Abstract
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Regulation of reactive oxygen species by p53: implications for nitric oxide-mediated apoptosis
Abstract
Nitric oxide (NO) induces vascular smooth muscle cell (VSMC) apoptosis in part through activation of p53. Traditionally, p53 has been thought of as the gatekeeper, determining if a cell should undergo arrest and repair or apoptosis following exposure to DNA-damaging agents, depending on the severity of the damage. However, our laboratory previously demonstrated that NO induces apoptosis to a much greater extent in p53−/− compared with p53+/+ VSMC. Increased reactive oxygen species (ROS) within VSMC has been shown to induce VSMC apoptosis, and recently it was found that the absence of, or lack of, functional p53 leads to increased ROS and oxidative stress within different cell types. This study investigated the differences in intracellular ROS levels between p53−/− and p53+/+ VSMC and examined if these differences were responsible for the increased susceptibility to NO-induced apoptosis observed in p53−/− VSMC. We found that p53 actually protects VSMC from NO-induced apoptosis by increasing antioxidant protein expression [i.e., peroxiredoxin-3 (PRx-3)], thereby reducing ROS levels and cellular oxidative stress. We also observed that the NO-induced apoptosis in p53−/− VSMC was largely abrogated by pretreatment with catalase. Furthermore, when the antioxidant protein PRx-3 and its specific electron acceptor thioredoxin-2 were silenced within p53+/+ VSMC with small-interfering RNA, not only did these cells exhibit greater ROS production, but they also exhibited increased NO-induced apoptosis similar to that observed in p53−/− VSMC. These findings suggest that ROS mediate NO-induced VSMC apoptosis and that p53 protects VSMC from NO-induced apoptosis by decreasing intracellular ROS. This research demonstrates that p53 has antioxidant functions in stressed cells and also suggests that p53 has antiapoptotic properties.
nitric oxide (NO) is known to have many beneficial effects in the vasculature. NO stimulates vascular smooth muscle cell (VSMC) relaxation, which leads to vessel vasodilatation (32). NO stimulates endothelial cell proliferation and prevents endothelial cell apoptosis, whereas it inhibits VSMC growth and migration and stimulates VSMC apoptosis (15, 21, 33, 45, 47). NO also inhibits platelet aggregation, adhesion, and activation; leukocyte adhesion and migration; and matrix formation (23, 24, 39). Favorable properties of NO on the vasculature are most apparent when considering pathologies that exist with impaired NO bioavailability. Examples of such disease include hypercholesterolemia, diabetes, hypertension, atherosclerosis, postangioplasty restenosis, and vein graft intimal hyperplasia (13, 38). Many of these disease states stem from endothelial dysfunction, and, in fact, the term “endothelial dysfunction” has now become synonymous with reduced biological activity of NO (46). Numerous experimental treatment options for these diseases aim at restoring NO bioavailability in the vasculature. For example, a large focus of our laboratory is the use of local NO therapy to prevent the neointimal hyperplasia and restenosis that follows many vascular interventions (1, 19, 36). One mechanism by which NO is able to inhibit the development of neointimal hyperplasia is via promoting VSMC apoptosis. However, the precise mechanism by which NO induces VSMC apoptosis has yet to be fully elucidated.
p53 has traditionally been thought of as the gatekeeper to survival or death, determining if a cell should undergo arrest and repair following DNA damage or if the cell should undergo apoptosis if the damage is severe. Prior studies have reported that NO increases p53 expression and stabilization in VSMC and that NO induces VSMC apoptosis in association with increased p53 expression (18). Thus it has been assumed that NO induces apoptosis in a p53-dependent manner. However, previous work in our laboratory sought to identify the mechanism by which NO induces VSMC apoptosis. We reported that p53−/− VSMC were much more sensitive to the proapoptotic effects of NO than were p53+/+ VSMC (21, 22). It was concluded that p53 actually protects VSMC against NO-mediated apoptosis, in part, through differential regulation of the mitogen-activated protein kinase (MAPK) pathways (21) and, more recently, through regulation of heme oxygenase-2 (HO-2) (22). These findings challenge the commonly accepted paradigm of p53 having primarily proapoptotic functions in cells.
Recently, Sablina et al. (41) suggested another role for p53 by demonstrating that cells with deficient or silenced p53 have increased reactive oxygen species (ROS) levels at baseline, physiological states (41). This finding is attractive since it is known that, within the vasculature, increased ROS and therefore oxidative stress has been shown to induce VSMC apoptosis (2, 28). Furthermore, it is known that NO can induce oxidative stress via inhibition of complexes I and IV of the mitochondrial electron transport chain, which leads to the leaking of electrons to molecular oxygen and the formation of the superoxide anion (O2−).(6) Once O2− is formed, it serves as the precursor to the majority of the other ROS. Because of our novel previous finding and the recent discovery that the absence of p53 leads to increased ROS, the aim of this study is to further illustrate the mechanism by which p53 protects VSMC and, conversely, how the absence of p53 renders VSMC more susceptible to NO-induced apoptosis. Our hypothesis is that p53 protects VSMC from NO-mediated apoptosis through the regulation of pro- and antioxidant proteins, and hence ROS generation. To examine this hypothesis, we focused on determining if differences in ROS levels existed between p53−/− and p53+/+ VSMC at baseline and after NO treatment. We aimed to explore differences in ROS levels by evaluating differences in antioxidant protein expression between p53−/− and p53+/+ VSMC. Finally, we sought to determine if differences in ROS production and antioxidant protein expression lead to the increased susceptibility to NO-induced apoptosis that is seen in p53−/− VSMC.
EXPERIMENTAL PROCEDURES
Chemicals and reagents.
Z-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1, 2-diolate (DETA/NO) was supplied by Dr. Larry Keefer (National Cancer Institute). S-nitroso-N-acetylpenicillamine (SNAP) was synthesized in our laboratory from the parent compound N-acetylpenicillamine. N-acetylcysteine (NAC), catalase, diphenyleneiodonium chloride (DPI), and vitamin C (Vit C) were purchased from Sigma-Aldrich (St. Louis, MO). Reduced l-glutathione (GSH) was purchased from Cayman Chemical (Ann Arbor, MI). Antibodies to superoxide dismutase (SOD)-1 and SOD-2 as well as small-interfering ribonucleic acid (siRNA) to peroxiredoxin-3 (PRx-3) and thioredoxin-2 (TRx-2) were purchased from Santa Cruz Technologies (Santa Cruz, CA). Antibodies to glutathione peroxidase-1 (GPx-1), TRx-1, TRx-2, PRx-1, PRx-2, PRx-3, and catalase were purchased from Abcam (Cambridge, MA). Antibody to β-smooth muscle cell (SMC)-actin was purchased from Dako (Carpinteria, CA). Antibodies to goat anti-rabbit and goat anti-mouse were purchased from Pierce Biotechnologies (Rockford, IL). The detection reagent CM-H2DCFDA was purchased from Invitrogen/Molecular Probes (Eugene, OR), and dihydroethidine (DHE) was purchased from Polysciences (Warrington, PA).
Cell culture.
Approval from the Northwestern University Institutional Animal Care and Use Committee was obtained for the use of animals in this project. Abdominal aortic VSMC were isolated and cultured from C57BL/6J (p53+/+) and B6.129S2-Trp53tm1Tyj/J (p53−/−) mouse strains using the collagenase method described by Ray et al. (40). Both strains of mice were purchased from The Jackson Laboratory (Bar Harbor, ME). We independently confirmed the lack of p53 expression in the p53−/− VSMC by Western blot analysis. Cultured VSMC had the characteristic hills-and-valleys appearance and were routinely >95% pure by α-SMC-actin staining. Cells were maintained in media containing equal volumes of DMEM-low glucose (SAFC Biosciences, Lenexa, KS) and Ham's F-12 (JRH, Lenexa, KS) supplemented with 10% FBS (Invitrogen; Carlsbad, CA), 100 U/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), and 4 mmol/l l-glutamine (VWR, West Chester, PA) and incubated at 37°C and 95% air-5% CO2. Cells used for experiments were between passages 4–9.
Western blot analysis.
Aortic VSMC were plated on 10-cm dishes and allowed to grow to 60–80% confluence. They were then exposed to media containing SNAP or DETA/NO (1 mM) for 24 h. SNAP was used in these experiments as an additional source of NO. The cells were then rinsed two times with ice-cold PBS and collected by scraping and then resuspended in buffer A [20 mM Tris with 1 mM phenylmethylsulfonyl fluoride (Sigma), 1 μg/ml leupeptin (Sigma), and 1 mM sodium orthovanadate (Sigma)]. Protein concentration was quantified with the bicinchoninic acid protein assay (Pierce). Samples (10–20 μg protein) were subjected to SDS-PAGE on 8 or 13% gels and transferred to nitrocellulose membranes (Pierce Biotechnology, Chicago, IL). Before all primary antibody incubation, the membranes were blocked with 5% milk overnight at 4°C. The membranes were rinsed three times with PBS-Tween 20 (PBST) for 5 min each just before addition of the primary antibody. Each primary antibody was added using the suggested concentrations by the manufacturer and incubated at room temperature for 1.5–2 h. The membranes were then rinsed with PBST before adding the appropriate secondary antibody at a concentration suggested by the manufacturer for 30–45 min. Protein expression was determined using chemoluminescence with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and imaged with the Kodak Image Station in vivo FX (Eastman Kodak, Rochester, NY). Densitometry was performed on all Western blots and normalized to protein loading.
ROS detection.
For intracellular ROS quantification, the detection reagents CM-H2DCFDA (detects H2O2, peroxynitrite, peroxyl radical, hydroxyl radical) and DHE (detects O2−) were used. CM-H2DCFDA enters into cells where it is then enzymatically deacetylated by intracellular esterases to dichlorofluorescein (DCFH). DCFH remains in the cytosol where it is oxidized to the fluorescent compound DCF by the ROS listed above. DHE enters cells where it is oxidized preferentially by O2− and then becomes intercalated into DNA.
Aortic VSMC were plated in six-well plates until they reached 60–80% confluence, after which they were exposed to media containing the NO donor DETA/NO (1 mM) for 24 h. Cells were rinsed with ice-cold PBS, trypsinized, collected, and pelleted. Cells were resuspended in either 5 μM CM-H2DCFDA or 5 μM DHE and incubated at 37°C for 30 or 5 min, respectively. After incubation, the cell samples (10,000 cells/sample) were analyzed on a Coulter Epics XLFlow Cytometer (Beckman Coulter, Fullerton, CA) using the fluorescence detector for fluorescein isothiocyanate or FL1 for CM-H2DCFDA and the red fluorescence channel FL3 for DHE.
Cell death and apoptosis.
Aortic VSMC were plated in six-well plates and allowed to reach 60–80% confluence. The cells were then exposed to media containing the NO donor DETA/NO (1 mM) for 24 h with or without preincubation with different exogenous antioxidants (NAC, GSH, Vit C, catalase). To measure cell death, the Guava ViaCount assay (Guava Technologies, Hayward, CA) was used according to the manufacturer's instructions. Briefly, media with all detached cells were collected. The plate was then rinsed with ice-cold PBS, and the cells were then trypsinized, collected, and pelleted. Cells were resuspended in 250 μl of PBS, and 40 μl of this suspension were added to 160 μl of Guava ViaCount reagent. Cell death was then quantified using the Guava PCA machine (Guava Technologies). The Guava ViaCount assay distinguishes between viable and nonviable cells based on the differential cell membrane permeability of DNA-binding dyes in the ViaCount Reagent.
Apoptosis was assessed using the Guava MultiCaspase Detection Kit (Guava Technologies). Cells were plated, treated, and collected in the same manner as described above for cell death. The samples were then prepared using the reagents from the MultiCaspase Detection Kit according to the manufacturer's instructions. In brief, 100 μl of resuspended cells were transferred to 2 ml Eppendorf tubes. Sulforhodamine-valyl-alanyl-asparyl-fluormethylketone (5 μl; binds to activated caspases) was added to each tube and mixed. Samples were then incubated for 1 h at 37°C and 95% air-5% CO2 and protected from light. Samples were then washed three times with 1 ml Apoptosis Washing Buffer. 7-Amino-actinomycin D reagent (5 μl) (excluded from live healthy cells, but permeates late-stage apoptotic and dead cells) was then added to each sample and incubated at room temperature for 10 min at which time samples were ready for assessment using the Guava PCA machine. This assay detects cells that have died or are dying via apoptosis via binding to specific activated caspases involved in the intrinsic (caspase 9) and extrinsic (caspase 8 and 10) pathways and the final common pathways (caspase 3 and 7) that are activated when cells undergo apoptosis. This assay also distinguishes between cells that are undergoing cell death via necrosis by utilizing a membrane-impermeable dye that is excluded from live healthy cells and cells undergoing apoptosis that have intact cell membranes.
siRNA transfection.
The protocol provided by Santa Cruz Technologies was used for VSMC transfection with siRNA to PRx-3 and TRx-2. Briefly, cells were plated in six-well plates in antibiotic-free media and allowed to grow until 50–60% confluence. The respective siRNAs were introduced into p53+/+ VSMC cells by using the Transfection Reagent provided (Santa Cruz) in 1 ml of medium containing no FBS or antibiotics at 37°C and 95% air-5% CO2. After 5–7 h of incubation, media containing 20% FBS and two times the normal amount of antibiotics were added to each well and allowed to incubate for 18–24 h. Adequacy of transfection was confirmed by Western blot analysis in the transfected VSMC.
Statistical analysis.
Results are expressed as means ± SE. Differences between multiple groups were analyzed using one-way ANOVA with the Student-Newman-Keuls post hoc test for all pairwise comparisons (SigmaStat; SPSS, Chicago, IL). Statistical significance was assumed when P < 0.05.
RESULTS
p53−/− VSMC experience heightened NO-induced cell death via apoptosis compared with p53+/+ VSMC.
Both p53−/− and p53+/+ VSMC were treated with 1 mM of the NO donor DETA/NO for 24 h. As can be seen on phase contrast imaging, p53−/− VSMC underwent appreciably more cell detachment upon exposure to DETA/NO than did matched p53+/+ VSMC (Fig. 1). To determine if the observed cell detachment represented cell death, the cells were analyzed using specific assays for cell death and apoptosis. Quantification of these findings revealed that, following exposure to DETA/NO (1 mM) for 24 h, p53−/− VSMC experienced significantly more cell death compared with p53+/+ VSMC using the Guava ViaCount assay (23.8 vs. 4.08%, P < 0.001) (Fig. 1E). The observed cell death was confirmed to be death via apoptosis and not necrosis with the Guava MultiCaspase Detection Kit (21.1% p53−/− DETA/NO vs. 2.2% p53+/+ DETA/NO, P < 0.05) (Fig. 2).
Phase contrast microscopy images showing the difference in cell death between p53−/− and p53+/+ vascular smooth muscle cells (VSMC) after 24 h of treatment with Z-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1, 2-diolate (DETA/NO, 1 mM). p53+/+ control (A), p53+/+ DETA/NO (B), p53−/− control (C), p53−/− DETA/NO (D), and p53−/− VSMC (E) experience heightened nitric oxide (NO)-induced cell death after exposure to DETA/NO (1 mM) for 24 h as measured utilizing Guava ViaCount. *P < 0.01 compared with wild-type (WT) DETA/NO. WT, p53+/+ VSMC; knockout (KO), p53−/− VSMC. Data are representative of at least 3 separate experiments.
Differences in apoptosis between p53−/− and p53+/+ VSMC. p53−/− VSMC experienced heightened apoptosis compared with p53+/+ VSMC. *P < 0.05 compared with WT DETA/NO. Data are representative of at least 3 separate experiments.
p53−/− VSMC have increased levels of ROS at baseline and following exposure to NO compared with p53+/+ VSMC.
Differences in ROS levels were examined at baseline and following different time points after exposure to the NO donor DETA/NO using the detection reagents CM-H2DCFDA and DHE. Consistently, CM-H2DCFDA mean fluorescence levels were ~2.7-fold higher in p53−/− compared with p53+/+ VSMC (P = 0.017) at baseline (Fig. 3A). Similarly, DHE mean fluorescence was consistently 1.4-fold higher in p53−/− compared with p53+/+ VSMC (P < 0.01) at baseline (Fig. 3B). Following exposure to the NO donor DETA/NO (1 mM), CM-H2DCFDA fluorescence increased 8.4-fold higher in the p53−/− compared with p53+/+ VSMC at 24 h (P < 0.001) (Fig. 3A). A smaller 3.2-fold (P = not significant) and 5.8-fold (P = 0.003) increase was noted at 3 and 6 h, respectively. After 24 h of exposure to the NO donor DETA/NO, DHE fluorescence was 1.9-fold higher in the p53−/− compared with p53+/+ VSMC (P < 0.001) (Fig. 3A). There was no increase in DHE fluorescence at the earlier 3- and 6-h time points.
Reactive oxygen species (ROS) levels in p53−/− vs. p53+/+ VSMC at baseline and after NO exposure. A: baseline CM-H2DCFDA (DCF) fluorescence was 2.7-fold higher in p53−/− VSMC vs. p53+/+ VSMC. τP = 0.017. NO-induced CM-H2DCFDA fluorescence was 3.2-, 5.8-, and 8.4-fold higher in p53−/− VSMC vs. p53+/+ VSMC. *P = 0.003 and **P < 0.001 vs. KO control. B: baseline mean dihydroethidine (DHE) fluorescence was ~1.4-fold higher in p53−/− VSMC vs. p53+/+ VSMC. *P < 0.01. NO-induced DHE fluorescence was 1.9-fold higher in p53−/− VSMC vs. p53+/+ VSMC at 24 h. *P < 0.01 vs. WT control. **P < 0.001 vs. KO control. Data are representative of at least 3 separate experiments.
p53−/− VSMC have decreased expression of antioxidant proteins compared with wild-type (WT) p53+/+ VSMC.
The cytoplasmic antioxidant proteins analyzed were SOD-1, GPx-1, PRx-1, PRx-2, and TRx-1. The mitochondrial antioxidant proteins analyzed were SOD-2, PRx-3, and TRx-2. Of the cytoplasmic antioxidant proteins, there was no difference in expression of the antioxidant protein GPx-1 (Fig. 4A). There was decreased expression at baseline of the antioxidant proteins PRx-1, PRx-2, TRx-1, and SOD-1 within the p53−/− VSMC. However, after 24 h of exposure to the NO donor DETA/NO or SNAP (1 mM), there were no observed differences in expression in any cytoplasmic antioxidant protein between the p53+/+ and p53−/− VSMC (Fig. 4A).
Western blot analysis of antioxidant proteins in p53+/+ and p53−/− VSMC. A: cytoplasmic antioxidant protein expression at baseline and after 24 h of NO exposure. B: mitochondrial antioxidant protein at baseline and after 24 h of NO exposure. Data are representative of 3 separate experiments. GPx, glutathione peroxidase; PRx, peroxiredoxin; TRx, thioredoxin; SOD, superoxide dismutase.
Among the mitochondrial antioxidant proteins, there was decreased expression at baseline of the antioxidant proteins PRx-3, TRx-2, and SOD-2 within the p53−/− VSMC. After exposure to the NO donor DETA/NO or SNAP (1 mM), only PRx-3 continued to have decreased expression within the p53−/− VSMC (Fig. 4B). We did not observe any changes in the expression of these proteins after only 3 and 6 h of NO exposure (data not shown).
Pretreatment with exogenous antioxidants decreases baseline and NO-induced ROS production, but only the exogenous addition of catalase protected p53−/− VSMC from NO-induced apoptosis.
To evaluate if the observed increased levels of ROS in p53−/− VSMC were responsible for their increased NO-induced apoptosis, p53−/− VSMC were pretreated with several exogenous antioxidants before exposure to NO. Although treatment with the antioxidants NAC, GSH, SOD, and the flavoprotein inhibitor DPI were able to decrease CM-H2DCFDA fluorescence levels at baseline and after NO exposure (Fig. 5), they did not decrease the amount of cell death induced by NO in the p53−/− VSMC (data not shown). Only pretreatment with catalase (500 and 1,000 units), which dismutates H2O2, before exposure to DETA/NO resulted in a 48% (P < 0.001) and 67% (P < 0.001) reduction in VSMC apoptosis (Fig. 5C). These data suggest that ROS, in particular H2O2, is responsible for NO-induced apoptosis.
Reduction of ROS levels and apoptosis in p53−/− VSMC after pretreatment with antioxidants. A: reduction in baseline CM-H2DCFDA fluorescence with the pretreatment with SOD, diphenyleneiodonium chloride (DPI), N-acetylcysteine (NAC), l-glutathione (Glut), and catalase (Cat) [time (t) = 3 h]. *P < 0.006 vs. KO control. B: reduction in NO-induced CM-H2DCFDA fluorescence (t = 24 h). *P < 0.005 vs. KO DETA/NO. C: reduction in NO-induced apoptosis following pretreatment with catalase in p53−/− VSMC (t = 24 h). *P < 0.001 vs. KO control. **P < 0.001 vs. KO DETA/NO. Data are representative of 3 separate experiments.
Transfection of p53+/+ VSMC with siRNA to PRx-3 and TRx-2 leads to increased ROS production and increased cell death similar to that of p53−/− VSMC.
The fact that pretreatment with the antioxidant catalase reduced NO-induced apoptosis in p53−/− VSMC suggests that H2O2 is the main ROS responsible for the observed finding. Because p53−/− VSMC exhibit decreased expression of the mitochondrial antioxidant enzyme PRx-3, and the mitochondria are the known location of NO-induced ROS production, we sought to knock down the expression of PRx-3 and its specific electron acceptor TRx-2 in p53+/+ VSMC using siRNA to determine if they would exhibit increased ROS and become more susceptible to NO-induced apoptosis, similar to the p53−/− VSMC. Although there was no change in baseline mean fluorescence, when p53+/+ VSMC were transfected with siRNA to PRx-3 and TRx-2 and then exposed to the NO donor DETA/NO, CM-H2DCFDA fluorescence increased ~2.5-fold more than p53+/+ VSMC alone (P < 0.05) (Fig. 6A). Moreover, after transfecting p53+/+ VSMC with siRNA to PRx-3 and TRx-2 and subsequently exposing them to DETA/NO for 24 h, they experienced increased cell death (6.96 vs. 26.54%, P < 0.001) compared with p53+/+ VSMC alone (Fig. 6B). Transfection of p53+/+ VSMC with TRx-2 and PRx-3 individually was performed, and this showed no change in mean fluorescence or cell death before or after exposure to DETA/NO (data not shown). These data indicate that PRx-3 and TRx-2 are necessary in the fight against oxidative stress in VSMC.
Transfection of p53+/+ VSMC with small-interfering RNA (siRNA) to PRx-3 and TRx-2. A: transfected p53+/+ VSMC showed a 2.5-fold increase in NO-induced CM-H2DCFDA fluorescence compared with p53+/+ alone. *P < 0.05. B: transfected p53+/+ VSMC showed a 3.8-fold increase in NO-induced cell death compared with p53+/+ VSMC alone. *P < 0.001. Data are representative of 3 separate experiments.
DISCUSSION
As in our previous work, this study further confirms that p53 protects VSMC from NO-induced apoptosis, since VSMC harvested from p53−/− mice underwent increased apoptosis when exposed to NO compared with p53+/+ VSMC (21, 22). In the previous studies, VSMC apoptosis was assessed by evaluating the characteristic nuclear changes observed with apoptosis, PARP cleavage, lactate dehydrogenase release, fluorescence-activated cell sorter analysis with propidium iodide staining, and reversal of apoptosis with caspase inhibitors (21, 22). This finding of increased NO-induced cell death in p53−/− VSMC is novel in that it suggests that p53 has antiapoptotic properties in VSMC, which is contrary to the commonly accepted model for p53.
Herein we find that the susceptibility of p53−/− VSMC to NO-induced apoptosis is related to their decreased expression of certain antioxidant proteins and subsequently increased levels of oxidative stress at baseline and following NO exposure. Our findings suggest that p53−/− VSMC have increased ROS at baseline secondary to the decreased expression of several cytoplasmic and mitochondrial antioxidant proteins. After NO exposure, however, our data imply that the mitochondria are the main source of the increased ROS production (specifically H2O2) leading to NO-induced apoptosis, since only the expression of the mitochondrial specific antioxidant protein PRx-3 was decreased in p53−/− VSMC at baseline and after NO exposure. Furthermore, when PRx-3 and its specific electron acceptor TRx-2 were silenced in the p53+/+ VSMC, these cells behaved similarly to p53−/− VSMC upon NO exposure and experienced increased NO-induced ROS generation and NO-induced apoptosis. Last, only the antioxidant catalase, which dismutates H2O2, reduced ROS levels and prevented NO-induced cell death. Thus we theorize that, following NO exposure in the p53−/− VSMC, the combined increase in SOD-2 expression with the decreased PRx-3 expression results in increased H2O2 buildup in the cell, leading to a greater ROS burden and more cell death. Rephrasing this logic, SOD is known to convert superoxide to H2O2. Thus increasing SOD would decrease superoxide but increase H2O2. NO increases superoxide at the mitochondrial level, yet superoxide cannot cross the mitochondrial membrane. Superoxide is converted to H2O2 by SOD-2 in the mitochondria, but because of the lack of PRx-3 (and TRx-2) the H2O2, which can cross the mitochondrial membrane, cannot be converted to water and oxygen and we presume this buildup of H2O2 is what leads to an increase in apoptosis in our model.
Apoptosis of VSMC is known to play an important role in the pathology of atherosclerosis, as well as to limit the hyperplastic response after vascular injury. NO is an interesting molecule in that it has both pro- and antiapoptotic properties depending on the concentration, duration of exposure, and cell type exposed (11). For example, NO has been shown to have proapoptotic effects on macrophages, pancreatic islet cells, neuronal cells, certain tumor cells, and VSMCs, whereas it has antiapoptotic effects on hepatocytes, human B lymphocytes, endothelial cells, splenocytes, eosinophils, and PC12 cells (6, 10, 11). In regard to VSMC, NO has been shown to primarily have an antiproliferative and proapoptotic effect (14, 43). Although many studies have detailed the mechanism for NO-induced VSMC cell cycle arrest (43), the mechanism(s) by which NO mediates VSMC apoptosis has not been fully elucidated. It should also be noted that the concentrations of the NO donors used in this study are supraphysiological. However, in our prior report, we demonstrated that this phenomena occurs at much lower concentrations of NO, just over longer periods of time (21).
As the central regulator of apoptosis in all cell types, p53 itself has been implicated in NO-induced apoptosis. A study by Li et al. (25, 26) exposed WT p53+/+ TK6 cells or mutant p53−/− WTK-1 cells to different concentrations of NO. Whereas the cells with WT p53 experienced apoptosis largely through the mitochondrial pathway, the WTK-1 cells, despite extensive DNA damage, transcriptional activation of early genes, and release of apoptogenic factors, did not exhibit significant caspase activation or PARP cleavage (25, 26). In another study, thymocytes from p53-null mice were resistant or less sensitive to NO-induced apoptosis compared with their WT counterparts (16). Although it does seem reasonable to conclude that p53 is responsible for mediating NO-induced apoptosis in many cell types, as seen by our current and past research, p53 is not required for NO-induced VSMC apoptosis.
Past research has pointed to several different proposed mechanisms for NO-induced apoptosis. Both mitochondrial and nonmitochondrial apoptotic pathways have been shown to be activated during NO-induced apoptosis, although the mitochondrial pathway is favored as the principal mechanism (5). One such proposed mechanism for NO-induced mitochondrial apoptosis is that NO inhibits mitochondrial respiration, which can lead to increased intracellular ROS and contribute to membrane potential reduction and subsequent transition pore opening (3, 7). By permeabilizing the inner and outer mitochondrial membranes, proapoptotic factors are released in the cytosol (such as cytochrome c), which eventually leads to caspase 9 activation and apoptotic death (31).
Other apoptotic pathways have been implicated as well. Prior research in our laboratory and research by others has shown that NO can activate various members of the MAPK pathway such as c-Jun NH2-terminal kinase, p38 kinase, and p42/44, and their activation can lead to apoptosis in VSMC (21, 42). However, as pointed out in a review by Brune (5), the signaling networks involved in the MAPK pathway make it very difficult to predict causation and unequivocally position distinct kinases or phosphoproteins in the apoptotic cascade. More recently, Kim et al. (22) demonstrated a role for HO-2 in the protection of VSMC by p53 from NO-induced apoptosis. In this study, it was reported that p53−/− VSMC had little to no expression of HO-2 compared with p53+/+ VSMC. It was speculated that the iron and carbon monoxide produced by the catalytic activity of HO-2 on heme conferred protection from NO-induced apoptosis. Furthermore, when HO-2 was inhibited in p53+/+ VSMC, their resistance to NO-induced apoptosis was attenuated (22).
The fact that NO can induce apoptosis in a p53-independent manner is not itself novel (17). In fact, a study by Kawahara et al. (20) exposed MG5 microglial cells from p53-deficient mice to stimuli that induced endogenous NO production and to exogenous NO donors and found that these cells began to die via apoptosis. They found that the apoptosis was mediated through endoplasmic reticulum stress. The difference between this study and our current findings is that, although their cells underwent p53-independent cell death, the WT microglial cells did not experience protection from NO-induced apoptosis, suggesting involvement of other pathways.
A review of the literature for antiapoptotic effects of p53 identified a study by Mercer et al. (30) which found that endogenous levels of p53 protected VSMC from apoptosis. They exposed VSMC harvested from the aortas of p53−/− mice to different proapoptotic stimuli such as 0% FCS, etoposide, and ultraviolet irradiation. They found that, contrary to their expectations, the p53−/− VSMC experienced significantly more apoptosis compared with p53+/+ VSMC. They found that the p53−/− VSMC had increased activity of specific DNA damage response enzymes (ATM/ATR and Chk-1) and that reintroduction of p53 into those cells inhibited the activity of those enzymes and as well inhibited DNA damage-induced apoptosis. Their proposed mechanism of apoptosis in these p53−/− VSMC was mediated through DNA damage. A difference between Mercer's proposed mechanism and ours is that NO has been shown to be inefficient at generating DNA damage, and it is reasonable to conclude that NO is mediating apoptosis through another pathway.
In this study, we propose that NO induces apoptosis in p53−/− VSMC via increased oxidative stress. It has been well established that increased oxidative stress mediated by ROS can cause various cell types to undergo apoptosis, including VSMC (12, 14, 27, 28). In fact, one of the end results of p53 activation is the formation of ROS that eventually leads to apoptosis (37). It has also been well established that NO can cause increased cellular ROS by inhibiting mitochondrial respiration as well as through inhibiting several antioxidant proteins, thereby further increasing ROS (4). What was not clear is why the absence of p53 causes VSMC to be more susceptible to NO-induced apoptosis. A recent article by Sablina et al. (41) found that p53 itself has antioxidant properties because it is responsible for the transcription of several antioxidant proteins (41). Furthermore, it was found that cells with absent or nonfunctional p53 had increased ROS at baseline physiological states and were more susceptible to oxidative stress induced by H2O2. They concluded that this was because there was decreased expression of antioxidant proteins within these p53-deficient cells (41). We found a similar trend in our VSMC.
Of all the cytoplasmic and mitochondria antioxidant proteins that we evaluated, only the mitochondrial antioxidant protein PRx-3 exhibited decreased expression in p53−/− VSMC at baseline and after NO exposure. PRx-3 is a mitochondrial specific antioxidant that is thought to be one of the most important antioxidant enzymes in the mitochondria to dismutate H2O2 (8). As well, among all of the exogenous antioxidants and enzyme inhibitors we tested, only the H2O2 dismutating enzyme catalase reduced the amount of p53−/− VSMC undergoing NO-induced apoptosis. In addition, because NO is known to increase ROS at the level of the mitochondria, these findings implicate mitochondrial H2O2 as the main culprit in mediating NO-induced apoptosis, which is consistent with prior research (14). Further implicating the mitochondria as the source of NO-induced apoptosis is the fact that p53−/− VSMC exhibit decreased expression of the antiapoptotic protein Bcl-2 compared with p53+/+ VSMC before and after NO exposure (21).
Previous research has demonstrated the importance of PRx-3 in protecting cells from ROS-mediated apoptosis. Nonn et al. (35) overexpressed PRx-3 in WEHI7.2 thymoma cells and found that this overexpression led to decreased levels of cellular H2O2 and that it protected these cells from H2O2-induced apoptosis. Similarly, Mukhopadhyay et al. (34) studied PRx-3 in different cell lines in patients with Fanconi's Anemia (FA). They found that these cells, among other things, had a sevenfold decrease in PRx-3 expression compared with WT control cells. When PRx-3 was transiently overexpressed in the FA cells, they exhibited decreased sensitivity to H2O2 exposure (34). Finally, a study by Chang et al. (9) used RNAi to deplete PRx-3 levels in HeLa cells. They found that, not only did these cells have increased levels of H2O2, but they were also sensitized to apoptosis induced by staurosporine and tumor necrosis factor-α. These effects were reversed by ectopic expression of PRx-3 and with mitochondrial-targeted catalase (9). These studies lend support to our finding that the decrease in PRx-3 expression found in our p53−/− VSMC is responsible for their increased ROS generation and increased susceptibility to NO-induced apoptosis.
In summary, we demonstrate that NO induces VSMC apoptosis by increasing intracellular ROS. p53−/− VSMC undergo heightened NO-induced apoptosis secondary to the fact that they exhibit increased oxidative stress at baseline and after NO exposure compared with p53+/+ VSMC. This study suggests that, not only does p53 have antioxidant properties, but that it also has antiapoptotic functions in VSMC. Although these findings demonstrate a novel mechanism, one could speculate using this mechanism in clinical practice. Because the proapoptotic effect of NO is heightened in p53−/− cells, several therapeutic implications for its use can be considered. For example, ~50% of all cancers have mutated or deficient p53, and one could hypothesize utilizing NO as a chemotherapeutic agent in this setting. Several groups have already begun to examine the use of different NO-donating compounds to induce apoptosis in several different cancer cell lines in vitro and in vivo (29, 44).
GRANTS
This work was supported, in part, by funding from the National Heart, Lung, and Blood Institute contract no. 1K08HL0842-03 to M. R. Kibbe, the American Vascular Association (Mentored Clinical Scientist Development Award to M. R. Kibbe), and Lifeline Student Research Fellowship (N. B. Rossi), the American Heart Association (M. R. Kibbe), the Department of Veterans Affairs VA Merit Review Grant (M. R. Kibbe), the American Medical Association Foundation Seed Grant (2006 Seed Grant to O. O. Aalami; 2007 Seed Grant to D. A. Popowich), and the Northwestern University, Medical Student Summer Research Program (C. P. Walsh).
ACKNOWLEDGMENTS
We express thanks to the Northwestern University Institute for BioNanotechnology in Medicine, the Northwestern University Feinberg Cardiovascular Research Institute, and to Lynnette Dangerfield for administrative support.
REFERENCES
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Funding
Funders who supported this work.
NCRR NIH HHS (1)
Grant ID: UL1 RR025741
NHLBI NIH HHS (1)
Grant ID: 1K08HL0842-03