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Sp1 Phosphorylation Regulates Apoptosis via Extracellular FasL-Fas Engagement

Sp1 Phosphorylation Regulates Apoptosis via Extracellular FasL-Fas Engagement THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 7, Issue of February 16, pp. 4964 –4971, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Sp1 Phosphorylation Regulates Apoptosis via Extracellular FasL-Fas Engagement* Received for publication, October 10, 2000 Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M009251200 Mary M. Kavurma‡, Fernando S. Santiago‡, Emanuela Bonfoco§, and Levon M. Khachigian‡¶i From the ‡Centre for Thrombosis and Vascular Research, The University of New South Wales, Sydney NSW 2052, Australia and the §Scripps Research Institute, La Jolla, California 92037 Apoptosis of smooth muscle cells (SMC) in atheroscle- 3/CPP32, which is expressed in cells as the inactive 32-kDa rotic vessels can destabilize the atheromatus plaque and form is, in turn, cleaved by caspase-8/FLICE to produce two result in rupture, thrombosis, and sudden death. In ef- mature subunits (17 and 12 kDa). Active caspase-3/CPP32 forts to understand the molecular processes regulating cleaves nuclear mitotic apparatus protein and mediates DNA apoptosis in this cell type, we have defined a novel fragmentation, chromatin condensation, and the formation of mechanism involving the ubiquitously expressed tran- apoptotic bodies (4). scription factor Sp1. Subtypes of SMC expressing abun- FasL (5) and Fas (6, 7) are both expressed in arterial tissue, dant levels of Sp1 produce the death agonist, Fas ligand including the human atherosclerotic plaque. Immunohisto- (FasL) and undergo greater spontaneous apoptosis. Sp1 chemical analysis revealed FasL expression in 34 of 34 carotid activates the FasL promoter via a distinct nucleotide atherosclerotic plaques examined, with virtually all FasL pos- recognition element whose integrity is crucial for induc- itive-staining associated with intimal smooth muscle cells ible expression. Inducible FasL promoter activation is (SMCs) and little staining apparent in normal arterial tissue also inhibited by a dominant-negative form of Sp1. In- (5). Fas is also highly expressed in intimal SMCs of the plaque creased SMC apoptosis is preceded by Sp1 phosphoryl- (6, 7). FasL/Fas expression and apoptosis (8 –10) in normal ation, increased FasL transcription, and the autocrine/ artery and plaque has prompted speculation on the roles of these paracrine engagement of FasL with its cell-surface molecular mediators in vascular cells. Apoptosis in undiseased receptor, Fas. Inducible FasL transcription and apopto- z, tissue may inhibit arterial thickening by limiting cell prolifera- sis are blocked by dominant-negative protein kinase C- whose wild-type counterpart phosphorylates Sp1. Thus, tion and accumulation in the intima (6). In atherosclerotic tissue, Sp1 phosphorylation is a proapoptotic transcriptional apoptosis particularly of collagen-producing SMCs may substan- event in vascular SMC and, given the wide distribution tially weaken the plaque causing it to rupture, initiate thrombo- of this housekeeping transcription factor, may be a com- sis, and trigger acute coronary syndromes (11–12). Overexpres- mon regulatory theme in apoptotic signal transduction. sion of FasL in balloon-injured rat carotid arteries devoid of endothelium-induced apoptosis in medial SMCs and inhibited intimal hyperplasia (13, 14). However, recent evidence in a rabbit Apoptosis is a genetically regulated “programmed” form of model suggests that FasL may promote rather than retard cell death and is characterized by a number of specific biochem- atherogenesis. FasL overexpression in nondenuded arteries of ical and morphological changes, including nuclear chromatin hypercholesterolemic animals stimulated lesion formation in condensation, cytoplasmic condensation, membrane blebbing, these animals via increased cellularity (15). These observations and internucleosomal fragmentation of DNA (1, 2). Fas/APO-1 may be due to differences in artery and lesion cellular composi- (or CD95) is a 45-kDa cell surface glycoprotein that belongs to tion or cholesterol feeding between the two animal models. the tumor necrosis factor receptor superfamily and mediates Despite clear evidence for FasL and Fas expression in SMCs apoptosis in various normal and transformed cell types. Upon of the artery wall, the molecular mechanisms mediating FasL the engagement of Fas by Fas ligand (FasL), a highly con- production in vascular cells are presently not known. The pro- served, ubiquitously expressed 40-kDa glycoprotein, the apo- moter region of the FasL gene has recently been cloned and ptotic cysteine protease caspase-8/FLICE is recruited to the found to contain binding sites for a number of transcription receptor via FADD and activated by proteolysis (3). Caspase- factors including NF-kB (16), AP-1 (16), NFAT (17), ATF2 (18), Egr-2 (17), and Egr-3 (17). The promoter contains a single transcription initiator site, as well as positive and negative * This work was supported in part by grants from the Australian regulatory regions within a 2.3-kilobase portion of the 59-un- Research Council (to L. M. K.), National Health and Medical Research Council of Australia (NHMRC) (to L. M. K.), and an NSW Department translated genome (19). Analysis of the FasL promoter has of Health Infrastructure grant to the Centre for Thrombosis and Vas- mostly been confined to T cells. For example, T cell activation cular Research. The costs of publication of this article were defrayed in following CD4 cross-linking induces NFAT binding to the FasL part by the payment of page charges. This article must therefore be enhancer and gene transactivation (19). Similarly, cytotoxic hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. stress-induced FasL expression involves the activation of NF- Research Fellow of the NHMRC. kB, AP-1, and c-Jun N-terminal kinase, prior to cell death i To whom correspondence should be addressed. Tel.: 61-2-9385 2537; (16 –20). Activity of the FasL promoter is also regulated by Fax: 61-2-9385 1389; E-mail: l.Khachigian@unsw.edu.au. MEK kinase-1 (18). However, transcription factor phosphoryl- The abbreviations used are: FasL, Fas ligand; CIP, calf intestinal alkaline phosphatase; DN-PKC-z, dominant-negative protein kinase ation has not yet been directly demonstrated as a prerequisite C-z; SMC, smooth muscle cells; CAM, camptothecin; PCR, polymerase step in apoptosis. chain reaction; RT-PCR, reverse transcription-PCR; PBS, phosphate- The discovery and functional characterization of Sp1 as a buffered saline; ELISA, enzyme-linked immunosorbent assay; FACS, GC-rich binding nuclear protein has provided a useful para- fluorescence-activated cell sorter; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus. digm to our understanding of the regulation of transcriptional 4964 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Sp1 Phosphorylation Regulates FasL-dependent Apoptosis 4965 was used in the ELISA, which was performed in accordance with the activation in eukaryotic cells (21, 22). Sp1 is a broadly ex- manufacturer’s instructions and normalized to total cell number meas- pressed nuclear protein of ;100 kDa and contains three Krup- ured using a Coulter counter. Results are expressed as total internu- pel-like zinc fingers that contact DNA (21, 22). A nucleotide cleosomal DNA fragmentation as a proportion of the cell population. recognition element for Sp1 is located in the FasL promoter at Annexin V Staining/FACS Analysis—SMCs were washed twice with position 2281/2276 base pairs (GGGCGG) relative to the tran- ice-cold phosphate-buffered saline, pH 7.4 and resuspended in 13 bind- scriptional start site. Sp1 can influence gene expression by ing buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl )ata concentration of 1 3 10 cells/ml. One hundred microliters of the sus- changes in its nuclear concentration and interaction with the pension was transferred to 5-ml flat bottomed tubes where 5 mlof promoter, by providing architectural support, serving as a co- annexin V-fluorescein isothiocyanate and 10 ml of propidium iodide (50 factor, or by undergoing chemical modification. Sp1 phospho- mg/ml stock in PBS) was added. The cells were gently vortexed and rylation e.g. mediates inducible tissue factor expression in vas- incubated in the dark at 22 °C for 15 min. Four hundred microliters of cular endothelial cells exposed to fluid shear stress (23). The binding buffer was added to each tube, and annexin V staining was significance of Sp1 in the process of apoptosis in any cell type is analyzed by flow cytometry within 1 h. Results are expressed as an- nexin V staining as a percentage of the total cell population. presently unknown. This knowledge would advance our under- Propidium Iodide Nuclear Staining—SMCs were grown in chamber standing of the transcriptional basis of extrinsic apoptosis, slides (80% confluent) and incubated with CAM (1 mg/ml) for 24 h. The given the wide distribution of both Sp1 and FasL. cells were washed in PBS, pH 7.4, and fixed with methanol/acetone WKY12-22 and WKY3M-22 cells are well established sub- (80:20) for 10 min at 22 °C. Propidium iodide (50 mM) was added to each types of vascular smooth muscle cells that are phenotypically well and incubated for a maximum of 5 min followed by a second wash distinct (24, 25). WKY12-22 cells have a cobblestone morphol- with PBS. Cells undergoing apoptosis were visualized by confocal microscopy. ogy in culture, proliferate in plasma-derived serum (which lacks Nuclear Extract Preparation—SMCs treated with CAM for various vital growth factors), and spontaneously overexpress mRNA for times were washed and scraped in 10 ml of PBS and transferred to platelet-derived growth factor (PDGF) B-chain, elastin, and os- precooled centrifuge tubes. Samples were spun at 1300 rpm for 15 min teopontin (24, 25). In contrast, WKY3M-22 cells are typically at 4 °C. The pellet was resuspended in 100 ml (for two 100-mm dishes) spindle-shaped and do not express PDGF-B, elastin, or osteopon- of solution A (10 mM Hepes, pH 7.9, 1.5 mM MgCl ,10mM KCl) and tin mRNA, nor do they grow in plasma-derived serum. Both cell placed on ice for 5 min. Samples were spun at 14,000 rpm for 40 s. The pellet was resuspended in 20 ml of solution C (20 mM Hepes, pH 7.9, 1.5 subtypes are phenotypically stable in culture and can be pas- mM MgCl , 420 mM NaCl, 0.2 mM EDTA) and mixed gently for 20 min saged indefinitely. Therefore, WKY12-22 and WKY3M22 cells at 4 °C. The supernatant was transferred to precooled Eppendorf tubes represent important cells with which to delineate the molecular containing 20 ml of solution D (20 mM Hepes, pH 7.9, 1.5 mM KCl, 0.2 basis for differences in SMC phenotype and gene expression. mM EDTA, 20% glycerol) and stored at 280 °C until use. All buffers We recently reported that Sp1 is spontaneously expressed at contained protease inhibitors. greater levels in WKY12-22 cells than WKY3M-22 cells and that Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts (6 –10 mg) were incubated with P-labeled double-stranded oligonucleo- as a consequence, Sp1-dependent genes, such as PDGF-B, are tide (150,000 cpm, 40 fmol) in 20 ml containing 10 mM Tris-HCl, pH 8.0, overexpressed in WKY12-22 cells compared with its sister cell 50 mM MgCl ,1mM EDTA, 1 mM dithiothreitol, 5% glycerol, 1 mgof subtype (26). These observations provided important insight into salmon sperm DNA, 5% sucrose, 1 mg of poly(dI-dC) and 1 mM phenyl- the transcriptional basis for differential gene expression. Here methylsulfonyl fluoride. The mixture was incubated for 35 min at 22 °C. we explored the regulatory role of Sp1 in inducible FasL expres- In supershift experiments, nuclear extract was incubated with 2 mgof sion and apoptosis in two phenotypically distinct SMC subtypes. antibody prior to addition of the probe. Samples were resolved by 8% nondenaturing polyacrylamide gel electrophoresis, and binding com- EXPERIMENTAL PROCEDURES plexes were visualized by autoradiography at 280 °C. Transfections and Luciferase Assays—-SMCs were maintained in Western Blot for Sp1—Fifteen micrograms of nuclear extract was Waymouth’s medium (Life Technologies, Inc.), pH 7.4, containing 10% resolved by 8% SDS-polyacrylamide gel electrophoresis and then trans- fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO . ferred onto Immobilon-P transfer membranes (Millipore). The mem- Transient transfections were performed with cells at 60% confluence, branes were blocked overnight at 4 °C in PBS containing 5% skim milk and the indicated constructs together with 2 mg of the internal control and 0.05% Tween 20. Sp1 was detected with Sp1 polyclonal antibodies vector, pRL-TK, using FuGENE6 transfection agent (Roche Molecular (1:1000, Santa Cruz Biotechnology) and subsequent chemiluminescent Biochemicals). After 24 h, the transfected cells were incubated with or visualization. without CAM (1 mg/ml), and luciferase activity was quantified using the Sp1 Dephosphorylation Analysis by Western Blotting and EMSA— Dual Luciferase Assay System (Promega). Firefly luciferase activity Nuclear extracts (10 –15 mg) were incubated with or without 5 units of was normalized to Renilla data generated from pRL-TK. calf intestinal alkaline phosphatase (CIP, NEB) for1hat37 °Cina Plasmid Constructs—Various sized fragments of the FasL promoter total volume of 20 ml. The reaction was quenched by the addition of (2271FasLzhsLuc and 2296FasLzhsLuc) were amplified from the par- loading dye prior to 8% SDS-polyacrylamide gel electrophoresis and ent vector FasLzhsLuc (gift of Dr Shailaja Kasibhatla, La Jolla Inst. of Western blot analysis for Sp1. In EMSA, 8 mg of nuclear extract was Cellular Immunology) by PCR and blunt-end cloned into pGL3. The incubated with 5 milliunit of CIP (final concentration determined by mutant counterpart of FasLzhsLuc bearing a mutation in the Sp1 bind- CIP titration experiments with P-labeled FasL Oligo) at 33 °C for 5 ing site (mSp1FasLzhsLuc) was constructed using the QuikChange min and then on ice for 15 min. The reaction was stopped by the site-directed mutagenesis kit (Stratagene). pEBGNLS-Sp1 was ob- addition of phosphatase inhibitors at a final concentration of 10 nM tained from Gerald Thiel (Inst. for Genetics, University of Cologne), LiNaF, 10 nM sodium vanadate, 10 nM potassium pyrophosphate, and 5 CMV-Sp1 was obtained from Robert Tjian (Howard Hughes Medical nM sodium phosphate. EMSA was performed as described above. CIP Inst., University of California), and CMV-FLAGzDN-PKC-z was ob- treatment prior to EMSA has previously been used for the assessment tained from Debabrata Mukhopadhyay (Beth Israel-Deaconess Hospi- of Sp1 phosphorylation in nuclear extracts (30, 31). tal, Boston) and Alex Toker (Boston Biomedical Research Inst.). RT-PCR—Five micrograms of total RNA isolated using TRIzol reagent Quantitative Assessment of DNA Fragmentation—SMCs were grown (Life Technologies, Inc.) were treated with DNase I, and cDNA was in 96-well plates to 80% confluency in 100 ml of growth medium. Where generated using Superscript II reverse transcriptase (Life Technologies, indicated, the cells were incubated with Fas-Fc (R&D Systems) and/or Inc.) with random primers according to the manufacturer’s instructions. IgG Fc (R&D Systems) (50 mg/ml, final concentration) for 1 h prior to Sequences of the primers for FasL, Fas, and b-actin are as follows: 59-Fa- the addition of CAM. After 24-h exposure to CAM, apoptosis was quan- sL, 59-AAACCCTTTCCTGGGGC-39;39-FasL, 59-GTGTCTTCCCATTCC- Plus titated using the Cell Death Detection ELISA (Roche Molecular AG-39;59-Fas, 59-CTGTGGATCATGGCTGTCCTGCCT-39;39-Fas, 59-CT- Biochemicals). This assay, which measures cytoplasmic histone-associ- CCAGACTTTGTCCTTCATTTTTC-39;59-b-actin, 59-TGACGGGGTCAC- ated internucleosomal DNA fragmentation, has been used previously to CCACACTGTGCCCATCTA-39;39-b-actin, 59-CTAGAAGCATTTGCGGT- quantitate inducible apoptosis in cultured cells (27–29). Briefly, the GGACGATGGAGGG-39;59-glyceraldehyde-3-phosphate dehydrogenase, cells were washed gently in PBS and incubated with shaking in lysis 59-ACCACAGTCCATGCCATCAC-39;39-glyceraldehyde-3-phosphate buffer for 30 min at 22 °C. Lysates were transferred into Eppendorf dehydrogenase, 59-TCCACCACCCTGTTGCTGTA-39. tubes and spun at 14,000 rpm for 30 s. Twenty ml of the supernatant PCR was performed in a total volume of 50 ml containing 2 mM 4966 Sp1 Phosphorylation Regulates FasL-dependent Apoptosis FIG.1. Differences in spontaneous apoptosis and FasL expression in WKY12-22 and WKY3M-22 cells. A, spontaneous levels of apoptosis as determined by internucleosomal DNA fragmentation (left) and annexin V/FACS analysis (right) in WKY12-22 and WKY3M-22 cells. The y axis in the left and right panels represents total internucleosomal DNA fragmentation (as a proportion of the cell population) and annexin V staining (as a percentage of the total number of cells in the population), respectively. B, overexpression of Sp1 in WKY3M22 cells stimulates apoptosis. SMCs were transfected with 0, 1, 3, and 5 mg of CMV-Sp1 using FuGENE6 prior to quantification of apoptosis after 24 h by ELISA. Where required, the total amount of DNA transfected was supplemented to 5 mg with pcDNA3. C, dominant-negative Sp1 blocks apoptosis in WKY12-22 cells. SMCs were transfected with 40 mg of pEBGNLS or pEBGNLS-Sp1 using FuGENE6 prior to quantification of apoptosis after 24 h by ELISA. Results in B and C (ordinate axes) are expressed as total internucleosomal DNA fragmentation as a proportion of the total number of cells in the treatment population. D, WKY12-22 cells, but not WKY3M-22 cells, express FasL mRNA, and this is stimulated by CAM. Total RNA prepared from WKY12-22 cells incubated with CAM (1 mg/ml) for 24 h prior to reverse-transcription and PCR with the indicated primers. PCR cycle number is indicated in the figure. The densitometric assessment of the amplicons is represented histodiagramatically. E, comparative FasL mRNA expression in WKY12-22 cells and WKY3M-22 cells. Northern blot analysis was performed with total RNA of WKY3M-22 cells or WKY12-22 cells (with or without 24-h incubation with 1 mg/ml CAM. The data are representative of two independent determinations. Error bars represent S.E. Sp1 Phosphorylation Regulates FasL-dependent Apoptosis 4967 FIG.2. Sp1 activates the FasL promoter in SMCs. Transient cotransfection analysis in WKY12-22 cells overexpressing Sp1 with FasL-hsLuc (A) and derivatives of FasLzhsLuc bearing 59 deletions in the FasL promoter (B). 6 mg of pcDNA3 or CMV-Sp1 was used in B;15 mg of FasL promoter-reporter construct was used throughout. Firefly luciferase activity was normalized to Renilla activity and the results were plotted as -fold 32 32 increase relative to 2296FasLzhsLuc or 2271FasLzhsLuc, respectively. C, EMSA using [ P]FasL Oligo, [ P]mFasL Oligo, and nuclear extracts of WKY12-22 cells. EMSA was performed as described under “Experimental Procedures,” and nucleoprotein complexes were visualized by autora- diography. Arrows indicate nucleoprotein complexes, S denotes a supershift. Sequence of [ P]mFasL Oligo (2296/2265) is 59-ATCAGAAAATT- GTGGGCGGAAACTTCCAGG-39, and [ P]mFasL Oligo is 59-ATCAGAAAATTGTTTTCTTAAACTTCCAGG-39; the mutation is underlined). D, mutation of the Sp1 site in FasLzhsLuc abrogates activation of the FasL promoter. E, Sp1 activation of FasL promoter in WKY3M-22 cells. 3 or 6 mg of pcDNA3 or CMV-Sp1 were used in cotransfection experiments with 15 mg of FasL promoter-reporter construct throughout. Firefly luciferase activity was normalized to Renilla activity, and the results were plotted as -fold increase relative to the pcDNA3 control in the FasLzhsLuc and mSp1FasLzhsLuc contransfectant groups, respectively. Error bars represent S.E. The data are representative of two independent determinations. 4968 Sp1 Phosphorylation Regulates FasL-dependent Apoptosis FIG.3. CAM stimulates apoptosis in vascular SMCs and induces FasL ex- pression in an Sp1-dependent man- ner. A, CAM increases propidium iodide nuclear staining in WKY12-22 cells. The SMCs were exposed to CAM (1 mg/ml) for 24 h prior to fixation, incubation with pro- pidium iodide, and confocal microscopy. Effect of CAM (1 mg/ml) on luciferase ex- pression driven by FasLzhsLuc or a con- struct bearing a deletion of the Sp1 bind- ing element (2271FasLzhsLuc, B), or FasLzhsLuc cotransfected with 5 mgof dominant-negative Sp1 (pEBGNLS-Sp1) or the backbone alone (pEBGNLS, C). 15 mg of FasL promoter-reporter construct was used throughout. Firefly luciferase activity was normalized to Renilla activ- ity, and the results were plotted as -fold increase relative to FasLzhsLuc or 2271FasLzhsLuc, respectively, in B,or the pEBGNLS or pEBGNLS-Sp1 groups in C. Error bars represent S.E. The data are representative of two independent determinations. MgCl ,2mM dNTPs, 2.5 units of Taq DNA polymerase (Sigma), 5 mlof 2 tion (27–29) is greater in WKY12-22 cells than WKY3M-22 cDNA, and either 100 pmol of Fas primers, 100 pmol of FasL primers, cells (Fig. 1A, left), indicating higher levels of spontaneous or 20 pmol of b-actin primers using a Perkin Elmer thermocycler. apoptosis in the former cell subtype and providing further Amplication was performed by denaturing the sample at 94 °C for 2 evidence that these SMC subtypes are phenotypically distinct. min, then cycled at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and an extension at 72 °C for 4 min. The number of PCR cycles for Fas/b These observations were confirmed by annexin V-fluorescein iso- actin was 25. Twenty microliters of the reaction was visualized on 1.5% thiocyanate staining upon fluorescence-activated cell sorting, in- agarose gels with ethidium bromide staining. dicating greater disrupted membrane symmetry exposing phos- Northern Blot Analysis—Fifteen mg of total RNA isolated using TRIzol phatidylserine to the external environment (Fig. 1A, right). reagent (Life Technologies, Inc.) was loaded onto a 1% formaldehyde/ Similar findings were obtained qualitatively by DNA laddering agarose gel and resolved by electrophoresis. Northern blot was performed on ethidium bromide-stained agarose gels (data not shown). To as previously described (32). FasL and glyceraldehyde-3-phosphate dehy- drogenase cDNA amplified by PCR were used as probes. The cDNA was determine whether Sp1 could directly modulate apoptosis, Sp1 labeled by nick translation (Roche Molecular Biochemicals). cDNA was transiently overexpressed in WKY3M-22 cells using RESULTS AND DISCUSSION a cytomegaloviral promoter-driven expression vector (CMV- Sp1). Sp1 increased apoptosis in this cell type (Fig. 1B). Con- Sp1 Is Proapoptotic—To begin investigating a possible mech- versely, apoptosis was inhibited by 50% in WKY12-22 cells anistic role for Sp1 in programmed cell death, we compared following overexpression of a dominant-negative form of Sp1 apoptosis in two well established SMC subtypes isolated orig- (DNA binding domain) using pEBGNLS-Sp1 (33) (Fig. 1C), inally from the arteries of pup (2-week-old) and adult (3-month- old) rats (24, 25). Pup SMCs (WKY12-22 cells) are phenotypi- demonstrating profound inhibition effected by a single tran- cally distinct from their adult counterparts (WKY3M-22 cells) scription factor. These data provide the first demonstration of and express abundant levels of Sp1 (26). We found that cyto- the capacity of Sp1 to modulate apoptosis in any cell type. plasmic histone-associated internucleosomal DNA fragmenta- Inspection of the FasL promoter sequence revealed the ex- Sp1 Phosphorylation Regulates FasL-dependent Apoptosis 4969 istence of a putative recognition element for Sp1 (59-GGGCGG- 39) located at nucleotides 2281/2276. Because Sp1 is preferen- tially expressed in WKY12-22 cells (26), we hypothesized that FasL, if it is Sp1-dependent, would be more abundantly ex- pressed in WKY12222 cells than WKY3M-22 cells. We as- sessed FasL mRNA expression in WKY12222 and WKY3M-22 cells by semiquantitative RT-PCR. FasL was readily expressed in WKY12-22 cells but was weakly, if at all, expressed in WKY3M-22 cells (Fig. 1D). Northern blot analysis confirmed preferential FasL mRNA expression in WKY12-22 cells (Fig. 1E). These findings suggested that Sp1 may regulate cell death through its activation of the FasL promoter. Sp1 Binds and Activates the FasL Promoter—To determine whether FasL is under the transcriptional control of Sp1, tran- sient transfection analysis was performed with the construct FasLzhsLuc, a firefly luciferase-based reporter vector driven by 1.2 kilobases of the FasL promoter (16). Cotransfection of FasLzhsLuc with an Sp1 expression vector induced FasL pro- moter-dependent expression (Fig. 2A). To localize the Sp1 re- sponse element in the FasL promoter, we generated a series of reporter constructs derived from parent FasLzhsLuc bearing 59 deletions in the FasL promoter. Luciferase activity increased upon cotransfection of 2296FasLzhsLuc and CMV-Sp1 (Fig. 2B). In contrast, 2271FasLzhsLuc failed to respond to Sp1 overexpression (Fig. 2B). The 59-FasL promoter end points in these constructs (2296 and 2271) occur on either side of the putative Sp1 binding site (2281/2276). EMSA using a P- labeled double-stranded oligonucleotide spanning this region of the promoter ([ P]FasL oligonucleotide, 2296/2265) and nu- clear extracts prepared from WKY12-22 cells revealed the for- mation of a number of nucleoprotein complexes (Fig. 2C). The most intense band was supershifted with polyclonal antibodies directed to Sp1 (Fig. 2C). Identical amounts of Smad1 poly- FIG.4. Phosphorylation of endogenous Sp1 following expo- clonal antibodies used as a negative control had no influence on sure to CAM. A, EMSA using [ P]mFasL Oligo and nuclear extracts of nucleoprotein complex formation (Fig. 2C). To demonstrate WKY12-22 cells incubated with CAM (1 mg/ml). Where appropriate, the sequence specificity of complex formation, we prepared an oli- extracts were treated with CIP prior to EMSA. The amount of CIP (5 gonucleotide ([ P]mFasL oligonucleotide, 2296/2265) bearing milliunit) used in this assay is based on CIP titration experiments that previously defined the concentration of CIP unable to dephosphorylate a mutation that disrupts the Sp1 binding site (to 59-TTTCTT- the P-labeled probe. Sp1 nucleoprotein complex intensity (with or 39). This mutation no longer supported the interaction of Sp1 without CAM exposure for CIP treatment of extracts) was semi-quan- with this region of the FasL promoter (Fig. 2C). When intro- titated by densitometry. B, Western blot analysis using nuclear extracts duced into full-length FasLzhsLuc, producing construct of WKY12-22 cells exposed to CAM (1 mg/ml), with and without CIP treatment (5 units). Sp1-P indicates hyperphosphorylated Sp1. The mSp1FasLzhsLuc, luciferase expression inducible by Sp1 was Coomassie Blue-stained gel is shown. The data are representative of completely abolished (Fig. 2D). These findings demonstrate two independent determinations. that Sp1 positively regulates FasL transcription. Activation by M-22 exogenous Sp1 of the FasL promoter was greater in WKY3 FasLzhsLuc blocked CAM-inducible FasL transcription (Fig. cells than WKY12-22 cells (Fig. 2, E versus A), consistent with 3C), whereas the empty expression vector had no effect (Fig. higher endogenous Sp1 expression in the latter cell type (26). 3C). These data demonstrate that Sp1 is required for FasL Inducible Apoptosis Involves the Phosphorylation of Sp1 and promoter activation by extracellular stimuli. Induction of FasL—We next explored the effect of extracellular EMSA using [ P]FasL oligonucleotide and nuclear extracts apoptotic stimuli on the capacity of Sp1 to stimulate apoptosis of cells exposed to CAM revealed that this agent did alter Sp1 and transactivate the FasL promoter. CAM, an inhibitor of occupancy of the promoter (Fig. 4A, lane 4 versus lane 2). DNA topoisomerase I, has been reported to induce apoptosis in Incubation of these extracts with CIP (22), which hydrolyzes several cell types (34) although its effect on SMCs is not known. 59-phosphate groups, prior to EMSA decreased the intensity of Nuclear condensation of SMCs stained by propidium iodide both Sp1 binding complexes (Fig. 4A, lane 5 versus lane 3) but increased dramatically following 24-h exposure to CAM (Fig. was most profound in extracts of cells exposed to CAM. Densi- 3A). This agent also induced internucleosomal fragmentation of tometric assessment of the intensities of these complexes (Fig. DNA and annexin V staining (data not shown). To determine 4A, lower left and lower center) revealed that 12% of promoter- whether FasL expression is altered by CAM and define the bound Sp1 is basally phosphorylated and that Sp1 phosphoryl- involvement of Sp1 in this process, we performed RT-PCR and ation increases to 31% upon exposure to CAM (Fig. 4A, lower transient transfection analysis with FasL promoter constructs. right). This indirect determination of Sp1 phosphorylation was CAM stimulated FasL promoter activity (Fig. 3B) and endoge- supported by Western immunoblot analysis with antibodies to nous FasL gene expression (Fig. 1D) by 2– 4-fold (Fig. 3, B and Sp1. We observed the appearance of a hyperphosphorylated C). CAM failed to activate the construct 2271FasLzhsLuc (Fig. species following exposure to CAM (Fig. 4B). This effect was 3B), which lacks the Sp1 site and was unable to mediate Sp1- abolished by prior incubation of the extracts with CIP (Fig. 4B). inducible FasL promoter-dependent expression (Fig. 2B). Over- These findings thus show that Sp1 is phosphorylated during expression of dominant-negative Sp1 (20) together with CAM-inducible apoptosis. Sp1 phosphorylation regulates the 4970 Sp1 Phosphorylation Regulates FasL-dependent Apoptosis FIG.6. Autocrine/paracrine FasL-Fas engagement mediates CAM-inducible apoptosis in SMCs. A, determination of spontaneous Fas expression in WKY12-22 and WKY3M-22 cells by RT-PCR. B, CAM-inducible apoptosis is blocked by Fas-Fc. WKY12-22 cells were exposed to 50 mg/ml FaszFc or Fc for 1 h and then incubated with CAM (0.1 mg/ml) for 24 h with assessment of apoptosis by ELISA. Results (ordinate axis) are expressed as total internucleosomal DNA fragmen- tation as a proportion of the total number of cells in the treatment population. Error bars represent S.E. The data are representative of two independent determinations. expression vector (Fig. 5A), whereas overexpression of mutant PKC-z attenuated CAM-inducible FasL promoter-dependent reporter expression (Fig. 5A). Dominant-negative PKC-z also FIG.5. CAM induction of the FasL promoter and apoptosis are blocked internucleosomal fragmentation stimulated by CAM PKC-z-dependent processes. A, WKY12-22 cells were transfected (Fig. 5B). Overexpression of dominant negative PKC-z in cells with FasLzhsLuc and 3 mg of either CMV-FLAG or CMV-FLAGzDN- not exposed to CAM did not significantly modulate the level of PKC-z prior to determination of luciferase activity after 24 h. Firefly luciferase activity was normalized to Renilla activity, and the results apoptosis compared with cells transfected with the backbone were plotted as -fold increase relative to the CMV-FLAG or CMV- control (data not shown). This suggests that the capacity of FLAGzDN-PKC-z groups, respectively. B, WKY12-22 cells were trans- dominant-negative PKC-z to suppress apoptosis detectable in fected with 3 mg of either CMV-FLAG or CMV-FLAGzDN-PKC-z.15 mg our system is conditional upon the cells being induced to un- of FasL promoter-reporter construct was used throughout. After 24 h, dergo further cell death by exposure to apoptotic stimuli. This the cells were incubated with CAM (1 mg/ml) for a further 24 h, and apoptosis was assessed by ELISA. Results (ordinate axis) are expressed is likely a direct consequence of the low level of spontaneous as total internucleosomal DNA fragmentation as a proportion of the phosphorylation of Sp1 (Fig. 4A) making attenuation by dom- total number of cells in the treatment population. Error bars represent inant-negative PKC-z difficult to measure in a cotransfection S.E. The data are representative of two independent determinations. setting. These findings, nonetheless, indicate that PKC-z reg- ulates inducible FasL transcription and apoptosis. inducible expression of a number of other genes, including Fas receptor, unlike FasL (Fig. 1C), is expressed in both vascular permeability factor/vascular endothelial growth factor WKY12-22 and WKY3M-22 cells (Fig. 6A). Because CAM in- (31), a -integrin (35), and tissue factor (23). duces FasL expression (Figs. 3, B–D, and 5A), we hypothesized CAM-inducible FasL Promoter Activity and Apoptosis Are that the induction of apoptosis by this agent involves the se- Protein Kinase-z-dependent Processes—A kinase found to me- cretion and autocrine/paracrine engagement of FasL with Fas diate Sp1 phosphorylation is protein kinase-z (PKC-z) (31), a at the cell surface. To address this possibility, prior to the diacylglycerol- and Ca -independent atypical member of the addition of CAM, we incubated the cells with FaszFc chimera, PKC family (36). PKC-z is ubiquitously expressed and interacts in which the extracellular domain of Fas is fused to the Fc directly with Sp1 (31). To investigate the role of PKC-z in the portion of human IgG. FaszFc blocked SMC apoptosis induced regulation of FasL expression, we cotransfected WKY12-22 by CAM (Fig. 6B). In contrast, an identical amount of the Fc cells with an expression vector (CMV-FLAGzDN-PKC-z) gener- fragment alone had no effect (Fig. 6B). These findings thus ating a kinase-inactive dominant-negative mutant of PKC-z demonstrate that autocrine/paracrine extracellular Fas/FasL 275 275 bearing a Lys 3 Trp substitution (37– 40). The FasL pro- engagement is involved in SMC apoptosis. Sp1 is phosphoryl- moter was activated by CAM in cells harboring the empty ated and activates FasL in SMCs upon exposure to extracellu- Sp1 Phosphorylation Regulates FasL-dependent Apoptosis 4971 (1997) Arterioscler. Thromb. Vasc. Biol. 17, 2200 –2208 lar apoptotic stimuli. 7. Cai, W., Devaux, B., Schaper, W., and Schaper, J. (1997) Atherosclerosis 131, In this paper, we have defined a novel role for the ubiqui- 177–186 8. Kockx, M. M., and Herman, A. G. (1998) Eur. Heart J. 19, G23–G28 tously expressed transcription factor Sp1 in apoptotic signal 9. Kockx, M. M., de Meyer, G. R. Y., Muhring, J., Jacob, W., Bult, H., and transduction. Subtypes of SMC expressing abundant levels of Herman, A. G. (1998) Circulation 97, 2307–2315 Sp1 produce FasL and undergo greater spontaneous apoptosis. 10. Kockx, M. M. (1998) Arterioscler. Thromb. Vasc. Biol. 18, 1519 –1522 11. Flynn, P. D., Byrne, C. D., Baglin, T. P., Weissberg, P. L., and Bennett, M. R. EMSA and transient transfection analysis revealed that the (1997) Blood 89, 4378 – 4384 FasL promoter is activated by Sp1 via a distinct element whose 12. Kockx, M. M., and Herman, A. G. (2000) Cardiovasc. Res. 45, 736 –746 integrity is crucial for inducible expression. Inducible FasL 13. Sata, M., Perlman, H., Muruve, D. A., Silver, M., Ikebe, M., Libermann, T. A., Oettgen, P., and Walsh, K. (1998) Proc. Natl. Acad. Sci. U S A 95, transcription is inhibited by a mutant form of Sp1, which also 1213–1217 blocks apoptosis. Inducible SMC apoptosis is preceded by Sp1 14. Luo, Z., Sata, M., Nguyen, T., Kaplan, J. M., Akita, G. Y., and Walsh, K. (1999) Circulation 99, 1776 –1779 phosphorylation, increased FasL transcription, and the auto- 15. Schnieder, D. B., Vassalli, G., Wen, S., Driscoll, R. M., Sassani, A. B., DeYong, crine/paracrine engagement of FasL with Fas. Both inducible M. B., Linnemann, R., Zvirmani, R., and Dichek, D. A. (2000) Arterioscler. FasL transcription and apoptosis are blocked by dominant- Thromb. Vasc. Biol. 20, 298 –308 16. Kasibhatla, S., Brunner, T., Genestier, L., Echeverri, F., Mahboubi, A., and negative protein kinase C-z. These data demonstrate that ap- Green, D. R. (1998) Mol. Cell 1, 543–551 optotic signaling in SMCs involves Sp1 phosphorylation. 17. Xiao, S., Matsui, K., Fine, A., Zhu, B., Marshak-Rothstein, A., Widom, R. L., The present study is the first report of transcription factor and Ju, S.-T. (1999) Eur. J. Immunol. 29, 3456 –3465 18. Faris, M., Latinis, K. M., Kempiak, S. J., Koretzky, G. A., and Nel, A. (1998) phosphorylation as a prerequisite biochemical process in induc- Mol. Cell. Biol. 18, 5414 –5424 ible apoptotic cell death. We used CAM as a model effector of 19. Holtz-Heppelmann, C. J., Algeciras, A., Badley, A. D., and Paya, C. V. (1998) J. Biol. Chem. 273, 4416 – 4423 cell death; however, given the general cellular expression of 20. Faris, M., Kokot, N., Latinis, K., Kasibhatla, S., Green, D. R., Koretzky, G. A., Sp1, our observations are unlikely to be confined to this agent and Nel, A. (1998) J. Immunol. 160, 134 –144 alone nor are they likely to be cell type-specific. Okadaic acid, 21. Jackson, S. P., MacDonald, J. J., Lees-Miller, S., and Tjian, R. (1990) Cell 5, 155–165 a selective inhibitor of serine-threonine phosphatase PP2A, 22. Kadonaga, J. T., Carner, K. R., Masiarz, F. R., and Tjian, R. (1987) Cell 51, stimulates apoptosis in a wide variety of cell types including 1079 –1090 murine fibroblasts (41), rat kidney epithelial cells (42) amongst 23. Lin, M.-C., Almus-Jacobs, F., Chen, H.-H., Parry, G. C. N., Mackmann, N., Shyy, J. Y., and Chen, S. (1997) J. Clin. Invest. 99, 737–744 them. Okadaic acid, like CAM, stimulates Sp1 phosphorylation 24. Majesky, M. W., Giachelli, C. M., Reidy, M. A., and Schwartz, S. M. (1992) Circ. and apoptosis in SMCs (data not shown). Tat, the transcriptional Res. 71, 759 –768 25. Lemire, J. M., Covin, C. W., White, S., Giachelli, C. M., and Schwartz, S. M. activator of human immunodeficiency virus type 1 (HIV-1), stim- (1994) Am. J. Pathol. 144, 1068 –1081 ulates Sp1 phosphorylation (43), activates FasL expression (44), 26. Rafty, L. A., and Khachigian, L. M. (1998) J. Biol. 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Sp1 Phosphorylation Regulates Apoptosis via Extracellular FasL-Fas Engagement

Journal of Biological ChemistryFeb 1, 2001

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 7, Issue of February 16, pp. 4964 –4971, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Sp1 Phosphorylation Regulates Apoptosis via Extracellular FasL-Fas Engagement* Received for publication, October 10, 2000 Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M009251200 Mary M. Kavurma‡, Fernando S. Santiago‡, Emanuela Bonfoco§, and Levon M. Khachigian‡¶i From the ‡Centre for Thrombosis and Vascular Research, The University of New South Wales, Sydney NSW 2052, Australia and the §Scripps Research Institute, La Jolla, California 92037 Apoptosis of smooth muscle cells (SMC) in atheroscle- 3/CPP32, which is expressed in cells as the inactive 32-kDa rotic vessels can destabilize the atheromatus plaque and form is, in turn, cleaved by caspase-8/FLICE to produce two result in rupture, thrombosis, and sudden death. In ef- mature subunits (17 and 12 kDa). Active caspase-3/CPP32 forts to understand the molecular processes regulating cleaves nuclear mitotic apparatus protein and mediates DNA apoptosis in this cell type, we have defined a novel fragmentation, chromatin condensation, and the formation of mechanism involving the ubiquitously expressed tran- apoptotic bodies (4). scription factor Sp1. Subtypes of SMC expressing abun- FasL (5) and Fas (6, 7) are both expressed in arterial tissue, dant levels of Sp1 produce the death agonist, Fas ligand including the human atherosclerotic plaque. Immunohisto- (FasL) and undergo greater spontaneous apoptosis. Sp1 chemical analysis revealed FasL expression in 34 of 34 carotid activates the FasL promoter via a distinct nucleotide atherosclerotic plaques examined, with virtually all FasL pos- recognition element whose integrity is crucial for induc- itive-staining associated with intimal smooth muscle cells ible expression. Inducible FasL promoter activation is (SMCs) and little staining apparent in normal arterial tissue also inhibited by a dominant-negative form of Sp1. In- (5). Fas is also highly expressed in intimal SMCs of the plaque creased SMC apoptosis is preceded by Sp1 phosphoryl- (6, 7). FasL/Fas expression and apoptosis (8 –10) in normal ation, increased FasL transcription, and the autocrine/ artery and plaque has prompted speculation on the roles of these paracrine engagement of FasL with its cell-surface molecular mediators in vascular cells. Apoptosis in undiseased receptor, Fas. Inducible FasL transcription and apopto- z, tissue may inhibit arterial thickening by limiting cell prolifera- sis are blocked by dominant-negative protein kinase C- whose wild-type counterpart phosphorylates Sp1. Thus, tion and accumulation in the intima (6). In atherosclerotic tissue, Sp1 phosphorylation is a proapoptotic transcriptional apoptosis particularly of collagen-producing SMCs may substan- event in vascular SMC and, given the wide distribution tially weaken the plaque causing it to rupture, initiate thrombo- of this housekeeping transcription factor, may be a com- sis, and trigger acute coronary syndromes (11–12). Overexpres- mon regulatory theme in apoptotic signal transduction. sion of FasL in balloon-injured rat carotid arteries devoid of endothelium-induced apoptosis in medial SMCs and inhibited intimal hyperplasia (13, 14). However, recent evidence in a rabbit Apoptosis is a genetically regulated “programmed” form of model suggests that FasL may promote rather than retard cell death and is characterized by a number of specific biochem- atherogenesis. FasL overexpression in nondenuded arteries of ical and morphological changes, including nuclear chromatin hypercholesterolemic animals stimulated lesion formation in condensation, cytoplasmic condensation, membrane blebbing, these animals via increased cellularity (15). These observations and internucleosomal fragmentation of DNA (1, 2). Fas/APO-1 may be due to differences in artery and lesion cellular composi- (or CD95) is a 45-kDa cell surface glycoprotein that belongs to tion or cholesterol feeding between the two animal models. the tumor necrosis factor receptor superfamily and mediates Despite clear evidence for FasL and Fas expression in SMCs apoptosis in various normal and transformed cell types. Upon of the artery wall, the molecular mechanisms mediating FasL the engagement of Fas by Fas ligand (FasL), a highly con- production in vascular cells are presently not known. The pro- served, ubiquitously expressed 40-kDa glycoprotein, the apo- moter region of the FasL gene has recently been cloned and ptotic cysteine protease caspase-8/FLICE is recruited to the found to contain binding sites for a number of transcription receptor via FADD and activated by proteolysis (3). Caspase- factors including NF-kB (16), AP-1 (16), NFAT (17), ATF2 (18), Egr-2 (17), and Egr-3 (17). The promoter contains a single transcription initiator site, as well as positive and negative * This work was supported in part by grants from the Australian regulatory regions within a 2.3-kilobase portion of the 59-un- Research Council (to L. M. K.), National Health and Medical Research Council of Australia (NHMRC) (to L. M. K.), and an NSW Department translated genome (19). Analysis of the FasL promoter has of Health Infrastructure grant to the Centre for Thrombosis and Vas- mostly been confined to T cells. For example, T cell activation cular Research. The costs of publication of this article were defrayed in following CD4 cross-linking induces NFAT binding to the FasL part by the payment of page charges. This article must therefore be enhancer and gene transactivation (19). Similarly, cytotoxic hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. stress-induced FasL expression involves the activation of NF- Research Fellow of the NHMRC. kB, AP-1, and c-Jun N-terminal kinase, prior to cell death i To whom correspondence should be addressed. Tel.: 61-2-9385 2537; (16 –20). Activity of the FasL promoter is also regulated by Fax: 61-2-9385 1389; E-mail: l.Khachigian@unsw.edu.au. MEK kinase-1 (18). However, transcription factor phosphoryl- The abbreviations used are: FasL, Fas ligand; CIP, calf intestinal alkaline phosphatase; DN-PKC-z, dominant-negative protein kinase ation has not yet been directly demonstrated as a prerequisite C-z; SMC, smooth muscle cells; CAM, camptothecin; PCR, polymerase step in apoptosis. chain reaction; RT-PCR, reverse transcription-PCR; PBS, phosphate- The discovery and functional characterization of Sp1 as a buffered saline; ELISA, enzyme-linked immunosorbent assay; FACS, GC-rich binding nuclear protein has provided a useful para- fluorescence-activated cell sorter; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus. digm to our understanding of the regulation of transcriptional 4964 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Sp1 Phosphorylation Regulates FasL-dependent Apoptosis 4965 was used in the ELISA, which was performed in accordance with the activation in eukaryotic cells (21, 22). Sp1 is a broadly ex- manufacturer’s instructions and normalized to total cell number meas- pressed nuclear protein of ;100 kDa and contains three Krup- ured using a Coulter counter. Results are expressed as total internu- pel-like zinc fingers that contact DNA (21, 22). A nucleotide cleosomal DNA fragmentation as a proportion of the cell population. recognition element for Sp1 is located in the FasL promoter at Annexin V Staining/FACS Analysis—SMCs were washed twice with position 2281/2276 base pairs (GGGCGG) relative to the tran- ice-cold phosphate-buffered saline, pH 7.4 and resuspended in 13 bind- scriptional start site. Sp1 can influence gene expression by ing buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl )ata concentration of 1 3 10 cells/ml. One hundred microliters of the sus- changes in its nuclear concentration and interaction with the pension was transferred to 5-ml flat bottomed tubes where 5 mlof promoter, by providing architectural support, serving as a co- annexin V-fluorescein isothiocyanate and 10 ml of propidium iodide (50 factor, or by undergoing chemical modification. Sp1 phospho- mg/ml stock in PBS) was added. The cells were gently vortexed and rylation e.g. mediates inducible tissue factor expression in vas- incubated in the dark at 22 °C for 15 min. Four hundred microliters of cular endothelial cells exposed to fluid shear stress (23). The binding buffer was added to each tube, and annexin V staining was significance of Sp1 in the process of apoptosis in any cell type is analyzed by flow cytometry within 1 h. Results are expressed as an- nexin V staining as a percentage of the total cell population. presently unknown. This knowledge would advance our under- Propidium Iodide Nuclear Staining—SMCs were grown in chamber standing of the transcriptional basis of extrinsic apoptosis, slides (80% confluent) and incubated with CAM (1 mg/ml) for 24 h. The given the wide distribution of both Sp1 and FasL. cells were washed in PBS, pH 7.4, and fixed with methanol/acetone WKY12-22 and WKY3M-22 cells are well established sub- (80:20) for 10 min at 22 °C. Propidium iodide (50 mM) was added to each types of vascular smooth muscle cells that are phenotypically well and incubated for a maximum of 5 min followed by a second wash distinct (24, 25). WKY12-22 cells have a cobblestone morphol- with PBS. Cells undergoing apoptosis were visualized by confocal microscopy. ogy in culture, proliferate in plasma-derived serum (which lacks Nuclear Extract Preparation—SMCs treated with CAM for various vital growth factors), and spontaneously overexpress mRNA for times were washed and scraped in 10 ml of PBS and transferred to platelet-derived growth factor (PDGF) B-chain, elastin, and os- precooled centrifuge tubes. Samples were spun at 1300 rpm for 15 min teopontin (24, 25). In contrast, WKY3M-22 cells are typically at 4 °C. The pellet was resuspended in 100 ml (for two 100-mm dishes) spindle-shaped and do not express PDGF-B, elastin, or osteopon- of solution A (10 mM Hepes, pH 7.9, 1.5 mM MgCl ,10mM KCl) and tin mRNA, nor do they grow in plasma-derived serum. Both cell placed on ice for 5 min. Samples were spun at 14,000 rpm for 40 s. The pellet was resuspended in 20 ml of solution C (20 mM Hepes, pH 7.9, 1.5 subtypes are phenotypically stable in culture and can be pas- mM MgCl , 420 mM NaCl, 0.2 mM EDTA) and mixed gently for 20 min saged indefinitely. Therefore, WKY12-22 and WKY3M22 cells at 4 °C. The supernatant was transferred to precooled Eppendorf tubes represent important cells with which to delineate the molecular containing 20 ml of solution D (20 mM Hepes, pH 7.9, 1.5 mM KCl, 0.2 basis for differences in SMC phenotype and gene expression. mM EDTA, 20% glycerol) and stored at 280 °C until use. All buffers We recently reported that Sp1 is spontaneously expressed at contained protease inhibitors. greater levels in WKY12-22 cells than WKY3M-22 cells and that Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts (6 –10 mg) were incubated with P-labeled double-stranded oligonucleo- as a consequence, Sp1-dependent genes, such as PDGF-B, are tide (150,000 cpm, 40 fmol) in 20 ml containing 10 mM Tris-HCl, pH 8.0, overexpressed in WKY12-22 cells compared with its sister cell 50 mM MgCl ,1mM EDTA, 1 mM dithiothreitol, 5% glycerol, 1 mgof subtype (26). These observations provided important insight into salmon sperm DNA, 5% sucrose, 1 mg of poly(dI-dC) and 1 mM phenyl- the transcriptional basis for differential gene expression. Here methylsulfonyl fluoride. The mixture was incubated for 35 min at 22 °C. we explored the regulatory role of Sp1 in inducible FasL expres- In supershift experiments, nuclear extract was incubated with 2 mgof sion and apoptosis in two phenotypically distinct SMC subtypes. antibody prior to addition of the probe. Samples were resolved by 8% nondenaturing polyacrylamide gel electrophoresis, and binding com- EXPERIMENTAL PROCEDURES plexes were visualized by autoradiography at 280 °C. Transfections and Luciferase Assays—-SMCs were maintained in Western Blot for Sp1—Fifteen micrograms of nuclear extract was Waymouth’s medium (Life Technologies, Inc.), pH 7.4, containing 10% resolved by 8% SDS-polyacrylamide gel electrophoresis and then trans- fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO . ferred onto Immobilon-P transfer membranes (Millipore). The mem- Transient transfections were performed with cells at 60% confluence, branes were blocked overnight at 4 °C in PBS containing 5% skim milk and the indicated constructs together with 2 mg of the internal control and 0.05% Tween 20. Sp1 was detected with Sp1 polyclonal antibodies vector, pRL-TK, using FuGENE6 transfection agent (Roche Molecular (1:1000, Santa Cruz Biotechnology) and subsequent chemiluminescent Biochemicals). After 24 h, the transfected cells were incubated with or visualization. without CAM (1 mg/ml), and luciferase activity was quantified using the Sp1 Dephosphorylation Analysis by Western Blotting and EMSA— Dual Luciferase Assay System (Promega). Firefly luciferase activity Nuclear extracts (10 –15 mg) were incubated with or without 5 units of was normalized to Renilla data generated from pRL-TK. calf intestinal alkaline phosphatase (CIP, NEB) for1hat37 °Cina Plasmid Constructs—Various sized fragments of the FasL promoter total volume of 20 ml. The reaction was quenched by the addition of (2271FasLzhsLuc and 2296FasLzhsLuc) were amplified from the par- loading dye prior to 8% SDS-polyacrylamide gel electrophoresis and ent vector FasLzhsLuc (gift of Dr Shailaja Kasibhatla, La Jolla Inst. of Western blot analysis for Sp1. In EMSA, 8 mg of nuclear extract was Cellular Immunology) by PCR and blunt-end cloned into pGL3. The incubated with 5 milliunit of CIP (final concentration determined by mutant counterpart of FasLzhsLuc bearing a mutation in the Sp1 bind- CIP titration experiments with P-labeled FasL Oligo) at 33 °C for 5 ing site (mSp1FasLzhsLuc) was constructed using the QuikChange min and then on ice for 15 min. The reaction was stopped by the site-directed mutagenesis kit (Stratagene). pEBGNLS-Sp1 was ob- addition of phosphatase inhibitors at a final concentration of 10 nM tained from Gerald Thiel (Inst. for Genetics, University of Cologne), LiNaF, 10 nM sodium vanadate, 10 nM potassium pyrophosphate, and 5 CMV-Sp1 was obtained from Robert Tjian (Howard Hughes Medical nM sodium phosphate. EMSA was performed as described above. CIP Inst., University of California), and CMV-FLAGzDN-PKC-z was ob- treatment prior to EMSA has previously been used for the assessment tained from Debabrata Mukhopadhyay (Beth Israel-Deaconess Hospi- of Sp1 phosphorylation in nuclear extracts (30, 31). tal, Boston) and Alex Toker (Boston Biomedical Research Inst.). RT-PCR—Five micrograms of total RNA isolated using TRIzol reagent Quantitative Assessment of DNA Fragmentation—SMCs were grown (Life Technologies, Inc.) were treated with DNase I, and cDNA was in 96-well plates to 80% confluency in 100 ml of growth medium. Where generated using Superscript II reverse transcriptase (Life Technologies, indicated, the cells were incubated with Fas-Fc (R&D Systems) and/or Inc.) with random primers according to the manufacturer’s instructions. IgG Fc (R&D Systems) (50 mg/ml, final concentration) for 1 h prior to Sequences of the primers for FasL, Fas, and b-actin are as follows: 59-Fa- the addition of CAM. After 24-h exposure to CAM, apoptosis was quan- sL, 59-AAACCCTTTCCTGGGGC-39;39-FasL, 59-GTGTCTTCCCATTCC- Plus titated using the Cell Death Detection ELISA (Roche Molecular AG-39;59-Fas, 59-CTGTGGATCATGGCTGTCCTGCCT-39;39-Fas, 59-CT- Biochemicals). This assay, which measures cytoplasmic histone-associ- CCAGACTTTGTCCTTCATTTTTC-39;59-b-actin, 59-TGACGGGGTCAC- ated internucleosomal DNA fragmentation, has been used previously to CCACACTGTGCCCATCTA-39;39-b-actin, 59-CTAGAAGCATTTGCGGT- quantitate inducible apoptosis in cultured cells (27–29). Briefly, the GGACGATGGAGGG-39;59-glyceraldehyde-3-phosphate dehydrogenase, cells were washed gently in PBS and incubated with shaking in lysis 59-ACCACAGTCCATGCCATCAC-39;39-glyceraldehyde-3-phosphate buffer for 30 min at 22 °C. Lysates were transferred into Eppendorf dehydrogenase, 59-TCCACCACCCTGTTGCTGTA-39. tubes and spun at 14,000 rpm for 30 s. Twenty ml of the supernatant PCR was performed in a total volume of 50 ml containing 2 mM 4966 Sp1 Phosphorylation Regulates FasL-dependent Apoptosis FIG.1. Differences in spontaneous apoptosis and FasL expression in WKY12-22 and WKY3M-22 cells. A, spontaneous levels of apoptosis as determined by internucleosomal DNA fragmentation (left) and annexin V/FACS analysis (right) in WKY12-22 and WKY3M-22 cells. The y axis in the left and right panels represents total internucleosomal DNA fragmentation (as a proportion of the cell population) and annexin V staining (as a percentage of the total number of cells in the population), respectively. B, overexpression of Sp1 in WKY3M22 cells stimulates apoptosis. SMCs were transfected with 0, 1, 3, and 5 mg of CMV-Sp1 using FuGENE6 prior to quantification of apoptosis after 24 h by ELISA. Where required, the total amount of DNA transfected was supplemented to 5 mg with pcDNA3. C, dominant-negative Sp1 blocks apoptosis in WKY12-22 cells. SMCs were transfected with 40 mg of pEBGNLS or pEBGNLS-Sp1 using FuGENE6 prior to quantification of apoptosis after 24 h by ELISA. Results in B and C (ordinate axes) are expressed as total internucleosomal DNA fragmentation as a proportion of the total number of cells in the treatment population. D, WKY12-22 cells, but not WKY3M-22 cells, express FasL mRNA, and this is stimulated by CAM. Total RNA prepared from WKY12-22 cells incubated with CAM (1 mg/ml) for 24 h prior to reverse-transcription and PCR with the indicated primers. PCR cycle number is indicated in the figure. The densitometric assessment of the amplicons is represented histodiagramatically. E, comparative FasL mRNA expression in WKY12-22 cells and WKY3M-22 cells. Northern blot analysis was performed with total RNA of WKY3M-22 cells or WKY12-22 cells (with or without 24-h incubation with 1 mg/ml CAM. The data are representative of two independent determinations. Error bars represent S.E. Sp1 Phosphorylation Regulates FasL-dependent Apoptosis 4967 FIG.2. Sp1 activates the FasL promoter in SMCs. Transient cotransfection analysis in WKY12-22 cells overexpressing Sp1 with FasL-hsLuc (A) and derivatives of FasLzhsLuc bearing 59 deletions in the FasL promoter (B). 6 mg of pcDNA3 or CMV-Sp1 was used in B;15 mg of FasL promoter-reporter construct was used throughout. Firefly luciferase activity was normalized to Renilla activity and the results were plotted as -fold 32 32 increase relative to 2296FasLzhsLuc or 2271FasLzhsLuc, respectively. C, EMSA using [ P]FasL Oligo, [ P]mFasL Oligo, and nuclear extracts of WKY12-22 cells. EMSA was performed as described under “Experimental Procedures,” and nucleoprotein complexes were visualized by autora- diography. Arrows indicate nucleoprotein complexes, S denotes a supershift. Sequence of [ P]mFasL Oligo (2296/2265) is 59-ATCAGAAAATT- GTGGGCGGAAACTTCCAGG-39, and [ P]mFasL Oligo is 59-ATCAGAAAATTGTTTTCTTAAACTTCCAGG-39; the mutation is underlined). D, mutation of the Sp1 site in FasLzhsLuc abrogates activation of the FasL promoter. E, Sp1 activation of FasL promoter in WKY3M-22 cells. 3 or 6 mg of pcDNA3 or CMV-Sp1 were used in cotransfection experiments with 15 mg of FasL promoter-reporter construct throughout. Firefly luciferase activity was normalized to Renilla activity, and the results were plotted as -fold increase relative to the pcDNA3 control in the FasLzhsLuc and mSp1FasLzhsLuc contransfectant groups, respectively. Error bars represent S.E. The data are representative of two independent determinations. 4968 Sp1 Phosphorylation Regulates FasL-dependent Apoptosis FIG.3. CAM stimulates apoptosis in vascular SMCs and induces FasL ex- pression in an Sp1-dependent man- ner. A, CAM increases propidium iodide nuclear staining in WKY12-22 cells. The SMCs were exposed to CAM (1 mg/ml) for 24 h prior to fixation, incubation with pro- pidium iodide, and confocal microscopy. Effect of CAM (1 mg/ml) on luciferase ex- pression driven by FasLzhsLuc or a con- struct bearing a deletion of the Sp1 bind- ing element (2271FasLzhsLuc, B), or FasLzhsLuc cotransfected with 5 mgof dominant-negative Sp1 (pEBGNLS-Sp1) or the backbone alone (pEBGNLS, C). 15 mg of FasL promoter-reporter construct was used throughout. Firefly luciferase activity was normalized to Renilla activ- ity, and the results were plotted as -fold increase relative to FasLzhsLuc or 2271FasLzhsLuc, respectively, in B,or the pEBGNLS or pEBGNLS-Sp1 groups in C. Error bars represent S.E. The data are representative of two independent determinations. MgCl ,2mM dNTPs, 2.5 units of Taq DNA polymerase (Sigma), 5 mlof 2 tion (27–29) is greater in WKY12-22 cells than WKY3M-22 cDNA, and either 100 pmol of Fas primers, 100 pmol of FasL primers, cells (Fig. 1A, left), indicating higher levels of spontaneous or 20 pmol of b-actin primers using a Perkin Elmer thermocycler. apoptosis in the former cell subtype and providing further Amplication was performed by denaturing the sample at 94 °C for 2 evidence that these SMC subtypes are phenotypically distinct. min, then cycled at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and an extension at 72 °C for 4 min. The number of PCR cycles for Fas/b These observations were confirmed by annexin V-fluorescein iso- actin was 25. Twenty microliters of the reaction was visualized on 1.5% thiocyanate staining upon fluorescence-activated cell sorting, in- agarose gels with ethidium bromide staining. dicating greater disrupted membrane symmetry exposing phos- Northern Blot Analysis—Fifteen mg of total RNA isolated using TRIzol phatidylserine to the external environment (Fig. 1A, right). reagent (Life Technologies, Inc.) was loaded onto a 1% formaldehyde/ Similar findings were obtained qualitatively by DNA laddering agarose gel and resolved by electrophoresis. Northern blot was performed on ethidium bromide-stained agarose gels (data not shown). To as previously described (32). FasL and glyceraldehyde-3-phosphate dehy- drogenase cDNA amplified by PCR were used as probes. The cDNA was determine whether Sp1 could directly modulate apoptosis, Sp1 labeled by nick translation (Roche Molecular Biochemicals). cDNA was transiently overexpressed in WKY3M-22 cells using RESULTS AND DISCUSSION a cytomegaloviral promoter-driven expression vector (CMV- Sp1). Sp1 increased apoptosis in this cell type (Fig. 1B). Con- Sp1 Is Proapoptotic—To begin investigating a possible mech- versely, apoptosis was inhibited by 50% in WKY12-22 cells anistic role for Sp1 in programmed cell death, we compared following overexpression of a dominant-negative form of Sp1 apoptosis in two well established SMC subtypes isolated orig- (DNA binding domain) using pEBGNLS-Sp1 (33) (Fig. 1C), inally from the arteries of pup (2-week-old) and adult (3-month- old) rats (24, 25). Pup SMCs (WKY12-22 cells) are phenotypi- demonstrating profound inhibition effected by a single tran- cally distinct from their adult counterparts (WKY3M-22 cells) scription factor. These data provide the first demonstration of and express abundant levels of Sp1 (26). We found that cyto- the capacity of Sp1 to modulate apoptosis in any cell type. plasmic histone-associated internucleosomal DNA fragmenta- Inspection of the FasL promoter sequence revealed the ex- Sp1 Phosphorylation Regulates FasL-dependent Apoptosis 4969 istence of a putative recognition element for Sp1 (59-GGGCGG- 39) located at nucleotides 2281/2276. Because Sp1 is preferen- tially expressed in WKY12-22 cells (26), we hypothesized that FasL, if it is Sp1-dependent, would be more abundantly ex- pressed in WKY12222 cells than WKY3M-22 cells. We as- sessed FasL mRNA expression in WKY12222 and WKY3M-22 cells by semiquantitative RT-PCR. FasL was readily expressed in WKY12-22 cells but was weakly, if at all, expressed in WKY3M-22 cells (Fig. 1D). Northern blot analysis confirmed preferential FasL mRNA expression in WKY12-22 cells (Fig. 1E). These findings suggested that Sp1 may regulate cell death through its activation of the FasL promoter. Sp1 Binds and Activates the FasL Promoter—To determine whether FasL is under the transcriptional control of Sp1, tran- sient transfection analysis was performed with the construct FasLzhsLuc, a firefly luciferase-based reporter vector driven by 1.2 kilobases of the FasL promoter (16). Cotransfection of FasLzhsLuc with an Sp1 expression vector induced FasL pro- moter-dependent expression (Fig. 2A). To localize the Sp1 re- sponse element in the FasL promoter, we generated a series of reporter constructs derived from parent FasLzhsLuc bearing 59 deletions in the FasL promoter. Luciferase activity increased upon cotransfection of 2296FasLzhsLuc and CMV-Sp1 (Fig. 2B). In contrast, 2271FasLzhsLuc failed to respond to Sp1 overexpression (Fig. 2B). The 59-FasL promoter end points in these constructs (2296 and 2271) occur on either side of the putative Sp1 binding site (2281/2276). EMSA using a P- labeled double-stranded oligonucleotide spanning this region of the promoter ([ P]FasL oligonucleotide, 2296/2265) and nu- clear extracts prepared from WKY12-22 cells revealed the for- mation of a number of nucleoprotein complexes (Fig. 2C). The most intense band was supershifted with polyclonal antibodies directed to Sp1 (Fig. 2C). Identical amounts of Smad1 poly- FIG.4. Phosphorylation of endogenous Sp1 following expo- clonal antibodies used as a negative control had no influence on sure to CAM. A, EMSA using [ P]mFasL Oligo and nuclear extracts of nucleoprotein complex formation (Fig. 2C). To demonstrate WKY12-22 cells incubated with CAM (1 mg/ml). Where appropriate, the sequence specificity of complex formation, we prepared an oli- extracts were treated with CIP prior to EMSA. The amount of CIP (5 gonucleotide ([ P]mFasL oligonucleotide, 2296/2265) bearing milliunit) used in this assay is based on CIP titration experiments that previously defined the concentration of CIP unable to dephosphorylate a mutation that disrupts the Sp1 binding site (to 59-TTTCTT- the P-labeled probe. Sp1 nucleoprotein complex intensity (with or 39). This mutation no longer supported the interaction of Sp1 without CAM exposure for CIP treatment of extracts) was semi-quan- with this region of the FasL promoter (Fig. 2C). When intro- titated by densitometry. B, Western blot analysis using nuclear extracts duced into full-length FasLzhsLuc, producing construct of WKY12-22 cells exposed to CAM (1 mg/ml), with and without CIP treatment (5 units). Sp1-P indicates hyperphosphorylated Sp1. The mSp1FasLzhsLuc, luciferase expression inducible by Sp1 was Coomassie Blue-stained gel is shown. The data are representative of completely abolished (Fig. 2D). These findings demonstrate two independent determinations. that Sp1 positively regulates FasL transcription. Activation by M-22 exogenous Sp1 of the FasL promoter was greater in WKY3 FasLzhsLuc blocked CAM-inducible FasL transcription (Fig. cells than WKY12-22 cells (Fig. 2, E versus A), consistent with 3C), whereas the empty expression vector had no effect (Fig. higher endogenous Sp1 expression in the latter cell type (26). 3C). These data demonstrate that Sp1 is required for FasL Inducible Apoptosis Involves the Phosphorylation of Sp1 and promoter activation by extracellular stimuli. Induction of FasL—We next explored the effect of extracellular EMSA using [ P]FasL oligonucleotide and nuclear extracts apoptotic stimuli on the capacity of Sp1 to stimulate apoptosis of cells exposed to CAM revealed that this agent did alter Sp1 and transactivate the FasL promoter. CAM, an inhibitor of occupancy of the promoter (Fig. 4A, lane 4 versus lane 2). DNA topoisomerase I, has been reported to induce apoptosis in Incubation of these extracts with CIP (22), which hydrolyzes several cell types (34) although its effect on SMCs is not known. 59-phosphate groups, prior to EMSA decreased the intensity of Nuclear condensation of SMCs stained by propidium iodide both Sp1 binding complexes (Fig. 4A, lane 5 versus lane 3) but increased dramatically following 24-h exposure to CAM (Fig. was most profound in extracts of cells exposed to CAM. Densi- 3A). This agent also induced internucleosomal fragmentation of tometric assessment of the intensities of these complexes (Fig. DNA and annexin V staining (data not shown). To determine 4A, lower left and lower center) revealed that 12% of promoter- whether FasL expression is altered by CAM and define the bound Sp1 is basally phosphorylated and that Sp1 phosphoryl- involvement of Sp1 in this process, we performed RT-PCR and ation increases to 31% upon exposure to CAM (Fig. 4A, lower transient transfection analysis with FasL promoter constructs. right). This indirect determination of Sp1 phosphorylation was CAM stimulated FasL promoter activity (Fig. 3B) and endoge- supported by Western immunoblot analysis with antibodies to nous FasL gene expression (Fig. 1D) by 2– 4-fold (Fig. 3, B and Sp1. We observed the appearance of a hyperphosphorylated C). CAM failed to activate the construct 2271FasLzhsLuc (Fig. species following exposure to CAM (Fig. 4B). This effect was 3B), which lacks the Sp1 site and was unable to mediate Sp1- abolished by prior incubation of the extracts with CIP (Fig. 4B). inducible FasL promoter-dependent expression (Fig. 2B). Over- These findings thus show that Sp1 is phosphorylated during expression of dominant-negative Sp1 (20) together with CAM-inducible apoptosis. Sp1 phosphorylation regulates the 4970 Sp1 Phosphorylation Regulates FasL-dependent Apoptosis FIG.6. Autocrine/paracrine FasL-Fas engagement mediates CAM-inducible apoptosis in SMCs. A, determination of spontaneous Fas expression in WKY12-22 and WKY3M-22 cells by RT-PCR. B, CAM-inducible apoptosis is blocked by Fas-Fc. WKY12-22 cells were exposed to 50 mg/ml FaszFc or Fc for 1 h and then incubated with CAM (0.1 mg/ml) for 24 h with assessment of apoptosis by ELISA. Results (ordinate axis) are expressed as total internucleosomal DNA fragmen- tation as a proportion of the total number of cells in the treatment population. Error bars represent S.E. The data are representative of two independent determinations. expression vector (Fig. 5A), whereas overexpression of mutant PKC-z attenuated CAM-inducible FasL promoter-dependent reporter expression (Fig. 5A). Dominant-negative PKC-z also FIG.5. CAM induction of the FasL promoter and apoptosis are blocked internucleosomal fragmentation stimulated by CAM PKC-z-dependent processes. A, WKY12-22 cells were transfected (Fig. 5B). Overexpression of dominant negative PKC-z in cells with FasLzhsLuc and 3 mg of either CMV-FLAG or CMV-FLAGzDN- not exposed to CAM did not significantly modulate the level of PKC-z prior to determination of luciferase activity after 24 h. Firefly luciferase activity was normalized to Renilla activity, and the results apoptosis compared with cells transfected with the backbone were plotted as -fold increase relative to the CMV-FLAG or CMV- control (data not shown). This suggests that the capacity of FLAGzDN-PKC-z groups, respectively. B, WKY12-22 cells were trans- dominant-negative PKC-z to suppress apoptosis detectable in fected with 3 mg of either CMV-FLAG or CMV-FLAGzDN-PKC-z.15 mg our system is conditional upon the cells being induced to un- of FasL promoter-reporter construct was used throughout. After 24 h, dergo further cell death by exposure to apoptotic stimuli. This the cells were incubated with CAM (1 mg/ml) for a further 24 h, and apoptosis was assessed by ELISA. Results (ordinate axis) are expressed is likely a direct consequence of the low level of spontaneous as total internucleosomal DNA fragmentation as a proportion of the phosphorylation of Sp1 (Fig. 4A) making attenuation by dom- total number of cells in the treatment population. Error bars represent inant-negative PKC-z difficult to measure in a cotransfection S.E. The data are representative of two independent determinations. setting. These findings, nonetheless, indicate that PKC-z reg- ulates inducible FasL transcription and apoptosis. inducible expression of a number of other genes, including Fas receptor, unlike FasL (Fig. 1C), is expressed in both vascular permeability factor/vascular endothelial growth factor WKY12-22 and WKY3M-22 cells (Fig. 6A). Because CAM in- (31), a -integrin (35), and tissue factor (23). duces FasL expression (Figs. 3, B–D, and 5A), we hypothesized CAM-inducible FasL Promoter Activity and Apoptosis Are that the induction of apoptosis by this agent involves the se- Protein Kinase-z-dependent Processes—A kinase found to me- cretion and autocrine/paracrine engagement of FasL with Fas diate Sp1 phosphorylation is protein kinase-z (PKC-z) (31), a at the cell surface. To address this possibility, prior to the diacylglycerol- and Ca -independent atypical member of the addition of CAM, we incubated the cells with FaszFc chimera, PKC family (36). PKC-z is ubiquitously expressed and interacts in which the extracellular domain of Fas is fused to the Fc directly with Sp1 (31). To investigate the role of PKC-z in the portion of human IgG. FaszFc blocked SMC apoptosis induced regulation of FasL expression, we cotransfected WKY12-22 by CAM (Fig. 6B). In contrast, an identical amount of the Fc cells with an expression vector (CMV-FLAGzDN-PKC-z) gener- fragment alone had no effect (Fig. 6B). These findings thus ating a kinase-inactive dominant-negative mutant of PKC-z demonstrate that autocrine/paracrine extracellular Fas/FasL 275 275 bearing a Lys 3 Trp substitution (37– 40). The FasL pro- engagement is involved in SMC apoptosis. Sp1 is phosphoryl- moter was activated by CAM in cells harboring the empty ated and activates FasL in SMCs upon exposure to extracellu- Sp1 Phosphorylation Regulates FasL-dependent Apoptosis 4971 (1997) Arterioscler. Thromb. Vasc. Biol. 17, 2200 –2208 lar apoptotic stimuli. 7. 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Published: Feb 1, 2001

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