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Mol Cell Biol. Apr 2002; 22(7): 1981–1992.
PMCID: PMC133680

Stat1-Dependent, p53-Independent Expression of p21waf1 Modulates Oxysterol-Induced Apoptosis

Abstract

7-Ketocholesterol (7kchol) is prominent in atherosclerotic lesions where apoptosis occurs. Using mouse fibroblasts lacking p53, p21waf1, or Stat1, we found that optimal 7kchol-induced apoptosis requires p21waf1 and Stat1 but not p53. Findings were analogous in a human cell system. Apoptosis was restored in Stat1-null human cells when wild-type Stat1 was restored. Phosphorylation of Stat1 on Ser727 but not Tyr701 was essential for optimum apoptosis. A neutralizing antibody against beta interferon (IFN-β) blunted Ser727 phosphorylation and apoptosis after 7kchol treatment; cells deficient in an IFN-β receptor subunit exhibited blunted apoptosis. IFN-β alone did not induce apoptosis; thus, 7kchol-induced release of IFN-β was necessary but not sufficient for optimal apoptosis. In Stat1-null cells, expression of p21waf1 was much less than in wild-type cells; introducing transient expression of p21waf1 restored apoptosis. Stat1 and p21waf1 were essential for downstream apoptotic events, including cytochrome c release from mitochondria and activation of caspases 9 and 3. Our data reveal key elements of the cellular pathway through which an important oxysterol induces apoptosis. Identification of the essential signaling events that may pertain in vivo could suggest targets for therapeutic intervention.

In the last two decades, evidence has emerged that low-density lipoprotein (LDL), the levels of which in plasma strongly correlate with cardiovascular disease, may acquire disease-promoting properties subsequent to modification by oxidation (3, 15-17, 71). The process by which LDL becomes oxidized in vivo is the subject of vigorous research efforts (17); however, three lines of evidence support a lipoprotein oxidation theory of atherosclerosis. First, oxidized LDL exists in vivo: LDL fractions removed from arterial lesions are oxidized (78); antibodies that recognize oxidized LDL, but not native LDL, bind to epitopes in lesions (78); and lesions contain many of the lipid oxidation products formed during oxidation of LDL in vitro. There is also evidence that oxidized lipoproteins exist in plasma (63). Second, oxidized LDL alters cell functions in vitro in ways that mimic the properties of cells in lesions. As examples, oxidized LDL but not native LDL induces cell injury, smooth muscle cell proliferation, attraction of monocytes, and the engorgement of monocyte-derived macrophages with lipid (15). Third, certain antioxidants delay or inhibit the formation of arterial lesions in a variety of animal models (16, 67, 71).

Cell injury is a characteristic of the formation of arterial lesions in various contexts. For example, a prominent theory of atherosclerosis proposes that endothelial cell injury and dysfunction initiate disease (68). Furthermore, dead cell debris is a component of developing lesions, and apoptotic cells have been identified in lesions (27, 36). Oxidized LDL, but not native LDL, induces apoptosis in vitro in a variety of cells, leading to the hypothesis that it is responsible for apoptosis in atherosclerotic plaques. The oxidation of LDL leads to the formation of dozens of lipid by-products, including modified phospholipids, aldehydic breakdown products of unsaturated fatty acids, and numerous cytotoxic oxysterols. Prominent among the oxysterols formed upon LDL oxidation in vitro (6, 19, 37, 79), as well as among those accumulating in arterial lesions in humans (5), is 7-ketocholesterol (7kchol). 7kchol has been shown to induce apoptosis in vitro (33, 46, 49-53, 60-62) and is believed to be a contributor to apoptosis in arterial lesions.

Some features of 7kchol-induced apoptosis have been reported. 7kchol treatment reduced bcl-2 levels in vascular smooth muscle cells, and inhibition of CPP32 blunted apoptosis (62). bcl-2 overexpression inhibited 7kchol-induced apoptosis in P388-D1 (macrophage-like) cells and two promonocytic leukemia cell lines, U937 and U4 (33, 51). The current study was undertaken to define further the key signaling events in the cellular mechanism of 7kchol-induced apoptosis. We find that Stat1 expression and p21waf1 expression, which is dependent on Stat1 but not on p53, are critical upstream components of a 7kchol-induced apoptotic signaling cascade involving cytochrome c release into cytoplasm and the activation of caspase 3. We propose that the qualitative nature or form of p21waf1 is a determinant in the final outcome of cellular response, such as cell cycle arrest or apoptosis. Our results appear to have implications beyond oxysterol-induced apoptosis, since we also observed reduced apoptosis in cells deficient in p21waf1 or Stat1 exposed to multiple apoptotic stimuli.

MATERIALS AND METHODS

Reagents and cell culture.

7kchol was purchased from Steraloids, Inc. Neutralizing antibody to beta interferon (IFN-β) was purchased from R&D Systems. Wild-type and Stat1−/− mouse embryo fibroblasts (MEF) were gifts from David E. Levy (Kaplan Cancer Center, New York University, New York). Wild-type and Stat1−/− mouse fibroblasts were gifts from Robert D. Schreiber (Washington University School of Medicine, St. Louis, Mo.). These were cultured and maintained as described earlier (22, 58). MEF from p21waf1−/− and p53−/− mice were gifts from Greg Hannon (Genetica, Inc., Cold Spring Harbor, N.Y.) and Lawrence Donehower (International Agency for Research on Cancer, Lyon, France), respectively. Wild-type control cells were obtained from littermates. Primary cultures of MEF were obtained after trypsin digestion of wild-type (129/svev) and Stat1−/− mouse embryos (Taconic Laboratories, Germantown, N.Y.), terminated at 11.5 days. Western analysis (for p21waf1) was performed on these primary cells at passage 2. The human fibrosarcoma cell lines 2fTGH and U3A and U3A-derived cells stably transfected with cDNAs encoding Stat1 mutant proteins were prepared as described (42, 65). Human fibroblast LF-1 wild-type and H07.2-1 p21waf1−/− cells were gifts from John M. Sedivy (7) (Brown University, Providence, R.I.). These were maintained in Dulbecco modified Eagle medium (DMEM) and Ham's (1:1) supplemented F-12 medium with 10% fetal bovine serum (FBS; BioWhittaker). At the time of treatment with 7kchol, cells were subconfluent and maintained in 5% lipoprotein-deficient serum (LPDS) (18). An adenovirus (Ad) construct containing the cDNA for p21waf1 (Ad-p21) and control virus (Ad-CMV) were gifts from Joseph Nevins (47) (Duke University Medical Center; Durham, N.C.).

Acridine orange and ethidium bromide morphological staining assay to quantify apoptosis.

Acridine orange stains DNA bright green, allowing visualization of the nuclear chromatin pattern. Apoptotic cells have condensed chromatin that is uniformly stained. Ethidium bromide stains DNA orange but is excluded by viable cells. Dual staining allows separate enumeration of populations of viable-nonapoptotic, necrotic, viable-apoptotic, and nonviable-apoptotic cells. The assay was performed as described earlier (66, 74). Briefly, cells after treatment with 7kchol (15 μg/ml) for 18 h were trypsinized, and cell pellets were collected by centrifugation at 300 × g, resuspended in 200 μl of phosphate-buffered saline (PBS), and stained with the mixture of dyes just before quantification.

Assessment of apoptosis by quantifying fragmentation of [3H]thymidine-labeled DNA.

3H-labeled thymidine (0.6 μCi/ml) was added just after cells were seeded (105 cells/well) in six-well plates. After overnight labeling, cells were washed twice with ice-cold PBS and cultured overnight in medium containing 5% LPDS and 7kchol. Nonadherent and adherent cells were pooled and pelleted at 300 × g for 5 min. The resulting pellet was lysed in 200 μl of lysis buffer (0.2% Triton X-100; 10 mM Tris-HCl, pH 7.4; 1 mM EDTA) by gentle mixing and incubation at 37°C for 30 min. The lysate was pelleted at 13,500 × g for 30 min. The supernatant solution (cytoplasmic DNA fraction) was collected, and the pellet (intact nuclear DNA) was solubilized in 20 μl of 1 M sodium hydroxide. The amount of 3H-labeled thymidine in each fraction was determined by liquid scintillation counting. The relative amount of 3H-labeled thymidine released was calculated as the ratio of counts per minute in the cytosolic fraction to the sum of the counts per minute in the cytosolic fraction and intact chromatin (39, 64).

Quantification of apoptosis by TUNEL assay.

After treatment with various apoptotic inducers cells were trypsinized, and cell pellets were collected by centrifugation at 300 × g, fixed in 2% paraformaldehyde for 25 min on ice, collected again by centrifugation at 300 × g, and washed with PBS. Then, 1 ml of 75% ice-cold ethanol was added, and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining was performed as described previously (72). TUNEL-positive cells were quantified with a Becton Dickinson FACSCalibur.

Colony-forming efficiency after exposure to 7kchol.

Cells were treated for 18 h with 7kchol at 10, 15, 10, 25, and 30 μg/ml and then washed with PBS, trypsinized, and replated in fresh DMEM with 10% FBS, in equal numbers of 1,000 or 2,000 cells per 100-mm tissue culture plate. The plates were incubated at 37°C in 5% CO2. After 15 days, cells were washed with ice-cold PBS and fixed with 4% paraformaldehyde, and colonies were then stained with Giemsa (Sigma) and counted.

Western analyses.

Cells were stimulated with 7kchol (15 μg/ml) for the times indicated. Cell extracts were prepared by using radioimmunoprecipitation assay lysis buffer (150 mM NaCl, 50 mM Tris base, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1% NP-40) for the detection of p21waf1 and Stat1. For the detection of caspase 3, lysis buffer containing 100 mM HEPES, 10% sucrose, 0.1% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 1 μg of phenylmethylsulfonyl fluoride/ml, 10 mM dithiothreitol, protease inhibitors (Complete; Boehringer Mannheim), and 0.5 mM EDTA (pH 6.8) was used. Samples were run in SDS-12% gradient gels (Novex) for the detection of p21waf1, in SDS-8% gels for the detection of Stat1, and in SDS-10% polyacrylamide gels for the detection of caspase 3. Proteins were transferred to polyvinylidine difluoride (PVDF) membranes and probed with antibodies to p21waf1, Stat1 (Santa Cruz Biotechnology), and caspase 3 (Santa Cruz Biotech), followed by treatment with rabbit anti-goat or goat anti-rabbit secondary antibody coupled with horseradish peroxidase (HRP). In selected experiments, protein samples were run on 8 to 20% gradient gels (Novex), transferred to PVDF membranes by semidry transfer apparatus (Bio-Rad), blocked in 5% fat-free dry milk, and simultaneously incubated with HRP-conjugated polyclonal anti-p21waf1 and monoclonal anti-p53 antibodies and a Stat1 polyclonal antibody (Santa Cruz). These were washed for 15 min three times with PBS-Tween, followed by incubation with HRP-conjugated goat anti-rabbit secondary antibody to the anti-Stat1. Transfers were analyzed by using the Super Signal substrate chemiluminescence reagent (Pierce) after four washes with PBS-Tween. For analysis of cytochrome c release into the cytosol, cells were treated for the times indicated, and the extraction of cytosolic proteins, SDS-polyacrylamide gel electrophoresis, and Western blot analysis were performed by using anti-cytochrome c clone 7H8.2C12 (PharMingen) (4). Mitochondrial contamination was monitored by probing the membrane again with a mouse monoclonal antibody against human cytochrome oxidase unit 2 (Molecular Probes). To detect Stat1 phosphorylation, cells were stimulated with IFN-γ (Boehringer Mannheim) at 1,000 IU/ml for 30 min (as a positive control) or with 7kchol (15 μg/ml) for various times. Cell extracts were prepared as described previously (43). Samples were run in SDS-8% polyacrylamide gels, transferred to PVDF membranes, and probed with antibodies to Stat1 peptides containing phosphorylated Ser727 (Upstate Biotechnology), followed by treatment with anti-rabbit immunoglobulin G coupled to HRP (Boehringer Mannheim). The transfers were analyzed by using the Super Signal substrate chemiluminescence reagent (Pierce).

Neutralization of IFN-β.

Anti-human IFN-β polyclonal antibody (0.1 μg/ml) (R&D Systems) was used to neutralize IFN-β. Cells were pretreated for 2 h with 0.1 μg of antibody/ml and then treated with 7kchol (15 μg/ml) for the times indicated. Stat1 phosphorylation at Ser727 or apoptosis was assessed as described above.

Fluorescence-activated cell sorting analysis of cell cycle phase.

After the treatments indicated, cells were harvested by trypsinization and fixed in 75% ice-cold ethanol for 30 min on ice. Cells were washed in PBS and incubated with propidium iodide solution (100 μg of propidium iodide/ml and 0.1% sodium citrate) (41) for 25 min on ice prior to flow cytometric analysis with Becton Dickinson FACSCalibur. Cell cycle profiles were determined by using CellQuest and ModFit cell cycle analysis software.

p21waf1 gene delivery to cells by Ad infection.

Wild-type MEF, Stat1−/− MEF, and human 2fTGH and U3A cells were grown in DMEM containing 10% serum in triplicate in six-well tissue culture plates. Cells were infected with a control Ad (Ad-CMV) at a multiplicity of infection (MOI) of 125 focus-forming units (FFU) per cell or with an Ad with p21waf1 cDNA (Ad-p21) at an MOI of 100 FFU per cell. Cells were incubated for 24 h with Ad. The medium was changed to DMEM with 5% LPDS, and cells were incubated for 18 h further with 15 μg of 7kchol/ml. Cells were trypsinized, and apoptosis was quantified by acridine orange staining as described above.

Northern analyses.

Total RNA was prepared by using the TRIzol (Gibco-BRL) method according to the manufacturer's instructions. Northern transfers were analyzed with p21waf1 cDNA probes (23). The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) probe was from Ambion.

DEVD-AMC fluorogenic substrate assay for caspase 3 activity.

DEVD-AMC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; Biomol) is a synthetic tetrapeptide substrate specific for caspase 3. Cells treated with 7kchol for 18 h were lysed in 0.25 ml of lysis buffer as described above. Lysis was completed by two 10-s pulses of sonication (Sonicator XL; Heat Systems). Cellular debris was removed by centrifugation (10 min at 16,000 × g), and the cytosolic extract was stored at −20°C. The protein content of the lysate was determined by the Bio-Rad Protein Assay according to the manufacturer's instructions. Cytosolic protein (20 μg) and DEVD-AMC (50 μm) were incubated in a total volume of 500 μl in 100 mM HEPES-10% sucrose-0.1% CHAPS (pH 6.8) for 30 min on an orbital shaker at 37°C. Samples were diluted to a final volume of 1 ml immediately before the fluorescence was measured (excitation, 400 nm; emission, 505 nm).

Inhibition of 7kchol-induced apoptosis by caspase 3 inhibitors.

MEF were seeded in six-well plates (105 cells/well), in DMEM-F-12 supplemented with 10% FBS and incubated overnight at 37°C in 5% CO2. Cells were either pretreated or left untreated with the cell-permeable caspase 3 inhibitors DEVD-FMK (Z-Asp-Glu-Val-Asp-FMK; 100 μM) and DEVD-CHO (N-acetyl-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Asp-Glu-Val-Asp-CHO; 150 μM) in DMEM-F-12 and 5% LPDS. After 2 h of pretreatment, 7kchol (15 μg/ml) was added to the cells, followed by incubation overnight. After 18 h, the cells were stained with acridine orange and ethidium bromide, and apoptosis was quantified as described above.

Experiments were performed at least three times with consistent results.

RESULTS

7kchol-induced apoptosis requires p21waf1 independently of p53.

p53, a key tumor suppressor protein, regulates several different biological processes, including cell cycle arrest, apoptosis, angiogenesis, and DNA repair (1). An important transcriptional target of p53 is p21waf1, an inhibitor of cyclin-dependent kinases, the activation of which leads to either cell cycle arrest or apoptosis (8, 26). To investigate the mechanism of oxysterol-induced apoptosis, we treated MEF deficient in either p53 or p21waf1 with 15 μg of 7kchol/ml for 18 h. Apoptosis was quantified by staining cells with acridine orange-ethidium bromide. Normal MEF and MEF deficient in p53 underwent apoptosis upon treatment with 7kchol. In contrast, cells lacking p21waf1 exhibited much less apoptosis (Fig. (Fig.1A),1A), suggesting that 7kchol activates a p21waf1-dependent, p53-independent apoptosis signaling pathway.

FIG. 1.
7kchol-induced apoptosis depends on p21waf1 and Stat1. (A) Apoptosis in MEF. Subconfluent cells were treated with 7kchol (15 μg/ml) for 18 h, and apoptosis was quantified by staining with acridine orange and ethidium bromide. (B) Apoptosis in ...

Stat1 is required for 7kchol-induced apoptosis.

Since Stat-responsive elements have been reported to be present in the promoter of the p21waf1 gene (14) and since Stat1 has been shown to be required for optimal apoptosis in other cell systems (42, 44, 45, 69, 72), we investigated whether Stat1 influenced 7kchol-mediated apoptosis. Stat1−/− and wild-type MEF were treated with 7kchol for 18 h, and apoptosis was quantified. The number of apoptotic cells was dramatically reduced in response to 7kchol in Stat1-deficient fibroblasts compared to wild-type fibroblasts (Fig. (Fig.1B1B).

We confirmed 7kchol-induced apoptosis and the protective effects of p21waf1 and Stat1 in additional ways. After cells were grown in labeled thymidine, DNA was harvested and analyzed for fragmentation (64) (Fig. (Fig.1C).1C). We also confirmed the decrease in apoptosis in p21waf1−/− and Stat1−/− cells by using TUNEL (data not shown). Although precise quantification varies between these assays, the reduction in apoptosis in Stat1- and p21waf1-deficient cells after treatment with 7kchol for 18 h was consistent. In addition, we confirmed the effects of these two proteins on cell survival in response to 7kchol in a colony-forming assay (Fig. (Fig.1D1D and andE).E). Cell survival was compromised by 7kchol in concert with the apoptotic response of MEF and mouse fibroblasts, whereas the absence of p21waf1 or Stat1 significantly reduced cell death. These data are consistent with the concept that 7kchol kills fibroblasts by both necrotic and apoptotic mechanisms but that impairing the apoptotic pathway results in significantly increased long-term cell survival.

p21waf1 and Stat1 were required for optimal apoptosis induced by multiple stimuli and not only 7kchol. For example, apoptosis measured by TUNEL and confirmed by either caspase 3 activity or DNA laddering was markedly reduced in p21−/− and Stat1−/− MEF compared to wild-type cells after exposure to etoposide, staurosporine, cycloheximide, and cycloheximide plus dexamethasone (data not shown).

In performing dozens of experiments with 7kchol as an inducer of apoptosis in a variety of fibroblast-related cells, we have encountered some variability in the number of cells that can be rigorously designated to be apoptotic at a given time. Because the apoptotic response of these cells is protracted, occurring over a 24-h period after induction, we believe that the number of cells at any particular time undergoing the transient period of morphological changes that define apoptosis is smaller than if the apoptotic response were temporally more uniform and synchronous. We have performed many experiments in which the time courses and concentrations of 7kchol were varied, and we can make the following generalizations: (i) the fraction of cells displaying apoptotic characteristics is maximal at ca. 15 μg of 7kchol/ml and after ca. 18 h, and (ii) the percentage of apoptotic cells at this time and concentration typically ranged from 10 to 20% for the mouse cells and from 5 to 15% for the human cells. Our results are in general agreement with those of other studies of the level of apoptotic responses to 7kchol in U937 and smooth muscle cells (50, 61) and to a variety of agents in fibroblasts (9, 24).

Because of the profound effects of p21waf1 on cell cycle progression, we tested whether differences in distribution among control, Stat1−/−, and p21waf1−/− cells among the cell cycle phases, rather than decreased p21waf1, determined susceptibility to apoptosis. Specifically, we sought to determine whether cell cycle arrest, rather than another action of p21waf1, was responsible for facilitating apoptosis in oxysterol-exposed cells. In two separate experiments, we quantified the distribution of wild-type, Stat1−/−, and p21waf1−/− MEF among cell cycle phases by fluorescence-activated cell sorting analysis at the time cultures were exposed to 7kchol. The distribution of cells at the time of addition of 7kchol did not differ markedly among the cell types (wild-type cells: G0/G1 [63.7%], S [29.2%], and G2/M [7.3%]; Stat1−/− cells: G0/G1 [58.0%], S [28.6%], and G2/M [13.4%]; p21waf1−/− cells: G0/G1 [69.6%], S [28.0%], and G2/M [2.3%]; these values are all averages of results from two experiments). However, apoptosis after 7kchol was analogous to that shown in Fig. Fig.1A1A and andBB (data not shown). In addition, the application of etoposide (24 h, 10 μM) to arrest cells in G1 increased the proportions of cells in G0/G1 to 81.3% for the wild type, 82.9% for Stat1−/−, and 75.1% for p21waf1−/−. Despite the immobilization of cells in G1 and the uniformity of cell cycle phase among these three populations of MEF, apoptosis induced by 7kchol was significant in etoposide-treated wild-type cells (11.9%) and dramatically less in etoposide-treated Stat1−/− cells (2.4%) and p21waf1−/− cells (1.4%), again a finding qualitatively analogous to that shown in Fig. Fig.1A1A and andB.B. These data suggest that differences in cell cycle phase distribution, either in asynchronous cultures or in cell cycle-blocked cultures, did not correlate with the differences in vulnerability to apoptosis among the three cell populations. Moreover, observed differences in apoptosis sensitivity were not due to a preference of the oxysterol to kill arrested cells.

Etoposide is known to induce p21waf1 (25). Western analysis in the latter experiment confirmed this with the wild-type MEF (data not shown). Interestingly, etoposide raised the basal level of apoptosis in the absence of 7kchol (4.3% in etoposide-treated wild-type MEF compared to 0.3% in untreated wild-type MEF) and elevated the level of apoptosis after 7kchol over that observed with 7kchol in the absence of etoposide pretreatment (11.9% versus 6.5%, respectively).

p21waf1 expression is Stat1 dependent; restoration of p21waf1 in Stat1-deficient cells restores 7kchol-induced apoptosis.

We examined whether the effects of the deficiencies in Stat1 and p21waf1 were interrelated. Western analysis verified that Stat1 was missing in Stat1−/− MEF, that p21waf1 was missing in p21waf1−/− cells, and that Stat1 was present at comparable levels in p21waf1−/− and wild-type cells (Fig. (Fig.2A).2A). However, in Stat1−/− cells, the level of p21waf1 was found to be markedly reduced (Fig. (Fig.2A).2A). The cells lacking Stat1 and the control MEF were obtained from mice developed by Durbin and coworkers (22). To determine whether the absence of p21waf1 was specific to this line of Stat1 knockout mice, we obtained fibroblasts from the Stat1 knockout mice developed independently by Meraz and coworkers (58). The deficiency in apoptosis in response to 7kchol and the deficiency in expression of p21waf1 were virtually identical in cells from the two lines of Stat1 knockout mice (Fig. (Fig.2B).2B). In addition, we obtained primary cultures of wild-type and Stat1−/− MEF (prepared from mice obtained from Taconic Laboratories, which were derived from the Stat1−/− mice developed by R. D. Schreiber). These Stat1−/− primary cells also had markedly reduced p21waf1 levels as determined by Western analysis, indicating that the deficiency was not simply secondary to immortalization (Fig. (Fig.2C).2C). These data indicate that p21waf1 expression depends on Stat1 in cultured mouse fibroblasts.

FIG. 2.
p21waf1 deficiency in Stat1−/− cells. The data shown in panels A and B were from Stat1−/− mice developed in two independent laboratories. (A) p21waf1 is decreased in Stat1−/− MEF compared to wild-type cells; ...

To test further the requirement for p21waf1 for optimal apoptosis in response to 7kchol, we infected Stat1-deficient MEF (from D. E. Levy) with an Ad carrying p21waf1 cDNA or with a control virus. Transient reintroduction of p21waf1restored the apoptotic response of Stat1−/− cells to 7kchol (Fig. (Fig.2D).2D). Moderately enhanced expression of p21waf1 was demonstrated by Western analysis (data not shown). These data suggest that the principal defect in the apoptotic response of Stat1−/− cells to 7kchol is due to the level of p21waf1 expression and not to other Stat1-dependent deficiencies.

Stat1 and p21waf1 involvement in apoptosis is upstream of cytochrome c release and the activation of caspase 3.

Caspase 3 is involved in oxidized LDL- and oxysterol-mediated apoptosis in other cells (21). We sought to confirm the involvement of caspase 3 in 7kchol-induced apoptosis in mouse fibroblasts and to test whether the release of cytochrome c from mitochondria is also involved.

The role of caspase 3 was examined by using multiple approaches. First, mouse fibroblasts were pretreated with two irreversible inhibitors of caspase 3, DEVD-FMK (100 μM) and DEVD-CHO (150 μM), for 2 h. The cells were then treated with 7kchol for 18 h, and apoptosis was quantified with acridine orange-ethidium bromide staining. Both inhibitors significantly blocked 7kchol-induced apoptosis (Fig. (Fig.3A).3A). Wild-type MEF and MEF from Stat1−/− and p21waf1−/− mice were treated with 7kchol (15 μg/ml) for 15 h and incubated further for 30 min with a fluorescent substrate specific for caspase 3, DEVD-AMC, and the caspase 3 activity was measured. Whereas caspase 3 activity was increased threefold in response to 7kchol in cells from wild-type mice, Stat1- and p21waf1-deficient cells showed no detectable increase in caspase 3 activity (Fig. (Fig.3B).3B). Western analysis with an antibody to caspase 3 revealed that Stat1−/− and p21waf1−/− cells had approximately the same levels of caspase 3 protein as wild-type mouse embryo fibroblasts (Fig. (Fig.3C).3C). Our data show that caspase 3 activation is required for 7kchol-induced apoptosis and is downstream of Stat1 and p21waf1 function in mouse cells. In addition, we found that in mouse cells the presence of caspase 3 protein did not require Stat1 or p21waf1 but that caspase 3 activation in response to 7kchol did require p21waf1 and Stat1.

FIG.3.
7kchol-induced apoptosis involves cytochrome c release from mitochondria and activation of caspase 3. (A) Apoptosis induced by 7kchol in MEF was inhibited by caspase 3 inhibitors. Cells were pretreated for 2 h with DEVD-FMK (100 μM) and DEVD-CHO ...

To test whether the release of cytochrome c into the cytosol was required for apoptosis, cells were treated with 7kchol and Western analysis was performed with anti-cytochrome c. Cytochrome c was released into the cytosol in response to 7kchol in wild-type cells, but the release was less in p21waf1- and Stat1-deficient cells (Fig. (Fig.3D3D).

The roles of Stat1 and p21waf1 in 7kchol-induced apoptosis are similar in human and mouse cells.

Using the human fibrosarcoma HT1080 cells, we obtained evidence that 7kchol-induced apoptosis was also independent of p53 in human cells. p53 protein levels in HT1080 cells were not altered by 7kchol (data not shown). In addition, 7kchol did not induce activation of the p53 gene by a luciferase activity assay with a p53-responsive promoter (data not shown). These data were consistent with the lack of p53 involvement in 7kchol-induced apoptosis that we observed with mouse cells. 7kchol-induced apoptosis was also decreased in the human fibroblast cell line H07.2-1, which is deficient in p21waf1 (7) compared to control cells (Fig. (Fig.4A4A).

FIG. 4.
Deficiencies in p21waf1 and Stat1 in human cells is associated with impaired apoptosis after treatment with 7kchol. (A) Induction of apoptosis in LF-1 (control) human fibroblasts and p21-null H07.2-1 cells. Cells were incubated with 7kchol (15 μg/ml) ...

To test for the involvement of Stat1 in 7kchol-induced apoptosis in human cells, we used HT1080-derived 2fTGH cells, U3A cells (the Stat1-deficient mutant derived from 2fTGH), and U3AR cells (U3A cells into which wild-type Stat1 has been reintroduced by stable transfection) (42). 7kchol induced apoptosis in 2fTGH cells, and this response was significantly less in the Stat1-deficient U3A cells (Fig. (Fig.4B).4B). In contrast, apoptosis was restored toward control levels in U3AR cells.

Stat1 structural elements required for apoptosis.

To examine whether Stat1 was activated by 7kchol, we used an antibody that recognizes the phosphorylation site of Stat1 at Tyr701. Phosphorylation at this site was induced in 2fTGH cells treated with 7kchol (data not shown), suggesting that 7kchol activates Stat1. We then tested the apoptotic response to 7kchol in Stat1-deficient U3A cells stably transfected with different Stat1 mutant proteins (42). Surprisingly, cells expressing two mutations that block Stat1 activation in response to IFN-γ, i.e., U3A-701 and U3A-SH2 cells, remained permissive for 7kchol-induced apoptosis (Fig. (Fig.5).5). However, cells expressing the U3A-p84 or U3A-727 proteins, in which the response to IFN-γ is also blunted, were impaired in their apoptotic response to 7kchol. Since the apoptotic response to 7kchol was not impaired in cells expressing U3A-701 mutant protein, even though 7kchol may activate Stat1 by inducing the phosphorylation of Tyr701, this event is not required for apoptosis-related events downstream of Stat1. These data suggest that elements of Stat1 protein required for IFN-dependent Stat1 dimerization (80) are distinct from those that are required for 7kchol-induced apoptosis via the p21waf1- and caspase 3-dependent pathways.

FIG. 5.
Apoptosis in response to 7kchol is impaired in cells expressing Stat1 mutants with substituted Ser727 or a deleted C terminus. Apoptosis was quantified after 18 h treatment with 7kchol (15 μg/ml) in 2fTGH control cells, U3A cells, and U3A cells ...

7kchol-induced apoptosis involves the release and autocrine action of IFN-β.

Because 7kchol induced the tyrosine phosphorylation of Stat1, we also tested whether phosphorylation at Ser727 was induced by 7kchol. 7kchol enhanced Ser727 phosphorylation, although with a delayed time course (peak between 1 and 4 h) compared to that induced by IFN-γ (peak at 30 min [38]) (Fig. (Fig.6A).6A). This suggested the possibility of an IFN-dependent step. 2fTGH cells have been shown to synthesize IFN-β (54). A neutralizing antibody to IFN-β, added to the medium 2 h before 7kchol treatment, partially blocked the 7kchol-induced phosphorylation of Ser727 (Fig. (Fig.6A)6A) and partially blunted apoptosis (Fig. (Fig.6B).6B). The failure to block apoptosis completely may have been due to incomplete access of the antibody to IFN-β prior to the autocrine interaction of IFN-β with its receptor.

FIG. 6.
7kchol-induced apoptosis is dependent on an autocrine action of IFN-β. (A) Neutralizing antibody to IFN-β decreases Stat1 phosphorylation at Ser727. Cell extracts were prepared at the times indicated after treatment with 7kchol (15 μg/ml). ...

Using another approach, we exposed another mutant cell line derived from 2fTGH cells, U5A, to 7kchol. These cells lack the IFN receptor subunit, IFNAR-2 (54). 7kchol-induced apoptosis was reduced in U5A cells compared to that in 2fTGH cells (Fig. (Fig.6C).6C). However, IFN-β (10 μg/ml for 18 h) treatment of 2fTGH cells in the absence of 7kchol failed to induce apoptosis (Fig. (Fig.6B6B).

Western and Northern analyses revealed that the levels of p21waf1 protein and mRNA were reduced in U3A cells compared to 2fTGH cells (Fig. (Fig.7A7A and andB).B). The levels of p21waf1 mRNA and protein were also lower in the Stat1 mutant cell lines (U3A-SH2, U3A-p84, U3A-701, and U3A-727) than in 2fTGH cells (data not shown). However, in multiple experiments treatment with 7kchol failed to reveal a consistent pattern of change in p21waf1 mRNA or protein in these mutant cell lines that correlated with competence of these mutants in the apoptotic pathway shown in Fig. Fig.5.5. Nevertheless, apoptosis in response to 7kchol was restored to nearly the level of control 2fTGH cells in U3A cells infected with an Ad expressing p21waf1 but not in cells infected with control virus (Fig. (Fig.7C7C).

FIG. 7.
p21waf1 expression and apoptosis in response to 7kchol are decreased in Stat1-deficient U3A cells compared to 2fTGH control cells; enhanced p21waf1 expression restores apoptosis. (A) Western analysis for p21waf1 reveals decreased protein in Stat1-null ...

In agreement with our findings with mouse cells, cytochrome c release into the cytosol was detected in 2fTGH and U3AR cells, but not U3A cells, after 7kchol treatment (Fig. (Fig.8).8).

FIG. 8.
Cytochrome c release from mitochondria is impaired in Stat1-deficient U3A cells compared to control cells. Western analysis was done to evaluate the levels of cytochrome c and cytochrome c peroxidase in cytosolic fractions. Cytosolic extracts were prepared ...

DISCUSSION

Apoptotic cells populate atherosclerotic lesions (27, 36). Oxidized LDL and its constituent oxysterols, including 7kchol (5, 55), are prominent constituents of lesions, and 7kchol induces apoptosis in numerous cell types and species (53, 61). Understanding the mechanism by which the aberrant lipids formed during lipoprotein oxidation and accumulating in arterial lesions affect apoptosis could aid in the identification of the effector lipid(s) and the sequence of apoptotic events that pertain in vivo. This in turn may allow interference with apoptosis progression in order to evaluate whether the occurrence of apoptosis in vascular lesions is beneficial or detrimental and to manipulate the outcome accordingly (57). Our results progress toward that goal in that some requisite steps are defined in the apoptotic pathway induced by the predominant oxysterol in atherosclerotic lesions, 7kchol.

Apoptosis induced by 7kchol was independent of p53. Consistent with this observation, p53 deficiency did not alter apoptosis rates in the cells of atherosclerotic lesions in the apoE-deficient mouse model of atherosclerosis (30). Actions of Stat1 and p21waf1 appeared to be required for optimal apoptosis by this oxysterol in both mouse and human cells. We also observed retarded apoptosis in p21waf1- and Stat1-deficient MEF exposed to certain stimuli other than 7kchol, including etoposide, staurosporine, cycloheximide, and cycloheximide plus dexamethasone (unpublished data). Thus, this pathway has implications beyond the responses induced by oxysterols. We have also observed that still other apoptotic agents do not follow this pattern. Apoptosis in response to actinomycin D was not decreased in p21waf1−/− MEF compared to wild-type cells (unpublished data). We also confirmed the report by Kumar et al. (42) that apoptosis was not decreased in IFNAR-2-deficient (U5A) cells compared to control (2fTGH) cells after exposure to actinomycin D plus tumor necrosis factor alpha (TNF-α); this is distinct from the response of these cells to 7kchol (see Fig. Fig.6C6C).

Our data reveal an apoptosis pathway induced by 7kchol also requiring cytochrome c release and caspase 3 activation. This sequence is not surprising, given previous reports that CPP32 (caspase 3) inhibition and bcl-2 overexpression (which is known to block cytochrome c release) decreased 7kchol-induced apoptosis in other cell systems (33, 51, 62). However, our observations that fibroblasts lacking Stat1 express reduced p21waf1 protein and that the lack of either Stat1 or p21waf1 leads to markedly reduced apoptosis in response to 7kchol establish a connection between Stat1 and p21waf1 and also place cytochrome c release and the activation of caspase 3 downstream of the regulatory roles played by Stat1 and p21waf1. Optimal apoptosis appears to depend on both 7kchol and p21waf1; overexpression of p21waf1 via Ad did not induce apoptosis in the absence of 7kchol. We do not know the mechanism by which p21waf1 mediates the response to 7kchol that leads to cytochrome c release. Interestingly, a possible role of p21waf1 in facilitating cytochrome c release and caspase 3 activation in response to ursolic acid has been reported by Kim et al. (40) for HepG2 cells.

Although p21waf1 is not a transcription factor, it has profound regulatory influences on many proteins. It binds various proteins involved in cell cycle regulation to inhibit growth, including cyclin-dependent kinases and proliferating cell nuclear antigen. It is believed to be involved in mediating senescence (7, 10). Its roles in apoptosis have been reported to be both positive and negative (2, 29, 75); which of these is pertinent appears to depend on cell type and apoptotic stimulus. In our studies, p21waf1 played a clear role as an apoptotic mediator and was required for optimal apoptosis in response to 7kchol. That the pro- or antiapoptotic response is cell dependent and not solely stimulus dependent is suggested by comparing our results to those of Mahyar-Roemer and Roemer (56). When we treated MEF with etoposide, the level of p21waf1 increased (as predicted by a previous report [25]), as did apoptosis, in both untreated and 7kchol-treated cells. An apparently opposite influence of p21waf1 was observed by Mahyar-Roemer and Roemer (56) who, using colon cancer cells, found apoptosis elevated in response to etoposide in p21waf1-deficient cells compared to control cells.

The essential role for p21waf1 was clearly apparent in mouse and human cells in that, for both, cells deficient in p21waf1 showed significantly blunted apoptosis in response to 7kchol (Fig. (Fig.11 and and4).4). In cells from two distinct lines of Stat1−/− mice (22, 58), the level of p21waf1 was markedly reduced, yet it was readily apparent in the cells from wild-type mice. This result apparently does not apply to all cell types. Lee et al. (44) showed that in T-cell-enriched splenocytes p21waf1 levels were similar in Stat1−/− and wild-type mice. We do not believe the difference is attributable to cell transformation, since we also observed p21waf1 deficiency in primary MEF from Stat1−/− mice compared to those from wild-type mice. We do not know the nature of the role played by p21waf1 in apoptosis in response to 7kchol. It is not, however, an effect secondary to differences in cell cycle phase between Stat1−/−, p21waf1−/−, and wild-type cell populations during exposure to 7kchol, since cell cycle phase in asynchronously growing cells or in cells largely immobilized in G1 did not correlate with susceptibility to 7kchol-induced apoptosis.

The importance of Stat proteins in apoptosis regulation has been highlighted by recent findings with a variety of cell systems (11, 20, 44, 45, 59, 69, 72). IFN-γ- and TNF-α-induced apoptosis has been shown to be defective in Stat1-deficient U3A cells (13, 42). Kumar et al. reported decreased expression of various caspases in Stat1-deficient cells (42). Lee and colleagues reported lower basal caspase 3 in cells from Stat1−/− mice (44). Our Western analysis data show a deficit in caspase 9 basal expression in Stat1-deficient mouse cells (unpublished observations). Stat1 may thus influence the basal expression of multiple genes involved in apoptosis. Our results further the notion of the importance of Stat1 in certain apoptotic pathways and indicate that one aspect of the defect may be mediated by Stat1-dependent deficiencies in p21waf1. Recently, Stat1 influences on p21waf1 have been reported downstream of thrombin binding to its receptor, PAR-1, a role that mediated growth inhibitory and apoptotic responses to thrombin in CHRF cells (a human megakaryocyte tumor cell line) and murine fibroblasts (35). However, our results showing restoration of apoptosis in response to 7kchol in Stat1-deficient mouse and human cells by transient overexpression of p21waf1 suggest that the deficiencies we observe in apoptosis in the oxysterol-treated Stat1-null cells are restored by p21waf1 expression and are not due to a more direct influence of Stat1 on other apoptosis-related genes.

Specific mutations of the C-terminal portion of the transactivation domain of Stat1 are known to impair IFN-γ signaling; however, only some of these mutations also impaired the apoptotic response to 7kchol. We used the Stat1-deficient cell line U3A, into which four Stat1 mutants described earlier (42) had been stably introduced. U3A-701 cells expressed a mutant of Stat1 in which the Tyr701 residue is replaced with Phe701; U3A-SH2 cells expressed Stat1 with Arg602 replaced with Leu602, a change that disables the SH2 transactivation domain of Stat1 (34, 70). Each of these mutants blocks IFN-γ signaling and Stat1 dimerization due to either the prevention of Tyr701 phosphorylation or phosphotyrosine-SH2 interactions; however, both were permissive for 7kchol-induced apoptosis. In U3A-727 cells a Stat1 mutant is expressed in which Ser727 is replaced with Ala727. U3A-p84 expresses a Stat1 variant missing the 38 amino acids of the C-terminal transactivation domain, including Ser727. These mutations block certain IFN-γ-dependent signaling events (76). Apoptosis by 7kchol was significantly reduced in both U3A-727 and U3A-p84 cells, to nearly the same degree as in U3A cells. While interpretations from data using derivatized cells must be made with caution, our data are consistent with the idea that Ser727 phosphorylation, but not Tyr701 phosphorylation, is essential for an optimal apoptotic response to 7kchol. This is different from the results of Huang et al. (35), who showed an increased expression of p21waf1 induced by thrombin that was p53 independent and Stat1 dependent, but was associated with tyrosine phosphorylation and nuclear translocation of Stat1. It is consistent with two recent reports by Stephanou et al. (73) and Chatterjee-Kishore et al. (12) demonstrating signaling actions of Stat1, including those leading to apoptosis, that require Stat1 phosphorylation at Ser727 but not Tyr701.

A neutralizing antibody to IFN-β blunted both Ser727 phosphorylation and apoptosis in response to 7kchol, and U5A cells, which are deficient in an IFN receptor subunit (IFNAR-2) (54), exhibited decreased 7kchol-induced apoptosis. These data suggest an autocrine response to 7kchol mediated by IFN-β release. The incompleteness of the block by the antibody may be related to its inability to interact with released IFN-β faster than IFN-β found its receptor.

We hypothesize the following: 7kchol causes release of IFN-β by an unknown mechanism. IFN-β, in turn, induces phosphorylation of Stat1. The phosphorylation at Ser727, but not that at Tyr701, is required for optimal apoptosis via a pathway requiring p21waf1, cytochrome c release, and caspase 3 activation. It is important to emphasize that the role of 7kchol in apoptosis goes beyond mediating the release of IFN-β, since IFN-β alone did not induce apoptosis. The other role of 7kchol is unknown but likely occurs upstream of cytochrome c release. The nature of the influence of Stat1 on p21waf1 is not known. Activated Stat proteins have been shown to recognize Stat-responsive elements in the p21waf1 promoter (14). Stat1 may be required for appropriate basal expression of p21waf1, and/or Stat1 phosphorylated at Ser727 may indirectly alter the actions of p21waf1. The latter might involve altering the fate of p21waf1 in binding among its various protein-binding partners (10) or decreasing p21waf1 degradation (28).

Whether 7kchol is an effector of apoptosis in vivo and whether apoptosis in arterial lesions is beneficial or detrimental are unknowns. This study and recent ones showing roles for Stat proteins as apoptotic regulators may facilitate an experimental approach to resolving these questions. The collaborative roles of 7kchol and IFN implied above are particularly interesting in the context of lesion development. T cells and IFN are known components of lesions (32), and there is growing evidence for a role for IFN-γ in atherosclerosis (31, 77). Quantifying atherosclerotic lesion development in Stat1- or p21waf1-deficient mice, for example, could provide clues.

Acknowledgments

This work was supported in part from NIH grants P01 HL29582 (G.M.C.) and P01 CA62220 (G.R.S.).

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