Sp1 Phosphorylation Regulates Apoptosis via Extracellular FasL-Fas Engagement*

Apoptosis of smooth muscle cells (SMC) in atherosclerotic vessels can destabilize the atheromatus plaque and result in rupture, thrombosis, and sudden death. In efforts to understand the molecular processes regulating apoptosis in this cell type, we have defined a novel mechanism involving the ubiquitously expressed transcription factor Sp1. Subtypes of SMC expressing abundant levels of Sp1 produce the death agonist, Fas ligand (FasL) and undergo greater spontaneous apoptosis. Sp1 activates the FasL promoter via a distinct nucleotide recognition element whose integrity is crucial for inducible expression. Inducible FasL promoter activation is also inhibited by a dominant-negative form of Sp1. Increased SMC apoptosis is preceded by Sp1 phosphorylation, increased FasL transcription, and the autocrine/paracrine engagement of FasL with its cell-surface receptor, Fas. Inducible FasL transcription and apoptosis are blocked by dominant-negative protein kinase C-ζ, whose wild-type counterpart phosphorylates Sp1. Thus, Sp1 phosphorylation is a proapoptotic transcriptional event in vascular SMC and, given the wide distribution of this housekeeping transcription factor, may be a common regulatory theme in apoptotic signal transduction.

3/CPP32, which is expressed in cells as the inactive 32-kDa form is, in turn, cleaved by caspase-8/FLICE to produce two mature subunits (17 and 12 kDa). Active caspase-3/CPP32 cleaves nuclear mitotic apparatus protein and mediates DNA fragmentation, chromatin condensation, and the formation of apoptotic bodies (4).
FasL (5) and Fas (6,7) are both expressed in arterial tissue, including the human atherosclerotic plaque. Immunohistochemical analysis revealed FasL expression in 34 of 34 carotid atherosclerotic plaques examined, with virtually all FasL positive-staining associated with intimal smooth muscle cells (SMCs) and little staining apparent in normal arterial tissue (5). Fas is also highly expressed in intimal SMCs of the plaque (6,7). FasL/Fas expression and apoptosis (8 -10) in normal artery and plaque has prompted speculation on the roles of these molecular mediators in vascular cells. Apoptosis in undiseased tissue may inhibit arterial thickening by limiting cell proliferation and accumulation in the intima (6). In atherosclerotic tissue, apoptosis particularly of collagen-producing SMCs may substantially weaken the plaque causing it to rupture, initiate thrombosis, and trigger acute coronary syndromes (11)(12). Overexpression 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 model suggests that FasL may promote rather than retard atherogenesis. FasL overexpression in nondenuded arteries of hypercholesterolemic animals stimulated lesion formation in these animals via increased cellularity (15). These observations may be due to differences in artery and lesion cellular composition or cholesterol feeding between the two animal models.
Despite clear evidence for FasL and Fas expression in SMCs of the artery wall, the molecular mechanisms mediating FasL production in vascular cells are presently not known. The promoter region of the FasL gene has recently been cloned and found to contain binding sites for a number of transcription factors including NF-B (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 regulatory regions within a 2.3-kilobase portion of the 5Ј-untranslated genome (19). Analysis of the FasL promoter has mostly been confined to T cells. For example, T cell activation following CD4 cross-linking induces NFAT binding to the FasL enhancer and gene transactivation (19). Similarly, cytotoxic stress-induced FasL expression involves the activation of NF-B, AP-1, and c-Jun N-terminal kinase, prior to cell death (16 -20). Activity of the FasL promoter is also regulated by MEK kinase-1 (18). However, transcription factor phosphorylation has not yet been directly demonstrated as a prerequisite step in apoptosis.
The discovery and functional characterization of Sp1 as a GC-rich binding nuclear protein has provided a useful paradigm to our understanding of the regulation of transcriptional activation in eukaryotic cells (21,22). Sp1 is a broadly expressed nuclear protein of ϳ100 kDa and contains three Kruppel-like zinc fingers that contact DNA (21,22). A nucleotide recognition element for Sp1 is located in the FasL promoter at position Ϫ281/Ϫ276 base pairs (GGGCGG) relative to the transcriptional start site. Sp1 can influence gene expression by changes in its nuclear concentration and interaction with the promoter, by providing architectural support, serving as a cofactor, or by undergoing chemical modification. Sp1 phosphorylation e.g. mediates inducible tissue factor expression in vascular endothelial cells exposed to fluid shear stress (23). The significance of Sp1 in the process of apoptosis in any cell type is presently unknown. This knowledge would advance our understanding of the transcriptional basis of extrinsic apoptosis, given the wide distribution of both Sp1 and FasL.
WKY12-22 and WKY3M-22 cells are well established subtypes of vascular smooth muscle cells that are phenotypically distinct (24,25). WKY12-22 cells have a cobblestone morphology in culture, proliferate in plasma-derived serum (which lacks vital growth factors), and spontaneously overexpress mRNA for platelet-derived growth factor (PDGF) B-chain, elastin, and osteopontin (24,25). In contrast, WKY3M-22 cells are typically spindle-shaped and do not express PDGF-B, elastin, or osteopontin mRNA, nor do they grow in plasma-derived serum. Both cell subtypes are phenotypically stable in culture and can be passaged indefinitely. Therefore, WKY12-22 and WKY3M22 cells represent important cells with which to delineate the molecular basis for differences in SMC phenotype and gene expression.
We recently reported that Sp1 is spontaneously expressed at greater levels in WKY12-22 cells than WKY3M-22 cells and that as a consequence, Sp1-dependent genes, such as PDGF-B, are overexpressed in WKY12-22 cells compared with its sister cell subtype (26). These observations provided important insight into the transcriptional basis for differential gene expression. Here we explored the regulatory role of Sp1 in inducible FasL expression and apoptosis in two phenotypically distinct SMC subtypes.

EXPERIMENTAL PROCEDURES
Transfections and Luciferase Assays--SMCs were maintained in Waymouth's medium (Life Technologies, Inc.), pH 7.4, containing 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO 2 . Transient transfections were performed with cells at 60% confluence, and the indicated constructs together with 2 g of the internal control vector, pRL-TK, using FuGENE6 transfection agent (Roche Molecular Biochemicals). After 24 h, the transfected cells were incubated with or without CAM (1 g/ml), and luciferase activity was quantified using the Dual Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla data generated from pRL-TK.
Plasmid Constructs-Various sized fragments of the FasL promoter (Ϫ271FasL⅐hsLuc and Ϫ296FasL⅐hsLuc) were amplified from the parent vector FasL⅐hsLuc (gift of Dr Shailaja Kasibhatla, La Jolla Inst. of Cellular Immunology) by PCR and blunt-end cloned into pGL3. The mutant counterpart of FasL⅐hsLuc bearing a mutation in the Sp1 binding site (mSp1FasL⅐hsLuc) was constructed using the QuikChange site-directed mutagenesis kit (Stratagene). pEBGNLS-Sp1 was obtained from Gerald Thiel (Inst. for Genetics, University of Cologne), CMV-Sp1 was obtained from Robert Tjian (Howard Hughes Medical Inst., University of California), and CMV-FLAG⅐DN-PKC-was obtained from Debabrata Mukhopadhyay (Beth Israel-Deaconess Hospital, Boston) and Alex Toker (Boston Biomedical Research Inst.).
Quantitative Assessment of DNA Fragmentation-SMCs were grown in 96-well plates to 80% confluency in 100 l of growth medium. Where indicated, the cells were incubated with Fas-Fc (R&D Systems) and/or IgG Fc (R&D Systems) (50 g/ml, final concentration) for 1 h prior to the addition of CAM. After 24-h exposure to CAM, apoptosis was quantitated using the Cell Death Detection ELISA Plus (Roche Molecular Biochemicals). This assay, which measures cytoplasmic histone-associated internucleosomal DNA fragmentation, has been used previously to quantitate inducible apoptosis in cultured cells (27)(28)(29). Briefly, the cells were washed gently in PBS and incubated with shaking in lysis buffer for 30 min at 22°C. Lysates were transferred into Eppendorf tubes and spun at 14,000 rpm for 30 s. Twenty l of the supernatant was used in the ELISA, which was performed in accordance with the manufacturer's instructions and normalized to total cell number measured using a Coulter counter. Results are expressed as total internucleosomal DNA fragmentation as a proportion of the cell population.
Annexin V Staining/FACS Analysis-SMCs were washed twice with ice-cold phosphate-buffered saline, pH 7.4 and resuspended in 1ϫ binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl 2 ) at a concentration of 1 ϫ 10 6 cells/ml. One hundred microliters of the suspension was transferred to 5-ml flat bottomed tubes where 5 l of annexin V-fluorescein isothiocyanate and 10 l of propidium iodide (50 g/ml stock in PBS) was added. The cells were gently vortexed and incubated in the dark at 22°C for 15 min. Four hundred microliters of binding buffer was added to each tube, and annexin V staining was analyzed by flow cytometry within 1 h. Results are expressed as annexin V staining as a percentage of the total cell population.
Propidium Iodide Nuclear Staining-SMCs were grown in chamber slides (80% confluent) and incubated with CAM (1 g/ml) for 24 h. The cells were washed in PBS, pH 7.4, and fixed with methanol/acetone (80:20) for 10 min at 22°C. Propidium iodide (50 M) was added to each well and incubated for a maximum of 5 min followed by a second wash with PBS. Cells undergoing apoptosis were visualized by confocal microscopy.
Nuclear Extract Preparation-SMCs treated with CAM for various times were washed and scraped in 10 ml of PBS and transferred to precooled centrifuge tubes. Samples were spun at 1300 rpm for 15 min at 4°C. The pellet was resuspended in 100 l (for two 100-mm dishes) of solution A (10 mM Hepes, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl) and placed on ice for 5 min. Samples were spun at 14,000 rpm for 40 s. The pellet was resuspended in 20 l of solution C (20 mM Hepes, pH 7.9, 1.5 mM MgCl 2 , 420 mM NaCl, 0.2 mM EDTA) and mixed gently for 20 min at 4°C. The supernatant was transferred to precooled Eppendorf tubes containing 20 l of solution D (20 mM Hepes, pH 7.9, 1.5 mM KCl, 0.2 mM EDTA, 20% glycerol) and stored at Ϫ80°C until use. All buffers contained protease inhibitors.
Western Blot for Sp1-Fifteen micrograms of nuclear extract was resolved by 8% SDS-polyacrylamide gel electrophoresis and then transferred onto Immobilon-P transfer membranes (Millipore). The membranes were blocked overnight at 4°C in PBS containing 5% skim milk and 0.05% Tween 20. Sp1 was detected with Sp1 polyclonal antibodies (1:1000, Santa Cruz Biotechnology) and subsequent chemiluminescent visualization.
Sp1 Dephosphorylation Analysis by Western Blotting and EMSA-Nuclear extracts (10 -15 g) were incubated with or without 5 units of calf intestinal alkaline phosphatase (CIP, NEB) for 1 h at 37°C in a total volume of 20 l. The reaction was quenched by the addition of loading dye prior to 8% SDS-polyacrylamide gel electrophoresis and Western blot analysis for Sp1. In EMSA, 8 g of nuclear extract was incubated with 5 milliunit of CIP (final concentration determined by CIP titration experiments with 32 P-labeled FasL Oligo) at 33°C for 5 min and then on ice for 15 min. The reaction was stopped by the addition of phosphatase inhibitors at a final concentration of 10 nM LiNaF, 10 nM sodium vanadate, 10 nM potassium pyrophosphate, and 5 nM sodium phosphate. EMSA was performed as described above. CIP treatment prior to EMSA has previously been used for the assessment of Sp1 phosphorylation in nuclear extracts (30,31).

RESULTS AND DISCUSSION
Sp1 Is Proapoptotic-To begin investigating a possible mechanistic role for Sp1 in programmed cell death, we compared apoptosis in two well established SMC subtypes isolated originally from the arteries of pup (2-week-old) and adult (3-monthold) rats (24,25). Pup SMCs (WKY12-22 cells) are phenotypically distinct from their adult counterparts (WKY3M-22 cells) and express abundant levels of Sp1 (26). We found that cytoplasmic histone-associated internucleosomal DNA fragmenta-tion (27)(28)(29) is greater in WKY12-22 cells than WKY3M-22 cells (Fig. 1A, left), indicating higher levels of spontaneous apoptosis in the former cell subtype and providing further evidence that these SMC subtypes are phenotypically distinct. These observations were confirmed by annexin V-fluorescein isothiocyanate staining upon fluorescence-activated cell sorting, indicating greater disrupted membrane symmetry exposing phosphatidylserine to the external environment (Fig. 1A, right). Similar findings were obtained qualitatively by DNA laddering on ethidium bromide-stained agarose gels (data not shown). To determine whether Sp1 could directly modulate apoptosis, Sp1 cDNA was transiently overexpressed in WKY3M-22 cells using a cytomegaloviral promoter-driven expression vector (CMV-Sp1). Sp1 increased apoptosis in this cell type (Fig. 1B). Conversely, apoptosis was inhibited by 50% in WKY12-22 cells following overexpression of a dominant-negative form of Sp1 (DNA binding domain) using pEBGNLS-Sp1 (33) (Fig. 1C), demonstrating profound inhibition effected by a single transcription factor. These data provide the first demonstration of the capacity of Sp1 to modulate apoptosis in any cell type.
Inspection of the FasL promoter sequence revealed the ex-

FIG. 3. CAM stimulates apoptosis in vascular SMCs and induces FasL expression in an Sp1-dependent manner.
A, CAM increases propidium iodide nuclear staining in WKY12-22 cells. The SMCs were exposed to CAM (1 g/ml) for 24 h prior to fixation, incubation with propidium iodide, and confocal microscopy. Effect of CAM (1 g/ml) on luciferase expression driven by FasL⅐hsLuc or a construct bearing a deletion of the Sp1 binding element (Ϫ271FasL⅐hsLuc, B), or FasL⅐hsLuc cotransfected with 5 g of dominant-negative Sp1 (pEBGNLS-Sp1) or the backbone alone (pEBGNLS, C). 15 g of FasL promoter-reporter construct was used throughout. Firefly luciferase activity was normalized to Renilla activity, and the results were plotted as -fold increase relative to FasL⅐hsLuc or Ϫ271FasL⅐hsLuc, 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.
istence of a putative recognition element for Sp1 (5Ј-GGGCGG-3Ј) located at nucleotides Ϫ281/Ϫ276. Because Sp1 is preferentially expressed in WKY12-22 cells (26), we hypothesized that FasL, if it is Sp1-dependent, would be more abundantly expressed in WKY12Ϫ22 cells than WKY3M-22 cells. We assessed FasL mRNA expression in WKY12Ϫ22 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, transient transfection analysis was performed with the construct FasL⅐hsLuc, a firefly luciferase-based reporter vector driven by 1.2 kilobases of the FasL promoter (16). Cotransfection of FasL⅐hsLuc with an Sp1 expression vector induced FasL promoter-dependent expression ( Fig. 2A). To localize the Sp1 response element in the FasL promoter, we generated a series of reporter constructs derived from parent FasL⅐hsLuc bearing 5Ј deletions in the FasL promoter. Luciferase activity increased upon cotransfection of Ϫ296FasL⅐hsLuc and CMV-Sp1 (Fig.  2B). In contrast, Ϫ271FasL⅐hsLuc failed to respond to Sp1 overexpression (Fig. 2B). The 5Ј-FasL promoter end points in these constructs (Ϫ296 and Ϫ271) occur on either side of the putative Sp1 binding site (Ϫ281/Ϫ276). EMSA using a 32 Plabeled double-stranded oligonucleotide spanning this region of the promoter ([ 32 P]FasL oligonucleotide, Ϫ296/Ϫ265) and nuclear extracts prepared from WKY12-22 cells revealed the formation 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 polyclonal antibodies used as a negative control had no influence on nucleoprotein complex formation (Fig. 2C). To demonstrate sequence specificity of complex formation, we prepared an oligonucleotide ([ 32 P]mFasL oligonucleotide, Ϫ296/Ϫ265) bearing a mutation that disrupts the Sp1 binding site (to 5Ј-TTTCTT-3Ј). This mutation no longer supported the interaction of Sp1 with this region of the FasL promoter (Fig. 2C). When introduced into full-length FasL⅐hsLuc, producing construct mSp1FasL⅐hsLuc, luciferase expression inducible by Sp1 was completely abolished (Fig. 2D). These findings demonstrate that Sp1 positively regulates FasL transcription. Activation by exogenous Sp1 of the FasL promoter was greater in WKY3M-22 cells than WKY12-22 cells (Fig. 2, E versus A), consistent with higher endogenous Sp1 expression in the latter cell type (26).
Inducible Apoptosis Involves the Phosphorylation of Sp1 and Induction of FasL-We next explored the effect of extracellular apoptotic stimuli on the capacity of Sp1 to stimulate apoptosis and transactivate the FasL promoter. CAM, an inhibitor of DNA topoisomerase I, has been reported to induce apoptosis in several cell types (34) although its effect on SMCs is not known. Nuclear condensation of SMCs stained by propidium iodide increased dramatically following 24-h exposure to CAM (Fig.  3A). This agent also induced internucleosomal fragmentation of DNA and annexin V staining (data not shown). To determine whether FasL expression is altered by CAM and define the involvement of Sp1 in this process, we performed RT-PCR and transient transfection analysis with FasL promoter constructs. CAM stimulated FasL promoter activity (Fig. 3B) and endogenous FasL gene expression (Fig. 1D) by 2-4-fold (Fig. 3, B and  C). CAM failed to activate the construct Ϫ271FasL⅐hsLuc (Fig.  3B), which lacks the Sp1 site and was unable to mediate Sp1inducible FasL promoter-dependent expression (Fig. 2B). Overexpression of dominant-negative Sp1 (20) together with FasL⅐hsLuc blocked CAM-inducible FasL transcription (Fig.  3C), whereas the empty expression vector had no effect (Fig.  3C). These data demonstrate that Sp1 is required for FasL promoter activation by extracellular stimuli. EMSA using [ 32 P]FasL oligonucleotide and nuclear extracts of cells exposed to CAM revealed that this agent did alter Sp1 occupancy of the promoter (Fig. 4A, lane 4 versus lane 2). Incubation of these extracts with CIP (22), which hydrolyzes 5Ј-phosphate groups, prior to EMSA decreased the intensity of both Sp1 binding complexes (Fig. 4A, lane 5 versus lane 3) but was most profound in extracts of cells exposed to CAM. Densitometric assessment of the intensities of these complexes (Fig.  4A, lower left and lower center) revealed that 12% of promoterbound Sp1 is basally phosphorylated and that Sp1 phosphorylation increases to 31% upon exposure to CAM (Fig. 4A, lower  right). This indirect determination of Sp1 phosphorylation was supported by Western immunoblot analysis with antibodies to Sp1. We observed the appearance of a hyperphosphorylated species following exposure to CAM (Fig. 4B). This effect was abolished by prior incubation of the extracts with CIP (Fig. 4B). These findings thus show that Sp1 is phosphorylated during CAM-inducible apoptosis. Sp1 phosphorylation regulates the FIG. 4. Phosphorylation of endogenous Sp1 following exposure to CAM. A, EMSA using [ 32 P]mFasL Oligo and nuclear extracts of WKY12-22 cells incubated with CAM (1 g/ml). Where appropriate, the extracts were treated with CIP prior to EMSA. The amount of CIP (5 milliunit) used in this assay is based on CIP titration experiments that previously defined the concentration of CIP unable to dephosphorylate the 32 P-labeled probe. Sp1 nucleoprotein complex intensity (with or without CAM exposure for CIP treatment of extracts) was semi-quantitated by densitometry. B, Western blot analysis using nuclear extracts of WKY12-22 cells exposed to CAM (1 g/ml), with and without CIP treatment (5 units). Sp1-P indicates hyperphosphorylated Sp1. The Coomassie Blue-stained gel is shown. The data are representative of two independent determinations. inducible expression of a number of other genes, including vascular permeability factor/vascular endothelial growth factor (31), ␣ 2 -integrin (35), and tissue factor (23).
CAM-inducible FasL Promoter Activity and Apoptosis Are Protein Kinase--dependent Processes-A kinase found to mediate Sp1 phosphorylation is protein kinase-(PKC-) (31), a diacylglycerol-and Ca 2ϩ -independent atypical member of the PKC family (36). PKC-is ubiquitously expressed and interacts directly with Sp1 (31). To investigate the role of PKC-in the regulation of FasL expression, we cotransfected WKY12-22 cells with an expression vector (CMV-FLAG⅐DN-PKC-) generating a kinase-inactive dominant-negative mutant of PKCbearing a Lys 275 3 Trp 275 substitution (37)(38)(39)(40). The FasL promoter was activated by CAM in cells harboring the empty expression vector (Fig. 5A), whereas overexpression of mutant PKC-attenuated CAM-inducible FasL promoter-dependent reporter expression (Fig. 5A). Dominant-negative PKC-also blocked internucleosomal fragmentation stimulated by CAM (Fig. 5B). Overexpression of dominant negative PKC-in cells not exposed to CAM did not significantly modulate the level of apoptosis compared with cells transfected with the backbone control (data not shown). This suggests that the capacity of dominant-negative PKC-to suppress apoptosis detectable in our system is conditional upon the cells being induced to undergo further cell death by exposure to apoptotic stimuli. This is likely a direct consequence of the low level of spontaneous phosphorylation of Sp1 (Fig. 4A) making attenuation by dominant-negative PKC-difficult to measure in a cotransfection setting. These findings, nonetheless, indicate that PKC-regulates inducible FasL transcription and apoptosis.
Fas receptor, unlike FasL (Fig. 1C), is expressed in both WKY12-22 and WKY3M-22 cells (Fig. 6A). Because CAM induces FasL expression (Figs. 3, B-D, and 5A), we hypothesized that the induction of apoptosis by this agent involves the secretion and autocrine/paracrine engagement of FasL with Fas at the cell surface. To address this possibility, prior to the addition of CAM, we incubated the cells with Fas⅐Fc chimera, in which the extracellular domain of Fas is fused to the Fc portion of human IgG. Fas⅐Fc blocked SMC apoptosis induced by CAM (Fig. 6B). In contrast, an identical amount of the Fc fragment alone had no effect (Fig. 6B). These findings thus demonstrate that autocrine/paracrine extracellular Fas/FasL engagement is involved in SMC apoptosis. Sp1 is phosphorylated and activates FasL in SMCs upon exposure to extracellu- In this paper, we have defined a novel role for the ubiquitously expressed transcription factor Sp1 in apoptotic signal transduction. Subtypes of SMC expressing abundant levels of Sp1 produce FasL and undergo greater spontaneous apoptosis. EMSA and transient transfection analysis revealed that the FasL promoter is activated by Sp1 via a distinct element whose integrity is crucial for inducible expression. Inducible FasL transcription is inhibited by a mutant form of Sp1, which also blocks apoptosis. Inducible SMC apoptosis is preceded by Sp1 phosphorylation, increased FasL transcription, and the autocrine/paracrine engagement of FasL with Fas. Both inducible FasL transcription and apoptosis are blocked by dominantnegative protein kinase C-. These data demonstrate that apoptotic signaling in SMCs involves Sp1 phosphorylation.
The present study is the first report of transcription factor phosphorylation as a prerequisite biochemical process in inducible apoptotic cell death. We used CAM as a model effector of cell death; however, given the general cellular expression of Sp1, our observations are unlikely to be confined to this agent alone nor are they likely to be cell type-specific. Okadaic acid, a selective inhibitor of serine-threonine phosphatase PP2A, stimulates apoptosis in a wide variety of cell types including murine fibroblasts (41), rat kidney epithelial cells (42) amongst them. Okadaic acid, like CAM, stimulates Sp1 phosphorylation and apoptosis in SMCs (data not shown). Tat, the transcriptional activator of human immunodeficiency virus type 1 (HIV-1), stimulates Sp1 phosphorylation (43), activates FasL expression (44), and can induce apoptosis (44,45). Sp1 phosphorylation may be an important theme in apoptotic signaling. However, this process alone may not account for FasL transactivation because Sp1 physically interacts and functionally cooperates with a large number of other transcription factors. These include NF-B p65/RelA (46,47) and AP1 (48), which each induce the FasL promoter in other cell types (16,19).
A common pathophysiologic setting in which Sp1 phosphorylation may be relevant is atherosclerosis. Sudden death in patients with unstable angina and myocardial infarction is associated with atherosclerotic plaque rupture (49,50). This could arise from SMC apoptosis, because SMCs are the only cells in the plaque capable of producing collagen fibers types I and III, which maintain tensile strength (8 -10, 12, 51). Loss of SMCs as a consequence of apoptosis could weaken the cap and result in plaque rupture (12). Consistent with this, SMCs located in vulnerable regions of the plaque, such as the fibrous cap and shoulders (52,53) undergo apoptosis (56,54) and express Fas (5,7). Indeed, SMCs isolated from atherosclerotic plaques undergo greater spontaneous apoptosis than cells derived from the normal artery wall (55). Moreover, SMC depletion in human aortic aneurysms is accompanied by biochemical and morphological changes consistent with SMC apoptosis (56). Sp1 phosphorylation may thus be an important biochemical mediator of cell death in a number of vascular disease settings.