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1Department of Pharmacology, University of South Alabama College of Medicine, Mobile, Alabama 36688; and 2Department of Clinical Evaluation of Medicine and Therapeutics, Osaka University, Osaka 565-0871, Japan
Submitted 15 May 2003 ; accepted in final form 17 July 2003
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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NADPH oxidase; peroxynitrite; p53; Bax; Bcl-2; mitochondrial DNA; inducible nitric oxide synthase
One of the putative AT2-linked death cascades requires the inactivation of ERK MAPK by a tyrosine phosphatase (35, 42). The observed decrease in ERK activity is thought to reduce the phosphorylation state and activity of the antiapoptotic factor Bcl-2. In PC12W cells, the inhibition of nerve growth factor-mediated Bcl-2 phosphorylation by ANG II leads to the induction of apoptosis (15). However, in the same cell line, a tyrosine phosphatase also promotes ceramide synthesis, an event that is linked to apoptosis (20). A tyrosine phosphatase has also been shown to regulate the phosphatidylinositol 3-kinase/Akt survival pathway (5).
Another AT2-mediated death cascade proceeds through p38 MAPK. Miura and Karnik (28) have reported that, whereas AT2-mediated reductions in ERK activity do not alter the number of A7r5 cells that undergo apoptosis, the inhibition of p38 MAPK reduces apoptosis caused by the overstimulation of the AT2 receptor. A role for the proapoptotic factor Bax in ANG II-mediated apoptosis also deserves consideration because the AT2 receptor has been reported to upregulate Bax (15).
In both glomerular visceral epithelial cells and rat blood vessels, ANG II-mediated apoptosis involves both AT1 and AT2 receptors (6, 7). Although the mechanism by which both receptors contribute to apoptosis has not been established, ANG II-mediated apoptosis in the epithelial cell appears to involve the upregulation of Bax by both AT1 and AT2 receptors and a reduction in Bcl-2 content by the AT1 receptor (7). In contrast, AT1-mediated elevations in Bax appear to be more important in blood vessel apoptosis (6).
In the cardiomyocyte, the upregulation of Bax appears to be solely AT1 mediated (33). In 1998, Leri et al. (21) found that stretched myocytes undergo apoptosis in an AT1-dependent manner. Cell stretching was found to increase p53 and Bax content, leading to the suggestion that ANG II might activate p53, which in turn would upregulate Bax. However, in a subsequent study, Leri et al. (22) found that the activation of p53 was upstream from ANG II generation in the stretched myocyte. Therefore, it was suggested that ANG II might promote apoptosis by activating Ca2+-dependent DNase I, an effect dependent on the activation of PKC and an elevation in intracellular Ca2+ concentration (18, 22). AT1-mediated apoptosis in human coronary artery endothelial cells and lung alveolar epithelial cells also appears to require the activation of PKC (23, 29).
Therefore, some key steps in the signaling pathways for both AT1- and AT2-mediated apoptosis have been identified; however, the actual sequence of events leading to programmed cell death is poorly defined. The present study introduces a logical cascade for AT1-mediated apoptosis in the isolated rat cardiomyocyte. The proposed cascade takes into consideration the potential role of PKC in both the stimulation of NADPH oxidase and induction of apoptosis (18, 23, 29, 40). Moreover, the hypothesis recognizes the central role of NADPH oxidase and free radical-mediated DNA damage in initiating apoptosis.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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10 x 106 cells/dish (10 cm diameter). They were then placed in serum-free medium containing minimal essential medium for a period of 3 days. The cells were then exposed to medium supplemented with either no addition (control), 10 µM diphenylene iodonium (DPI), 1 nM ANG II, 1 nM ANG II + 10 µM DPI, 0.5 mM aminoguanidine (AG), or 0.5 mM aminoguanidine + 1nMANG II. The concentrations of ANG II, DPI, and AG were chosen based on their abilities to induce apoptosis (18), inhibit NADPH oxidase (40), and selectively inhibit inducible nitric oxide (NO) synthase (iNOS) (26). At the appropriate time, the cells were either used for analysis of apoptosis, Western blot analysis, or Southern blot analysis. Annexin V and propidium iodide procedures. To assess the number of apoptotic and dead cells, the TACS Annexin V-FITC kit was used. After 6 h of exposure to control medium or medium supplemented with ANG II, AG, DPI, or a combination of these factors, the culture medium was removed from the cells. The cells were first rinsed with cold 1x PBS buffer and then placed in 100 µl of annexin V incubation reagent consisting of 10 µl of 10x binding buffer, 10 µl of propidium iodide, 1 µl of annexin V conjugate, and 79 µl of distilled water. After a 15-min incubation in the dark with the annexin V reagent, the cells were washed for 2 min with an excess of 1x binding buffer [100 mM HEPES (pH 7.4), 1.5 M NaCl, 50 mM KCl, 10 mM MgCl2, and 18 mM CaCl2]. Annexin V and propidium iodide staining were observed by fluorescence microscopy using an Olympus IX 70 inverted microscope.
Western blot analyses. Cellular content of Bax, Bcl-2, p53, p53 (phosphorylated Ser15), p53 (phosphorylated Ser20), and caspase 9 were determined by Western blot analysis using a modification of methods described previously (37, 38). To dislodge the cells from the bottom of the petri dishes, the cells were incubated for 5–7 min with medium containing trypsin. They were then centrifuged at 500 g for 5 min. After the cells were washed with PBS, they were suspended in lysis buffer [62.5 mM Tris (pH 6.8) containing 2% (wt/vol) SDS and 10% glycerol, to which was added 1 mM orthovanadate, 0.1 mg PMSF, and a 1/100 dilution of protease inhibitor cocktail (Calbiochem)]. The cells were disrupted by drawing them into a pipette and then discharging them several times. The samples were then heated to 95–100°C for 5 min and placed on ice for 15 min. The samples were subjected to SDS-PAGE (12% for Bcl-2, Bax, and caspase 9; 8% for p53 and the phosphorylated forms of p53). The proteins were then transferred to nitrocellulose membranes, where they were blocked. After incubation with the appropriate antibody (purchased from Santa Cruz and Cell Signaling), the membranes were washed and then incubated with a secondary antibody, goat anti-rabbit IgG. The Western blots were detected by the enhanced chemiluminescence reaction. All data were analyzed by densitometry using ChemiImage 4400 (Alpha Innotech).
Detection of mitochondrial DNA damage using quantitative Southern blots. Isolated neonatal cardiomyocytes were exposed to 1 nM ANG for various periods of time. In some experiments, 1 nM ANG was added to the cells in combination with 10 µM DPI or 0.5 mM AG. After the cells were lysed, high-molecular-weight DNA was phenol extracted, precipitated with ammonium acetate, and then exposed to 2 volumes of cold ethanol. The DNA samples were resuspended in water and then digested with the restriction endonuclease BamHII (10 U/µg DNA) at the same time it was treated with DNAse-free RNase (1.0 /ml) for 12–16 h at 37°C. After digestion, the samples were precipitated with ammonium acetate and resuspended in Tris-EDTA (TE) buffer (10 mM Tris and 1 mM EDTA; pH 8.0). The DNA was precisely quantified using a Hoefer TKO 100 Mini-Fluorometer and a TKO standards kit (Hoefer Scientific Instruments; San Francisco, CA). Samples containing 5 µg DNA were heated at 65°C for 20 min and then cooled at room temperature for an additional 20 min. After NaOH was added to a final concentration of 0.1 N, samples were incubated for 15 min at 37°C. This produced single-strand breaks at any abasic or sugar-modified site in the DNA. Next, samples were combined with 5 µl of loading dye, loaded onto a 0.6% alkaline agarose gel, and electrophoresed at 30 V (1.5 V/cm gel length) for 16 h in an alkaline buffer consisting of 23 mM NaOH and 1 mM EDTA. The gels were stained with ethidium bromide to confirm equal loading. After the gels were washed, DNA was transferred to a Zeta-Probe GT nylon membrane (Bio-Rad; Hercules, CA). The membranes were cross-linked and hybridized with a 32P-labeled rat mitochondrial DNA-specific PCR-generated probe. Hybridization and subsequent washes were performed according to the manufacturer's recommendations. DNA damage was assessed as follows: break frequency = –ln- (band intensity after ANG II)/(band intensity of control).
Assay for detecting mitochondrial DNA damage at the nucleotide level by ligation-mediated PCR. After treatment, extraction, digestion, and quantitation, 5- to 10-µg aliquots of the DNA samples were heated at 70°C for 20 min and, after being cooled, incubated with 0.1 N NaOH for 15 min. After alkali treatment, DNA samples were precipitated with 0.1 volume of 3 M sodium acetate (pH 5.2) and cold ethanol and then were dissolved in TE buffer. Untreated and alkali-treated DNA (1 µg) from each sample were subjected to ligation-mediated PCR (LM-PCR). Primer extension and ligation were all performed according to Pfeifer et al. (30, 31) with modifications (13). After ligation, the reaction products were precipitated with 0.1 volume of 3 M sodium acetate (pH 5.2), 6.6 mM EDTA (pH 8.0), and 20 µg glycogen and then resuspended in 50 µl H2O. PCR amplification was performed as previously described (30, 31); the exception being the use of 5 pmol/µl primer and a longer oligonucleotide in the asymmetric linker. The increase in the amount of primer used in the LM-PCR was designed to compensate for the increased number of mitochondrial DNA templates present in cells compared with single-copy nuclear genes. After PCR, all samples were precipitated by adding a 25-µl mixture containing 60 mM EDTA (pH 8.0), 1.6 M sodium acetate (pH 5.2), and 2 volumes of cold ethanol. Pellets were air dried and resuspended in a formamide dye solution containing 57% deionized formamide, 7 mM EDTA, 0.7 mg/ml xylene cyanol FF, and 0.7 mg/ml bromphenol blue. Samples were electrophoresed using regular sequencing gels, blotted, and UV cross-linked to the membrane as previously described (13). Hybridization was performed with a single-strand PCR-generated probe using primer for the appropriate strand. After exposure of the membrane to Kodak XAR, the film density of individual hybridization bands corresponding to nucleotide bases of interest was evaluated by scanning of the radioautographs.
Statistical analysis. The statistical significance of the data was determined using Student's t-test for comparison with groups and ANOVA combined with Tukey's post hoc test for comparison between groups. Values of P < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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13% of control cardiomyocytes incubated with medium lacking ANG II and DPI exhibited positive staining for the apoptotic marker annexin V and for the cell death marker propidium iodide. The addition of 10 µM DPI to the incubation medium lacking ANG II had no significant influence on the number of apoptotic, dead cells (17%). However, the NADPH oxidase inhibitor completely prevented ANG II-induced apoptosis. Whereas the number of annexin V-positive cells found after 6 h of ANG II treatment was 31.6 ± 3.0%, the number was reduced to 16.0 ± 1.7% when both 1 nM ANG II and 10 µM DPI were included in the incubation medium.
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Inhibition of NADPH oxidase with DPI also blocked the ANG II-mediated activation of caspase 9 (Fig. 2). In the absence of DPI, ANG II increased the caspase 9-to-procaspase 9 ratio over 70%. However, no significant elevation in the ratio was observed when cells were exposed to both 1 nM ANG II and 10 µM DPI.
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Caspase 9 is an initiator caspase that is activated by the aptosome, a complex formed when cytochrome c is released from the mitochondria. One of the major regulators of mitochondrial cytochrome c release is the Bcl-2 family of proteins, with Bax promoting the release of cytochrome c and Bcl-2 preventing its release (10, 43). Both Bax and Bcl-2 have been previously implicated in ANG II-induced apoptosis (25, 43). Therefore, the effect of DPI on ANG II-induced alterations in the Bax-to-Bcl-2 ratio was examined. In agreement with a previous study (39), it was found that the addition of 1 nM ANG II to the culture medium caused a fairly rapid but modest increase in the Bax content of the isolated cardiomyocyte (Fig. 3). ANG II also mediated a modest decrease in the levels of the antiapoptotic factor Bcl-2 (Fig. 3). Treatment of the cells with DPI prevented both the 18% increase in Bax content and the decrease in Bcl-2 content mediated by ANG II (Fig. 4). The net effect of ANG II action was a 40% increase in the Bax-to-Bcl-2 ratio, an action that was completely prevented by the administration of DPI (Fig. 5).
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Another regulator of apoptosis that is located upstream of Bax and Bcl-2 is the oncosuppressor gene p53. Several factors regulate the activity of p53 through posttranslational modifications, reduced turnover, and protein induction (19, 36). Two of the posttranslational phosphorylation sites of p53 are Ser15 and Ser20. The time course for ANG II-mediated phosphorylation of both Ser15 and Ser20 is shown in Fig. 6. Whereas ANG II-treated cells showed a >50% increase in the amount of phosphorylated Ser15 content after 6 h of ANG II exposure, the elevation in phosphorylated Ser20 content was somewhat less. In contrast, ANG II had no effect on the cellular levels of the total p53 pool (Fig. 6). The addition of DPI to the incubation medium lacking ANG II reduced the levels of phosphorylated Ser20 by 40%, suggesting that the basal rates of NADPH oxidase-mediated superoxide production regulates the phosphorylation state of Ser20. When cells were cotreated with DPI and ANG II, the content of phosphorylated Ser20 was reduced to the level seen in control cells treated with DPI (Fig. 7). Because these levels were 40% below the control, it is clear that NADPH oxidase is involved in the regulation of Ser20 phosphorylation. Although it is likely that NADPH oxidase also regulates the phosphorylation status of Ser15, this could not be ascertained because DPI was found to interfere with the Western blot of phosphorylated Ser15 (see Fig. 12).
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It is widely accepted that DNA damage activates protein kinases that phosphorylate p53, with two of the favored phosphorylated sites being Ser15 and Ser20. Having already shown that ANG II enhanced the phosphorylation state of p53, we tested the hypothesis that oxidative stress arising from the activation of NADPH oxidase might cause DNA damage. DNA samples from control and ANG II-treated cells were first treated with BamHII and NaOH and then subjected to Southern blot analysis. As seen in Fig. 8, DNA obtained from ANG II-treated cells exhibited significant reductions in the intensity of the prominent 10-kb band. Because NaOH treatment cleaves DNA at sites of base modification, the decreased intensity of the 10-kb band was attributed to both preexisting breaks and NaOH-mediated DNA cleavage. According to the analysis of Grishko et al. (13), the frequency of DNA cleavage after 1 h of ANG II exposure was 1.0 ± 0.045 fragments/10 kb. A similar degree of damage was observed after 3 h of ANG II treatment. However, after 24 h of ANG II exposure, considerably less DNA damage was observed, with the decline likely caused by DNA repair. Figure 9A reveals that treatment of the cells with the NADPH oxidase inhibitor DPI prevented the damage at both 1 and 3 h.
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Cai et al. (2) found that hydrogen peroxide derived from NADPH oxidase can promote NO production in bovine aortic endothelial cells. Because NO is also capable of causing DNA damage, the effect of a NOS inhibitor, AG, on ANG II-mediated mitochondrial DNA damage was determined. Interestingly, AG, like DPI, completely blocked ANG II-mediated DNA damage (Fig. 9B).
Grishko et al. (13) have shown that NO modifies DNA by causing the deamination of purines, whereas superoxide-linked ROS cause extensive modification of thymine and guanine. To identify the type of nucleotide damaged, as well as the pattern of ANG II-mediated DNA damage, LM-PCR was performed on a 200-bp stretch of mitochondrial DNA isolated from ANG II-treated cardiomyocytes. This stretch was studied because it contains the common deletion sequence associated with many chronic diseases. For comparison, the pattern of DNA damage mediated by NO (PAPA/NO), superoxide (xanthine oxidase/xanthine), and peroxynitrite was also determined. DNA treated with NaOH contains strands with preexisting breaks and NaOH-induced cleavage sites, whereas DNA treated without NaOH only shows preexisting breaks. On the basis of the Maxim-Gilbert sequencing ladder, most of the damage caused by NO involved guanine, with adenine also being affected (Figs. 10 and 11). By comparison, peroxynitrite and ANG II preferentially modified guanine and adenine, although some thymine and cytosine residues were also modified. Thymine and guanine served as the favored targets of superoxide. Despite the preferential oxidation of guanine by ANG II and NO, the intensity of damage to specific bases differed between the two agents (Fig. 11). Nonetheless, the pattern of damage was virtually identical for ANG II and peroxynitrite, suggesting that peroxynitrite is the oxidizing agent responsible for ANG II-mediated DNA damage.
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Ghafourifar et al. (10) found that stimulation of mitochondrial NOS with calcium is associated with mitochondrial cytochrome c release and the induction of apoptosis, a sequence of events prevented by the SOD mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin. Because calcium-induced apoptosis was also prevented by the NOS inhibitor N
-monomethyl-L-arginine, it was proposed that peroxynitrite might initiate the apoptotic cascade. In a recent review article, Brown and Borutaite (1) proposed that reactive nitrogen species might promote cytochrome c release and apoptosis through a p53-linked pathway. Having already confirmed that peroxynitrite was the oxidizing agent responsible for ANG II-mediated DNA damage, we decided to examine the effect of the iNOS inhibitor AG on the phosphorylation status of p53. As shown in Fig. 12, AG had no major effect on the phosphorylation state of Ser15 in the absence of ANG II. However, the iNOS inhibitor partially prevented the elevation in phosphorylated p53 (Ser15) content seen in the ANG II-treated cell (Fig. 12). Associated with the attenuation in p53 phosphorylation was a reduction in the degree of ANG II-induced apoptosis. Whereas ANG increased the number of annexin V-positive cells from 14.4 ± 1.8% to 31.6 ± 3.0%, only 19.7 ± 1.8% of the AG-treated cells became apoptotic after 6 h of ANG II exposure (Fig. 1).
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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It is widely accepted that ANG II activates PKC in the cardiomyocyte (18). Evidence that inhibitors of PKC block ANG II-induced ROS production in vascular smooth muscle cells places PKC upstream from NADPH oxidase (40). In support of this notion, it has been shown that NADPH oxidase can be phosphorylated and activated by PKC in the neutrophil (11). Therefore, the involvement of NADPH oxidase in ANG II-mediated apoptosis is consistent with the view that activation of PKC is a required step in ANG II-induced apoptosis (18).
In 2000, Griendling and Ushio-Fukai (12) proposed that ANG II-mediated ROS could trigger apoptosis. However, the steps between ROS generation and cell death remained unclear. The present study was designed to clarify some of the steps in this death cascade. One of the initial steps is the formation of peroxynitrite from NO and superoxide. Although the study did not directly examine the effect of ANG II on iNOS activity, it was found that the iNOS inhibitor AG blocked ANG II-induced DNA damage, p53 phosphorylation, and apoptosis. Whether ANG II increased iNOS activity through a signaling pathway that did not involve NADPH oxidase was not determined. Nonetheless, it is significant that Ikeda et al. (16) have shown that ANG II augments interleukin-1
-mediated NO synthesis, an effect associated with an increase in iNOS mRNA accumulation. Moreover, Cai et al. (2) reported that ANG II-mediated H2O2 generation induces endothelial NOS, causing a release of NO. Therefore, it is likely that NADPH oxidase is involved in ANG II-mediated activation of iNOS in the neonatal cardiomyocyte. In other words, NADPH oxidase is responsible for the generation of both factors that combine to form peroxynitrite. Although previous studies have reported on the oxidation of proteins and lipids by peroxynitrite, this is the first report showing that peroxynitrite oxidizes the purine and pyrimidine residues of mitochondrial DNA (Fig. 10). In fact, the pattern of DNA damage by ANG II was virtually identical to that seen with peroxynitrite. As previously reported by Grishko et al. (13), certain guanines are oxidized by PAPA/NO but not by the alkylating agent methylnitrosourea. On the basis of the pattern of DNA damage, it is clear that neither superoxide-derived ROS nor NO directly contribute to ANG II-induced DNA damage. While there are certain similarities between NO-induced DNA damage and ANG II-induced damage, the frequency of individual base damage is most consistent with a role of peroxynitrite in ANG II-induced DNA damage.
The link between DNA damage and p53 activation has been the focus of numerous studies (19, 36). In response to DNA damage, several protein kinases are activated that phosphorylate one or more serine residues of p53. Two of the sites phosphorylated by these protein kinases and by ANG II treatment are Ser15 and Ser20. In addition to promoting the phosphorylation of these regulatory sites on p53, DNA damage also indirectly facilitates the acetylation of lysine residues on the COOH terminus of p53. While the phosphorylation and acetylation reactions also stabilize and reduce the turnover of p53, a p53-linked increase in transcriptional activity can occur in the absence of a large increase in p53 protein content. Indeed, in the present study, ANG II promoted the phosphorylation of p53 but had no significant effect on the protein content of p53. Nonetheless, the increase in the phosphorylation state of Ser15 and Ser20 should cause the DNA binding activity of p53 to rise, resulting in transcriptional activation.
ANG II was found to modestly elevate the levels of Bax in the cardiomyocyte. Although both AT1 and AT2 receptors are capable of elevating Bax levels (7), the elevation of Bax in the cardiomyocyte appears to depend on the cascade initiated by the AT1 receptor (22, 39). Indeed, the AT1 receptor blocker losartan, but not the AT2 receptor blocker PD-123319, is an effective inhibitor of ANG II-mediated apoptosis and the upregulation of Bax in both neonatal and adult cardiomyocytes (3, 18, 22). The elevation in Bax content is most likely caused by p53-dependent transcriptional activation of the proapoptotic factor, an event that is blocked by DPI (25, 43). Nonetheless, an upregulation of Bax is not necessarily a requirement for p53-mediated apoptosis. Regula and Kirshenbaum (34) found that in ventricular myocytes overexpressing wild-type p53, Bax levels were elevated. However, Bax was not elevated in cells that overexpressed a mutant form of p53 defective in gene transcription and that ultimately underwent apoptosis. Moreover, Ding et al. (8) found that p53 participated in radiation-induced apoptosis of myc/ras-transformed rat embryo fibroblasts without the involvement of Bax.
Besides its ability to regulate cellular Bax levels, p53 also downregulates myocardial Bcl-2 levels (32). In the present study, 6 h of ANG II exposure resulted in an 18% decrease in Bcl-2 levels and a 40% increase in the Bax-to-Bcl-2 ratio. Although these changes are modest, the increase in the number of apoptotic cells is also modest. Nonetheless, it is likely that events independent of Bcl-2 family members contribute to ANG II-induced apoptosis. A logical candidate for the initiation of the p53-dependent apoptotic cascade is the impairment in mitochondrial function (27).
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DISCLOSURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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