase activity was normalized to Renilla. Error bars represent the mean ± S.E. The data are representative of two independent determinations.

The GGAA Element between 395 and 322 Is Required for Ets-1 Activation of the FasL Promoter-- We next performed 5' deletion and transient transfection analysis to delineate the actual region in the FasL promoter mediating Ets-1 activation. Ets-1 induced the reporter activity in smooth muscle cells transfected with construct 756FasL-hsLuc and 395FasL-hsLuc (Fig. 5A). In contrast, constructs 322FasL-hsLuc and 262FasL-hsLuc failed to respond to Ets-1 activation (Fig. 5A). These findings indicate that Ets-1 activation of the FasL promoter is mediated by bps spanning the region 395 and 322. Inspection of the promoter sequence revealed an Ets site with a core motif 5'-GGAA-3' (Fig. 5B, 356/352). Fig. 5C demonstrates that recombinant Ets-1 protein (DNA-binding domain, residues Pro-386 to Glu-494) protects the putative Ets-1 binding site from partial DNase I digestion in a dose-dependent manner. This protection was abolished by the presence of 5-fold molar excess of unlabeled oligonucleotide 381/346 (Oligo381/346) (Fig. 5A), which includes the putative ³⁵⁶GGAA³⁵² Ets response element. To demonstrate the functionality of this element, we introduced a transverse mutation at this site (³⁵⁶GGAA³⁵² to ³⁵⁶TTCC³⁵²) in construct 756FasL-hsLuc, generating construct 756mGGAAFasL-hsLuc, and then performed co-transfection experiments with Ets-1 and Sp1. Fig. 5D demonstrates the positive activation by Ets-1 and Sp1 either alone or together on the FasL promoter. In contrast, when the same experiments were performed using the GGAA mutant (756mGGAAFasL-hsLuc), Sp1 transactivation was attenuated, and Ets-1 induction either alone or together with Sp1 was abolished (Fig. 5D). These findings demonstrate that Ets-1 and Sp1 activation of the FasL promoter requires an intact GGAA binding motif.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Ets-1 activation of the FasL promoter is determined between regions spanning from 395 and 322. A, transfection analysis using FasL-hsLuc and 5' derivatives. 3 µg of Ets-1-pKCR3 and pKCR3 were used. B, sequence from human FasL promoter. One core GGAA (EBS) has been identified. The Sp1 binding element and the EBS are italicized. Underlined sequences indicate probes used in EMSA, Oligo381/346 and Oligo296/265. C, DNase I footprint analysis of the FasL promoter. 2 and 8 µl of recombinant Ets-1 were used. Unlabeled 5× molar excess Oligo381/346 was used in competition assays. Putative transcription factor binding sites are noted. D, mutation of 365/362 element abrogates Ets-1 activation of the FasL promoter. Unless indicated, 3 µg of Ets-1-pKCR3 and CMV-Sp1 are used throughout with relative backbones. 15 µg of FasL-hsLuc, 5' derivatives of FasL-hsLuc, and 756mFasL-hsLuc were used. Luciferase activity was normalized to Renilla. Error bars represent the mean ± S.E. The data are representative of two independent determinations.

Ets-1 and Sp1 Physically Interact and Bind the FasL Promoter-- The cooperative activation of the FasL promoter by Ets-1 and Sp1 in smooth muscle cells suggest that these factors may indeed physically interact. We performed immunoprecipitation experiments to determine whether in smooth muscle cells these factors physically associate with one another. Magnetic beads coated with Sp1 antibodies were incubated with smooth muscle cell nuclear extract prior to Western blot analysis with antibodies to Ets-1. This produced readily detectable Ets-1 immunoreactivity sourced from the nuclear extracts (Fig. 6A). When the blot was stripped and reprobed with antibodies targeting Sp1, the immunoreactivity of the expected size was detected (Fig. 6A). These findings demonstrate that Ets-1 physically interacts with Sp1 in smooth muscle cells.

View larger version (55K): [in this window] [in a new window]   Fig. 6.   Ets-1 and Sp1 form a complex. A, immunoprecipitation studies demonstrate a physical association between Ets-1 and Sp1. B, Ets-1 binding is abrogated by the mutation of GGAA element. EMSA using indicated double-stranded 32P-labeled Oligo and recombinant Ets-1 protein is shown. Sequence of Oligo-Ets is 5'-GAT CTC GAG CAG GAA GTT CGA-3' (29). Sequence of Oligo381/346 is 5'-CGT CTG CAG GAT CCC AGG AAG GTG AGC ATA GCC TAC-3'. Sequence of mOligo381/346 is 5'-CGT CTG CAG GAT CCC ATT CCG GTG AGC ATA GCC TAC-3'; the putative EBS is underlined. C, Ets-1 binding is abrogated by the addition of cold excess Oligo381/346. The NFB oligonucleotide sequence is 5'-GGA TGC CAT TGG GGA TTT CCT CTT TAC TGG AGT T-3'. D, Ets-1 and Sp1 are involved in a nucleoprotein complex at the FasL promoter. Supershift studies using [32P]Oligo381/346, WKY12-22 nuclear extracts, and indicated antibodies are illustrated. EMSA was performed as described under "Experimental Procedures," and nucleoprotein complexes were visualized by autoradiography. NE, nuclear extracts.

We next performed electrophoretic mobility shift analysis to determine whether the Ets response element in the FasL promoter could support the interaction of Ets-1 and Sp1. EMSA using recombinant Ets-1 and a consensus Ets-1 oligonucleotide supported the formation of a specific nucleoprotein complex (Fig. 6B). This complex was also apparent with ³²P-labeled Oligo381/346 (Fig. 6B). The interaction was no longer supported when the GGAA motif in [³²P]Oligo381/346 was mutated to TTCC (Fig. 6B). EMSA from nuclear extracts produced three specific nucleoprotein complexes (Fig. 6C, complexes I-III) whose formation was blocked by 50-fold molar excess of unlabeled probe but was unaffected by the same fold excess of an unrelated oligonucleotide bearing the NFB binding site (Fig. 6C). Supershift analysis using antibodies to Ets-1, Sp1, and pCNA identified the protein components of complex I to contain Ets-1 and Sp1, complex II to contain Sp1, and complex III to be neither Ets-1 nor Sp1 (Fig. 6D). These results indicate that Sp1 and Ets-1 co-occupy this region of the FasL promoter (complex I).

The positive supershift using Sp1 antibodies suggested that complex II (Fig. 6, C and D) contains Sp1. However, Oligo381/346 does not bear a consensus Sp1 binding site (Fig. 5B). To determine whether Sp1 can directly bind this region of the promoter, we performed EMSA using [³²P]Oligo381/346 and recombinant Sp1. [³²P]Oligo381/346 did not support a Sp1·DNA complex (Fig. 7A), whereas [³²P]Oligo296/265 containing a functional Sp1 site (13) produced a nucleoprotein complex (Fig. 7A). Thus, it appears that the FasL promoter Ets response element hosts a complex between Ets-1 and Sp1, although Sp1 alone cannot directly contact DNA. Sp1 may associate with other factor(s) that interact with DNA at this site.

View larger version (42K): [in this window] [in a new window]   Fig. 7.   Sp1 does not physically bind to 381/346 region. A, EMSA was performed using [32P]Oligo381/346, [32P]Oligo295/265 and recombinant Sp1 (Promega). B, Ets-1 does not interact with double-stranded [32P]295/265. C, mutation in the Sp1 site abrogates Sp1 binding. Sequence of [32P]Oligo296/265 is 5'-ATC AGA AAA TTG TGG GCG GAA ACT TCC AGG-3'. Sequence of [32P]mOligo296/265 is 5'-ATC AGA AAA TTG TTT TCT TAA ACT TCC AGG-3'. The Sp1 element is underlined. EMSA was performed as described under "Experimental Procedures," and nucleoprotein complexes were visualized by autoradiography. NE, nuclear extracts.

The functional studies in Fig. 3A revealed that Ets-1 activation of the FasL promoter requires the existence of an intact Sp1 element (²⁸²GGGCGG²⁷⁷). We used EMSA to determine whether Ets-1 physically associates with Sp1 at this downstream site (Fig. 5B). [³²P]Oligo296/265 as expected (13) produced an Sp1 supershift from nuclear extracts (Fig. 7B). This was abolished when the Sp1 binding site was mutated from GGGCGG to TTTCTT (Fig. 7C). However, unlike [³²P]Oligo381/346 (Fig. 6D), an Ets-1 supershift was not observed with [³²P]Oligo296/265 (Fig. 7B). These findings indicate that Ets-1 physically associates with Sp1 at the 381/346 site but not the 296/265 site in the FasL promoter.

Atherosclerotic plaque rupture can lead to acute coronary syndromes including myocardial infarction and death. The underlying factors and mechanisms precipitating plaque rupture are poorly understood. However, accumulating evidence suggests that instability may arise from smooth muscle cell apoptosis. For example, comparison of coronary atherectomy specimens from patients with unstable and stable angina revealed a significantly lower content of smooth muscle cells and greater apoptosis in unstable versus stable lesions (25). Similarly, human plaque smooth muscle cells show higher rates of apoptosis in culture than smooth muscle cells from normal vessels (27). Although the mechanism is not entirely known, FasL gene expression has been implicated in this process (28). We recently demonstrated a proapoptotic role for Sp1 by activating FasL expression in smooth muscle cells via the ²⁸²GGGCGGG²⁷⁷ element in a PKC-dependent manner (13). This study now shows that Ets-1 in collaboration with Sp1 binds and activates the FasL promoter via the ³⁵⁶GGAA³⁵² element and induces FasL expression. Whether the existence of a second putative Ets motif (³⁷²GGAT³⁶⁹) also mediates FasL promoter expression is presently under investigation in our laboratory. Dominant-negative experiments revealed that the structural integrity of Ets-1 and Sp1 is critical for the cooperative activation of the FasL promoter by these factors. Our demonstration that Ets-1, Sp1, and FasL are expressed in apoptotic smooth muscle cells in atherosclerotic lesions supports the main finding of this study that Ets-1 and Sp1 alone and together regulate FasL in smooth muscle cells.

Ets factors modulate the transcription via cooperative interactions with nuclear factors. These include NFB (18), AP-1 (19), Pax (20), and Tax (22). This study is the first to demonstrate that Ets-1/Sp1 cooperativity mediates the expression of FasL. Such cooperativity has been observed in other promoters. For example, Ets-1/Sp1 activates MRG1 expression in fibroblasts (26), glycoprotein IIb gene expression in megakaryocytes (23), and together with Tax facilitates PTHrP promoter transactivation (21). Based on these findings, we propose that the Ets-1/Sp1 activation of FasL transcription involves a direct physical association between these factors upstream in the promoter where Sp1 can also bind independently of Ets-1 but does not contact DNA (Fig. 8). In contrast, Ets-1 does not appear to associate with the Sp1 complex at the proximal site. Because the Sp1 binding element is located in close proximity to sites for NFAT and NFB, it is probable that these other factors may play a role in the stereo-specific enhancer complex involving Ets-1 and Sp1.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Schematic of proposed interactions between regulatory elements involved in the transcriptional regulation of FasL. Sp1 physically associates with Ets-1, and the nucleoprotein complex at 381/346 can be supershifted with antibodies to either Sp1 or Ets-1. Sp1 binds this region independently of Ets-1 but does not directly contact the promoter. Ets-1 in contrast is unable to associate with promoter-bound Sp1 at 296/265. The 296/265 site is a "hot spot" with putative recognition elements for NFB and NFAT in close proximity.

In summary, this study introduces a new player, Ets-1, in the complex transcriptional regulation of FasL gene expression. Ets-1 activation of the FasL promoter is critically dependent upon physical and cooperative interactions with the zinc finger nuclear protein Sp1. Mutations that perturb the capacity of Sp1 or Ets-1 to bind recognition elements in the FasL promoter impair the capacity of either factor to induce gene expression.

	ACKNOWLEDGEMENTS

We thank Drs. Ian Cassidy for the Ets-1 expression vector (University of Queensland), Melissa Holmes (University of New South Wales) and Denis Hickstein (University of Washington) for the Fli-1 expression vector, Robert Tjian (University of California) for the Sp1 expression vector, Gerald Thiel (University of Cologne) for the dominant-negative Sp1 vector, Robert Passey (University of New South Wales) for the PU.1 expression vector, and Alex Toker (Harvard Medical School) for the PKC-Thr⁴¹⁰ antibody.

	FOOTNOTES

^* This work was supported by grants from the New South Wales State Department of Health and by National Health and Medical Research Council of Australia (NHMRC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Principal Research Fellow of the NHMRC. To whom correspondence should be addressed: Centre for Thrombosis and Vascular Research, Dept. of Pathology, The University of New South Wales, Sydney NSW 2052, Australia. Tel.: 61-2-9385-2537; Fax: 61-2-9385-1389; E-mail: L.Khachigian@unsw.edu.au.

Published, JBC Papers in Press, April 22, 2002, DOI 10.1074/jbc.M200463200

	ABBREVIATIONS

The abbreviations used are: FasL, Fas Ligand; PKC, protein kinase ; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; EBS, Ets binding site; DN, dominant-negative.

	REFERENCES

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