Angiotensin Type 2 Receptor Dephosphorylates Bcl-2 by Activating Mitogen-activated Protein Kinase Phosphatase-1 and Induces Apoptosis*

We examined the cellular and signaling mechanism of angiotensin II (Ang II) type 2 (AT2) receptor-induced apoptosis in PC12W (rat pheochromocytoma cell line) cells that express abundant AT2 receptor but not Ang II type 1 receptor. In these cells, nerve growth factor (NGF) inhibited the internucleosomal DNA fragmentation induced by serum depletion, whereas Ang II antagonized this NGF cell survival action and induced apoptosis. We studied the mechanism of NGF and AT2 receptor interaction on apoptosis by examining their effects on the survival factor Bcl-2. AT2 receptor activation did affect intracellular Bcl-2 protein levels. Bcl-2 phosphorylation was stimulated by NGF, whereas AT2 receptor activation blocked this NGF effect. Pretreatment with antisense oligonucleotide of mitogen-activated protein (MAP) kinase phosphatase-1 enhanced the effects of NGF on MAP kinase activation and Bcl-2 phosphorylation but attenuated the inhibitory effects of AT2 receptor on MAP kinase, Bcl-2 phosphorylation, and apoptosis. Taken together, these results suggest that MAP kinase plays a critical role in inhibiting apoptosis by phosphorylating Bcl-2. The AT2 receptor inhibits MAP kinase activation, resulting in the inactivation of Bcl-2 and the induction of apoptosis.

We examined the cellular and signaling mechanism of angiotensin II (Ang II) type 2 (AT2) receptor-induced apoptosis in PC12W (rat pheochromocytoma cell line) cells that express abundant AT2 receptor but not Ang II type 1 receptor. In these cells, nerve growth factor (NGF) inhibited the internucleosomal DNA fragmentation induced by serum depletion, whereas Ang II antagonized this NGF cell survival action and induced apoptosis. We studied the mechanism of NGF and AT2 receptor interaction on apoptosis by examining their effects on the survival factor Bcl-2. AT2 receptor activation did affect intracellular Bcl-2 protein levels. Bcl-2 phosphorylation was stimulated by NGF, whereas AT2 receptor activation blocked this NGF effect. Pretreatment with antisense oligonucleotide of mitogen-activated protein (MAP) kinase phosphatase-1 enhanced the effects of NGF on MAP kinase activation and Bcl-2 phosphorylation but attenuated the inhibitory effects of AT2 receptor on MAP kinase, Bcl-2 phosphorylation, and apoptosis. Taken together, these results suggest that MAP kinase plays a critical role in inhibiting apoptosis by phosphorylating Bcl-2. The AT2 receptor inhibits MAP kinase activation, resulting in the inactivation of Bcl-2 and the induction of apoptosis.
The processes of cell survival and cell death involve highly regulated signaling pathways that are currently the subject of intense investigation. Apoptosis is a ubiquitous, evolutionally conserved, physiological mechanism of cell death that regulates tissue mass and architecture in many tissues (1). The rat PC12W pheochromocytoma cell line is widely used to examine the molecular and cellular mechanism of apoptosis. Xia et al. (2) demonstrate, using PC12W cells, that signaling through mitogen-activated protein (MAP) 1 kinases plays a critical role in cell survival and death. Extracellular signal-regulated kinases (ERK) (p42 and p44 MAP kinases known as p42 MAPK / ERK2 and p44 MAPK /ERK1) act as survival signals, whereas c-JUN NH 2 -terminal protein kinase and p38 exert cell death signaling. In the presence of nerve growth factor (NGF), the survival signal pathway is activated, whereas the cell death signaling pathway is suppressed.
Angiotensin II (Ang II) exerts various actions in its diverse target tissues controlling vascular tone, hormone secretion, tissue growth, and neuronal activities primarily via Ang II type 1 receptor. Recently, a second receptor subtype known as AT2 receptor has been cloned (3,4). We and others have demonstrated that the AT2 receptor stimulates a tyrosine phosphatase (5-8) that inhibits MAP kinase (p42 MAPK /ERK2 and p44 MAPK /ERK1) activation and induces apoptosis in PC12W cells and confluent R3T3 cells (mouse fibroblast cell line) (7). In this study, we hypothesize that this inactivation of MAP kinase plays a pivotal role in mediating apoptosis via the inactivation of the cell survival factor Bcl-2. Bcl-2 can prevent or delay apoptosis induced by a wide variety of stimuli and insults, suggesting that Bcl-2 controls a distal step in the final common pathway for cell death (9). Recent data support that the posttranslational modification of Bcl-2 such as phosphorylation is important for the regulation of Bcl-2 function (10 -12). Here, we demonstrated that MAP kinase phosphorylated Bcl-2 and AT2 receptor stimulation activated MAP kinase phosphatase 1 (MKP-1) and inhibited the phosphorylation of Bcl-2 in PC12W cells, resulting in the induction of apoptosis.

EXPERIMENTAL PROCEDURES
Cells and Treatment-PC12W cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% horse serum, 5% fetal bovine serum. Cell number was counted by Coulter counter.
Measurement of Bcl-2 Phosphorylation and Immunoblot Assay-PC12W cells were seeded onto a 10-cm dish (Becton Dickinson) at 2 ϫ 10 Ϫ6 cells/dish. The cells were first grown in serum-fed Dulbecco's modified Eagle's medium and then kept in serum-free medium for 12 h. The cells were washed three times in serum-free and phosphate-free medium and equilibrated with [ 32 P]orthophosphoric acid (Amersham Life Science, Inc.) at the concentration of 100 Ci/ml in phosphate-free medium for 12 h. The radiolabeled cells were treated with NGF (20 ng/ml), Ang II (10 Ϫ8 and 10 Ϫ7 M), and/or PD123319 (10 Ϫ5 M) at 37°C for 30 min. The cells were washed with HEPES-buffered saline and lysed in 0.5 ml of radioimmune precipitation buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 10 g/ml aprotinin. Cell lysates were centrifuged at 8,500 ϫ g for 20 min, and the supernatant was incubated with 10 g of Bcl-2 antibody (Santa Cruz Biotechnology) at 4°C for 12 h. After precipitation with protein A/Gagarose (Santa Cruz Biotechnology), samples were boiled in Laemmli loading buffer for 3 min and resolved by 12% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue, dried, and analyzed by autoradiography. The bands corresponding to the Bcl-2 were cut, and their radioactivity was measured.
For immunoblotting, cell lysates (100 g) were run on 12% SDS-PAGE, electroblotted onto nitrocellulose membrane, and immunoblotted with Bcl-2 antibody or MKP-1 antibody (Santa Cruz Biotechnology). Antibodies were detected by horse radish peroxidase-linked * This work was supported by National Institutes of Health Grants HL46631, HL35252, HL35610, HL48638, and HL07708, the American Heart Association Bugher Foundation Center for Molecular Biology in the Cardiovascular System, and by a grant from Ciba-Geigy, Basel, Switzerland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
MAP Kinase Activity Determination-MAP kinase activity was assayed by its ability to phosphorylate myelin basic protein as described previously (13) with a slight modification (6 -8).
MKP-1 Antisense Oligonucleotide Transfection-PC12W cells were transfected with 300 nM anti-MKP-1 antisense oligonucleotide (phosphorothioate modified) in Lipofectin (Life Technologies) according to the approach of Duff et al. (14). After transfection, the cells were maintained in the presence of 10% horse serum and 5% fetal bovine serum for 1 day. Then the medium was changed to serum-free medium with or without Ang II. Two days later, cells were harvested and subjected to analysis of DNA fragmentation. Anti-MKP-1 antisense transfected PC12W cells were also used for Bcl-2 phosphorylation, and MAP kinase activity determinations as described above. Oligonucleotide sequences (20 base pairs) are as follows: MKP-1 antisense, 5Ј GGAACTCAGTGGAACTCAGG 3Ј; MKP-1 sense, 5Ј CCTGAGTTC-CACTGAGTTCC 3Ј.
Statistical Analysis-All values are expressed as mean Ϯ S.D. Statistical significance was assessed by ANOVA followed by Scheffe's test. p Ͻ 0.05 was considered significant.

Bcl-2 Phosphorylation by NGF and Its Inhibition by AT2
Receptor-We recently demonstrated (7) that AT2 receptor antagonizes the anti-apoptotic effect of NGF and induces apoptosis in PC12W cells, which express abundant AT2 receptor and very low levels of Ang II type 1 receptor (17). In this study, we examined the possibility that the AT2 receptor regulates apoptosis via its influence on Bcl-2 ( Fig. 1). We first stimulated these cells with NGF and then studied the effect of Ang II on Bcl-2 protein levels. We observed that the Bcl-2 level was not changed with Ang II (Fig. 1B). Since phosphorylation of Bcl-2 is essential for its physiological function (10 -13), we then focused on the effect of AT2 receptor on the Bcl-2 phosphorylation.
PC12W cells were equilibrated with [ 32 P]orthophosphoric acid in phosphate-free medium, and the radiolabeled cells were treated with NGF (20 ng/ml), Ang II (10 Ϫ8 and 10 Ϫ7 M), and PD123319 (10 Ϫ5 M) at 37°C for 30 min. Cell lysates were immunoprecipitated with Bcl-2 antibody, analyzed by SDS-PAGE, and autoradiographed. As shown in Fig. 1, A and C, we observed that NGF stimulated the phophorylation of Bcl-2, whereas Ang II inhibited the NGF-induced Bcl-2 phosphorylation (Fig. 1B). Inhibition of the NGF-mediated phosphorylation of Bcl-2 by Ang II was restored by PD123319, a specific AT2 receptor antagonist, suggesting that this Ang II effect is exerted specifically via the AT2 receptor.
Regulation of Bcl-2 Phosphorylation by MAP Kinase and MKP-1-Since the signaling pathway through MAP kinases appears to play a critical role in the survival of PC12W cells (2), we postulated that MAP kinase enhances Bcl-2 phosphorylation and that the AT2 receptor inhibits this. MAP kinase activity is regulated by a dual-specificity phosphatase known as MKP-1 (18), leading us to hypothesize that the AT2 receptor activates MKP-1, inhibits NGF-mediated MAP kinase activation, and results in the inhibition of Bcl-2 phosphorylation.
We applied antisense oligonucleotide to block basal MKP-1 expression in PC12W cells. Due to the short half-life of MKP-1 mRNA and protein, MKP-1 is an ideal target molecule for studies with antisense inhibition. PC12W cells were trans-fected with 300 nM anti-MKP-1 antisense or sense oligonucleotides. Indeed, we observed that MKP-1 antisense oligonucleotide treatment reduced the level of MKP-1 protein ( Fig. 2A). In MKP-1 sense oligonucleotide-pretreated PC12W cells, NGF increased MAP kinase activity and AT2 receptor stimulation inhibited the NGF-mediated MAP kinase activation as well as the basal level of MAP kinase activity (Fig. 2B). We demonstrated that MKP-1 antisense pretreatment enhanced the effect of NGF on MAP kinase activation in PC12W cells (Fig. 2B). Moreover, we observed that the MKP-1 antisense pretreatment attenuated the antagonistic effect of the AT2 receptor on NGFmediated MAP kinase activation (Fig. 2B).
We next examined the effect of MKP-1 antisense oligonucleotide on Bcl-2 phosphorylation. As shown in Fig. 3, A-C, we observed that the NGF-mediated phosphorylation of Bcl-2 was enhanced by MKP-1 antisense oligonucleotide pretreatment, suggesting that MAP kinase activation is closely linked to phosphorylation of Bcl-2. Moreover, MKP-1 blockade attenuated the inhibitory effect of the AT2 receptor on Bcl-2 phosphorylation. Since MKP-1 possesses tyrosine phosphatase activity (18), we also examined the possibility that MKP-1 directly dephosphorylates the tyrosine residue of Bcl-2. Our data showed that NGF and AT2 receptor does not influence tyrosine phosphorylation (Fig. 4).
Regulation of Apoptosis by MAP Kinase and MKP-1-We next examined the effect of MKP-1 antisense oligonucleotide pretreatment on AT2 receptor-mediated DNA fragmentation ( Fig. 5A and B). The most striking biochemical feature of apoptosis is the activation of endonuclease, which cleaves cellular DNA between regularly spaced nucleosomal units of 180 base pairs or multiples thereof that are readily detected as a DNA ladder by electrophoresis (16). In MKP-1 sense oligonucleotidetreated cells, we observed that NGF inhibited apoptotic changes in PC12W cells after serum depletion and AT2 receptor antagonized the effect of NGF and induced apoptosis. We observed that MKP-1 antisense oligonucleotide pretreatment blocked AT2 receptor-mediated apoptosis (Fig. 5, A and B). As shown in Fig. 5C, serum deprivation alone induces DNA fragmentation in MKP-1 antisense oligonucleotide treated cells. DISCUSSION The highly abundant expression of AT2 receptor during embryonic and neonatal growth, the rapid disappearance after birth (19 -21), and the up-regulation of AT2 receptor in some diseased states such as in myocardial infarction (22), cardiac hypertrophy (23), and skin wounds (24) suggest that this receptor is closely involved with growth, development, and/or differentiation. Indeed, we have demonstrated that the AT2 receptor mediates the developmentally regulated decrease in rat aortic DNA synthesis at late gestation, and we have also demonstrated, using in vivo gene transfer of the AT2 receptor cDNA into injured rat carotid artery, that overexpression of the AT2 receptor transgene results in an attenuation of neointimal hyperplasia (6). Consistent with this in vivo observation, cultured vascular smooth muscle cells transfected with the AT2 receptor expression vector also exhibit decreased rates of DNA synthesis. Stoll et al. (25) also report an antiproliferative influence of the AT2 receptor on cultured coronary endothelial cells. Moreover, we have demonstrated that the AT2 receptor induces apoptosis in cultured PC12W and in R3T3 cells (7,16), both expressing abundant AT2 receptors (17,26,27). These results support the notion that AT2 receptor plays an important role in fetal development and in the pathogenesis of some diseases in which apoptosis is involved. However, the molecular and cellular mechanism of AT2 receptor-mediated apoptosis has not been defined.
MAP kinase mediates multiple cellular pathways. In neuronal cells, MAP kinase activity mediates the action of growth factors like epidermal growth factor, which stimulates cellular proliferation, as well as factors like NGF, which maintains neuronal survival and differentiation (28 -30). Activation of the p42 and p44 isoforms of MAP kinase requires dual phosphorylation on Thr-183 and Tyr-185 residues (31). It has been suggested that the inactivation of MAP kinase is a critical event that regulates the physiological response for cell growth (18). This inactivation is mediated by dephosphorylation of Thr-183 and Tyr-185 residues by a "dual specificity" phosphatase known as MKP-1, which is encoded by the mitogen-inducible gene 3CH134 (18). Recently, an isoform of MKP-1 was isolated from PC12W cells named MKP-2 (32). Cellular response to various stimuli may regulate MAP kinase activity by coordinating the action of the MAP kinase activation cascade (33) and MKPs, resulting in the cell differentiation and survival or cell death. Using MKP-1 antisense strategy, we demonstrated that MKP-1 is involved in the inactivation of MAP kinase by the AT2 receptor.
MKP-1 was discovered as an immediate early gene whose rapid transcription and subsequent translation have been suggested to provide a feedback loop to terminate growth factor signaling (18,33). However, the mechanism of activation of this phosphatase is not known. We previously reported that the dephosphorylation of MAP kinase was observed within 5 min after AT2 receptor stimulation in PC12W cells and that this effect was blocked by sodium vanadate and pertussis toxin (7). In contrast, Duff et al. (34) report that Ang II type 1 receptor stimulation rapidly induced MKP-1 mRNA (30 min maximum) in rat vascular smooth muscle cells. Therefore, we examined MKP-1 mRNA expression in PC12W cells after AT2 receptor stimulation and observed that AT2 receptor stimulation did not increase the MKP-1 mRNA in this cell line (data not shown). Taken together, these results suggest that AT2 receptor stimulation activates MKP-1 phosphatase activity without the apparent induction of MKP-1 expression.
Apoptosis is controlled in part by a family of cytoplasmic proteins, the Bcl-2 family. Bcl-2 can prevent or delay apoptosis induced by a wide variety of stimuli and insults (9). It has been reported that the Bcl-2 protein requires post-translational modification, specifically phosphorylation, to be functionally active (35,36). Recent data support the notion that phosphorylation of Bcl-2 is important for the regulation of Bcl-2 function and thereby apoptosis (10 -12). In this study, we demonstrated that NGF enhanced the phosphorylation of Bcl-2 and that the AT2 receptor inhibited the NGF-mediated Bcl-2 phosphorylation and induced apoptosis.
Our results suggest that NGF-mediated MAP kinase activation is closely linked to increases in the phosphorylation of Bcl-2, resulting in the cell survival signal. MAP kinase is a serine/threonine kinase, and the minimal consensus sequence of substrate specificity of this kinase is the (Ser/Thr)-Pro (37) and Ser/Pro sequence, conserved in murine and human Bcl-2 at the positions 70 and 71 (38,39). Indeed, the phophorylation of the serine residue in Bcl-2 has been reported (10 -12). These results suggest that MAP kinase phosphorylates the serine residue of Bcl-2. Moreover, we examined the possibility that MKP-1 could directly dephosphorylate the tyrosine residue of Bcl-2, since MKP-1 exerts tyrosine phosphatase activity (18). However, in PC12W cells, we did not observe any effect of NGF and AT2 receptor on tyrosine phophorylation of Bcl-2.
We also observed that inhibition of MKP-1 expression blocked the AT2 receptor-mediated DNA fragmentation. Based on these results, we propose that MAP kinase plays a critical role in suppressing apoptosis in PC12W cells by phosphorylating and activating Bcl-2. The AT2 receptor inhibits MAP kinase activation by activating MKP-1 and subsequently dephosphorylates Bcl-2, resulting in the development of apoptosis. PC12W cells were transfected with 300 nM anti-MKP-1 antisense or sense oligonucleotide. The cells were maintained in the presence of serum for 1 day and treated with NGF (20 ng/ml) and/or Ang II (10 Ϫ7 M) in serum-free medium. Two days later, cells were harvested, and DNA fragmentation was studied. The representative autoradiograph of DNA fragmentation is shown in (A). The amount of radiolabeled ddATP incorporated into low (Ͻ20 kilobase pairs) molecular weight DNA fractions was quantitated by cutting the respective fraction of DNA from the dried gel and counting in a ␤ counter. The results were expressed as a percentage of radioactivity counts in control samples (serum-starved, nontreated cells) (B). The effects of sense and antisense MKP-1 oligonucleotide treatment without Ang II treatment on the DNA fragmentation were also shown in C. The values are expressed as mean Ϯ S.D. and were obtained from four different cell culture dishes. * shows p Ͻ 0.01.