Ligand-independent signals from angiotensin II type 2 receptor induce apoptosis
2000; Springer Nature; Volume: 19; Issue: 15 Linguagem: Inglês
10.1093/emboj/19.15.4026
ISSN1460-2075
AutoresShin-ichiro Miura, Sadashiva S. Karnik,
Tópico(s)Apelin-related biomedical research
ResumoArticle1 August 2000free access Ligand-independent signals from angiotensin II type 2 receptor induce apoptosis Shin-ichiro Miura Shin-ichiro Miura Department of Molecular Cardiology/NB50, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 USA Search for more papers by this author Sadashiva S. Karnik Corresponding Author Sadashiva S. Karnik Department of Molecular Cardiology/NB50, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 USA Search for more papers by this author Shin-ichiro Miura Shin-ichiro Miura Department of Molecular Cardiology/NB50, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 USA Search for more papers by this author Sadashiva S. Karnik Corresponding Author Sadashiva S. Karnik Department of Molecular Cardiology/NB50, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 USA Search for more papers by this author Author Information Shin-ichiro Miura1 and Sadashiva S. Karnik 1 1Department of Molecular Cardiology/NB50, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:4026-4035https://doi.org/10.1093/emboj/19.15.4026 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Conventional models of ligand–receptor regulation predict that agonists enhance the tone of signals generated by the receptor in the absence of ligand. Contrary to this paradigm, stimulation of the type 2 (AT2) receptor by angiotensin II (Ang II) is not required for induction of apoptosis but the level of receptor protein expression is critical. We compared Ang II-dependent and -independent AT2 receptor signals involved in regulating apoptosis of cultured fibroblasts, epithelial cells and vascular smooth muscle cells. We found that induction of apoptosis—blocked by pharmacological inhibition of p38 mitogen-activated protein kinase and caspase 3—is a constitutive function of the AT2 receptor. Biochemical and genetic studies suggest that the level of AT2 receptor expression is critical for physiological ontogenesis and its expression is restricted postnatally, coinciding with cessation of developmental apoptosis. Re-expression of the AT2 receptor in remodeling tissues in the adult is linked to control of tissue growth and regeneration. Therefore, we propose that overexpression of the AT2 receptor itself is a signal for apoptosis that does not require the renin–angiotensin system hormone Ang II. Introduction Hormonal regulation of in vivo functions of the type 2 receptor (AT2) for the octapeptide angiotensin II [Ang II (Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-COO−)] is an enigma. Ang II is an important mediator of the renin–angiotensin system's functions. Almost all of the classical physiological effects attributed to Ang II regulation are mediated by the Ang II type 1 (AT1) receptor, leaving an unexplained role for the AT2 receptor in the renin–angiotensin system. Ang II is widely regarded as the physiological ligand for the AT2 receptor, although Ang II binding does not elicit the usual second-messenger responses or desensitization and down-regulation of the AT2 receptor, the biochemical responses considered hallmarks of ligand regulation of receptor functions (Bottari et al., 1991; Brechler et al., 1993; Hein et al., 1995, 1997; Ichiki et al., 1995; Matsubara, 1998; Horiuchi et al., 1999). Expression of the AT2 receptor is developmentally regulated; genetic defects in it are linked to attenuated apoptosis of mesenchymal cells, contributing to aberrant ontogenesis of the kidney and urinary tract (Nishimura et al., 1999). Apoptosis in cultured pheochromocytoma and fibroblast cell lines is accompanied by overexpression of the AT2 receptor (Yamada et al., 1996; Matsubara, 1998; Nishimura et al., 1999). AT2 receptor gene knockout leads to defective navigational control in mice (Hein et al., 1995; Ichiki et al., 1995). Transgenic cardiac overexpression leads to malfunctioning pacemaker cells and abnormal blood pressure regulation (Masaki et al., 1998). Besides contributing to apoptosis through signals, the AT2 receptor is reported to regulate activities of the mitogen-activated protein kinase (MAPK), inward rectifier potassium channel, T-type calcium channel, protein tyrosine phosphatase SHP-1 and protein phosphatase 2B (for a review see Matsubara, 1998). The AT2 receptor is a seven transmembrane (7TM) helical receptor that binds subtype selective ligand PD123319 and CGP42112 (Brechler et al., 1993). It is an atypical receptor with regard to agonist recognition, GTP analogue-dependent agonist affinity modulation, G-protein coupling, and the ability to activate second-messenger pathways (Bottari et al., 1991; Pucell et al., 1991; Brechler et al., 1993; Nakajima et al., 1995; Miura and Karnik, 1999). Although the AT2 receptor contains consensus sites for phosphorylation, it is not phosphorylated in response to Ang II and does not internalize (Hein et al., 1997). Abundant expression of the AT2 receptor in fetal tissues is essential for physiological ontogenesis. Its expression in adults is limited to few tissues, but re-expressed ∼5- to 25-fold over basal during remodeling of tissues where the AT2 receptor is thought to play a role in the apoptosis of smooth muscle cells, fibroblasts and endothelial cells (Bottari et al., 1991; Pucell et al., 1991; Stoll et al., 1995; Dimmeler et al., 1997; Matsubara, 1998; Horiuchi et al., 1999). Thus, the importance of AT2 receptor functions is beginning to be documented, but there is still no explanation of how Ang II regulates the receptor's functions. In an earlier study (Miura and Karnik, 1999) we elucidated that the chemical groups on Ang II essential for high affinity binding to the AT2 receptor each contributed only a small proportion (<10%) of the overall binding energy. It is generally believed that the high affinity of hormones is a consequence of their selective binding to receptors that are already active, so that the hormone shifts the equilibrium in favor of active receptors. Alternatively, hormones could bind to inactive receptors and facilitate isomerization to the active state and stabilize the active receptor (Gether and Kobilka, 1998; Karnik, 2000). In either mechanism, agonist analogs, differing in the ability to influence activation, also display large differences in affinity. The lack of affinity discrimination of Ang II analogs (Miura and Karnik, 1999) suggested to us that either the native AT2 receptor is already in an active state or Ang II is not a classical agonist for the AT2 receptor. The current study explores the specificity of the Ang II–AT2 receptor complex in the regulation of signals that contribute to apoptosis. Our results suggest that the AT2 receptor, without Ang II stimulation, generates signals that are critical for the cell death program. Also, consistent with Ang II-independent regulation, the AT2 receptor-induced apoptosis of R3T3 fibroblasts, Chinese hamster ovary (CHO) epithelial cells and the A7r5 vascular smooth muscle cells (VSMCs) is not further enhanced by Ang II. Results and discussion Apoptosis in R3T3 fibroblasts may not require Ang II Whether Ang II is essential in vivo for the AT2 receptor to mediate apoptosis during embryonic development or physiological and pathological remodeling is unclear. The R3T3 fibroblast cell line has been used as a model for study since apoptosis in these cells resembles the in vivo situation described in ovarian granulosa cells and other remodeling tissues (Pucell et al., 1991; Stoll et al., 1995; Yamada et al., 1996; Matsubara, 1998; Speth et al., 1999). AT2 receptor overexpression precedes apoptosis in confluent R3T3 cells, which express low levels of AT1 and AT2 receptors (2 ± 0.4 versus 9 ± 0.3 fmol/mg protein) when grown in serum. Serum starvation leads to an ∼10-fold increase in AT2 receptor density in the confluent state (see Figure 1). However, in non-confluent R3T3 cells, serum depletion itself was sufficient to induce apoptosis, which was not accompanied by an increase in AT2 receptor density. However, addition of Ang II was not required for induction of apoptosis and the apoptosis could not be blocked by the AT2 receptor-selective antagonist PD123319 (not shown). Thus, the up-regulation of AT2 receptor gene expression might be a signal for apoptosis in vivo. These results are qualitatively similar to those reported in several cultured cell models that lead to the conclusion that Ang II is required to induce apoptosis via the AT2 receptor. Since our finding differs significantly with regard to the specificity of apoptosis induction by the Ang II–AT2 receptor complex, we conclude that it is difficult to separate the critical effects of culture conditions and AT2 receptor overexpression on induction of apoptosis. Hence, the AT2 receptor-expressing native cell lines are not a suitable model to conclude the requirement of an Ang II–AT2 receptor complex for apoptosis and are even less appropriate to conclude a role for induced overexpression of AT2 receptor protein. Cell models where levels of AT2 receptor expression can be controlled are better suited for these studies. Figure 1.Apoptosis in R3T3 cells in the presence of 10% serum (A) and under serum-free conditions (B) without Ang II, shown by the TUNEL method. Nuclear stain (see speckled stain pattern) indicates apoptotic cells. The expression levels of AT1 (black bars) and AT2 receptors (white bars) under serum-free conditions for 72 versus 0 h (n = 3, *p <0.05) was measured by saturation binding of 125I-[Sar1,Ile8]Ang II (C). Download figure Download PowerPoint AT2 receptor expression induces apoptosis in CHO cells and VSMCs To link de novo expression of AT2 receptor to apoptosis, we used the CHO and A7r5 cells as surrogate models (Figure 2). CHO cells are an established epithelial lineage of non-transformed cells that are capable of growing under low-serum conditions with appropriate supplements (Kao and Puck, 1968). A7r5 is an established cell line that retains several smooth muscle characteristics (Kimes and Brandt, 1976). VSMCs, a key component of the blood vessels, is a relevant cell model because it is subjected to apoptotic and mitogenic stimulation, respectively, by AT2 and AT1 receptors in vivo (Kimes and Brandt, 1976; Stouffer and Owens, 1992; Duff et al., 1995; Horiuchi et al., 1999). Expression of the endogenous AT2 receptor is undetectable by 125I-[Sar1-Ile8]Ang II binding and RT–PCR using AT2 receptor mRNA-specific primers, under conditions of both serum-stimulated growth and serum deprivation in both these cell lines. The endogenous AT1 receptor (<18 ± 6 fmol/mg protein) does not elicit any significant inositol phosphate production and MAPK phosphorylation. However, transfection of AT1 receptor cDNA elicited both these responses, indicating that the signal transduction components are present in both cell lines. RT–PCR analysis indicated that these cells do not express angiotensinogen, a key source of intracellular Ang II production and paracrine/autocrine regulation (data not shown). Therefore, by introducing the AT2 receptor in these cells, its effects on apoptosis can be studied without the hypertrophic or mitogenic signaling from the AT1 receptor and/or autocrine influence of Ang II. Figure 2.Chromatin condensation and DNA strand break in A7r5 cells (A–D) and CHO cells (E–H) transiently transfected with the AT2 receptor expression vector. Cells transfected with pEYFP (A, B, E and F) or pAT2R-EYFP (C, D, G and H) were stained with DAPI to visualize nuclear morphology (shown in A, C, E and G). The arrows indicate nuclei of EYFP-positive cells without nuclear condensation (A and E) and with nuclear fragmentation (C and G). Download figure Download PowerPoint Exogenous overexpression of the AT2 receptor induced apoptosis in both CHO and A7r5 cells. Nuclear DNA condensation and fragmentation began ∼24 h (time at which growth arrest is established) after shifting to the low-serum growth condition and was complete after 72 h (Figure 2). Specifically in the enhanced yellow fluorescent protein (EYFP)-positive cells transfected with pAT2R-EYFP, morphological features associated with the apoptotic cells, such as irregular shaped nuclei in conjunction with chromatin condensation, appeared (Figure 2C and G). These cells also exhibited membrane blebbing, cytoplasmic shrinkage and inhibition of protein synthesis (data not shown). In contrast, 4′,6-diamidino-2-phenylindole (DAPI) staining of the pEYFP-transfected control cell nuclei showed sharp round edges and diffuse chromatin (Figure 2A and E). These cells did not exhibit apoptotic features, indicating that the observed apoptosis is specific for the AT2 receptor and is not an artifact of transfection, serum deprivation or EYFP expression. The APO-BRDU™ chromatin DNA fragmentation method was used to quantitate the percentage of cells in apoptosis, employing fluorescence activated cell sorter (FACS) analysis (Figures 3 and 4; see Materials and methods for details). Fluorescein-labeled DNA break points (green) shown in Figure 3A illustrate nuclei in apoptosis in an AT2 receptor-transfected A7r5 culture. Figure 3Ad and 3Ae shows early phase apoptotic nuclei that are characterized by preservation of the nuclear envelope (propidium iodide stained, red). The vector-transfected control cells were mostly not stained with fluorescein (Figure 3Aa and 3Ab). APO-BRDU staining of CHO cells yielded similar results (not shown). Figure 3.(A) TUNEL analysis of A7r5 cell nuclei visualized by propidium iodide staining in cells transfected with pcDNA3 (a–c) and pcDNA3-AT2R (d–f). Cells were fixed and stained with propidium iodide (red), then Br-dUTP labeled with terminal deoxynucleotide transferase and then stained with the fluorescein-R-1 antibody (green). Cells were imaged on a confocal microscope. Flow cytometric distribution of cells in apoptosis in each condition is shown in c and f. The upper right quadrant in each plot represents TUNEL-positive apoptotic cells (see legend to Figure 4 for details). (B) Influence of various culture conditions on apoptosis in mock, wild-type (WT) or mutant (N127G) AT2 receptor-transfected A7r5 and CHO cells. Effect measured by FACS analysis upon treatment with 0.2% fetal bovine serum (Sf), 10% fetal bovine serum (Se), 0.1 μM agonist [Sar1]Ang II and 10 μM non-peptide antagonist PD123319; n = 3, *p <0.05 versus mock, †p <0.05 versus serum condition. Download figure Download PowerPoint Figure 4.(A) Quantitation of the AT2 receptor's influence on A7r5 cell apoptosis. The percentage of cells in apoptosis in the samples shown was 2% in 12 μg (per 106 cells) pcDNA3 empty vector DNA-transfected control, 29% in a 1:1 mixture of pcDNA3 (6 μg) + pcDNA3-AT2 receptor (6 μg) and 92% in 12 μg pcDNA3-AT2 receptor expression vector-transfected sample. Transfected cells (1 × 106) were TUNEL stained and counterstained with propidium iodide (see Materials and methods for details). FACscan shows the distribution of cells within different cell cycle stages indicated on top. The upper right quadrant in each plot represents TUNEL-positive apoptotic cells. In each instance, 10 000 propidium iodide-stained cells were monitored and the percentage of apoptotic cells per dish was calculated. (B) Correlation between the expressed AT2 receptor number and the percentage of apoptotic A7r5 cells. Regression lines from independent transfection experiments are shown, y = 5.3 + 0.15x, n = 19, r = 0.97, p <0.05 (no treatment); y = 8.0 + 0.13x, n = 10, r = 0.94, p <0.05 (0.1 μM [Sar1]Ang II); y = 2.9 + 0.14x, n = 14, r = 0.96, p 100-fold over Kd) did not stimulate apoptosis in both A7r5 and CHO cells transfected with the N127G mutant, indicating that decreased agonist affinity is not the cause of the defect in this mutant. Thus, cell surface expression of the AT2 receptors is not sufficient, rather the observations suggest a specific conformation attained by the WT protein to induce apoptosis in a concentration-dependent manner. Apoptosis in the CHO cells correlated with the level of expression of the WT AT2 receptor. Expression of the N127G mutant did not induce apoptosis in CHO cells (Figure 3B). Thus, no unique characteristic feature of smooth muscle cells seems to be critical for AT2 receptor to induce apoptosis. AT2 receptor-induced apoptosis is mediated by p38 MAPK and caspase-3 Mechanisms for the recruitment of distinct members of the MAPK superfamily and caspase family do exist in the AT2 receptor. Pharmacological inhibition of p38 MAPK and caspase-3 blocked apoptosis induced by two different levels of AT2 receptor expression in A7r5 cells (Figures 5,6,7). The p38 MAPK-specific inhibitor SB203580 blocked AT2 receptor-induced apoptosis by ∼80%. The SB203580 inhibitor alone had no significant effect on A7r5 cells. The maximal inhibitory effect of SB203580 (10 μM) occurred when the inhibitor was present during serum starvation; this inhibitor was not effective when added 24 h after serum starvation, suggesting a small time window for its action. Immunoblot analysis indicated that a protein of ∼44 kDa that cross-reacted with p38 MAPK-specific antiserum is present in A7r5 cells (Figure 6A). VSMCs have also been shown to contain p38 MAPK (Kusuhara et al., 1998). In AT2 receptor-transfected cells, basal levels of both p38 MAPK and phospho-p38 MAPK were elevated. In contrast, cells transfected with empty vector or the N127G mutant contained lower levels of mostly non-phosphorylated p38 MAPK (Figure 6A). Treatment with [Sar1]Ang II did not further increase phospho-p38 MAPK levels but treatment with SB203580 decreased levels in these cells. PD123319 treatment had no effect. None of the treatments affected p38 MAPK levels (Figure 6B). These results suggest a significant functional role for p38 MAPK in AT2 receptor-mediated apoptotic signaling. Figure 5.Pharmacological intervention of apoptosis in AT2 receptor-transfected A7r5 cells. Effect measured by FACS analysis upon treatment with 30 μM MEK inhibitor PD98059, 10 μM p38 MAPK inhibitor SB203580, 1 μM caspase-3 inhibitor DEVD-cmk, 1 μM CGP42112, 10 μM PD123319 and 0.1 μM [Sar1]Ang II; n = 3, *p <0.05 versus no treatment. Download figure Download PowerPoint Figure 6.Immunoblot analysis of the p38 MAPK and phospho-p38 MAPK levels in A7r5 cells transfected with various expression vectors (A) and with various treatments (B) following serum starvation. An equal amount of total protein was loaded in each lane. *p <0.05 versus control shown in lane 1 in each group; †p <0.05 versus AT2 receptor control shown in lane 4 in each group. Results shown are of three experiments normalized; the control was set at 1.0 arbitrary unit. Download figure Download PowerPoint Figure 7.Caspase-3-like activity was measured in A7r5 cells transfected with pcDNA3 (Mock), pcDNA3-AT2R (WT), pcDNA3-AT2R N127G mutant plasmid. Cells were treated with [Sar1]Ang II (0.1 μM), PD123319 (10 μM) or caspase-3 Inh. (1 μM). The activity of the enzyme in the pcDNA3-transfected cell extract, 1.73 pmol of pNA liberated/μg protein/h at 37°C, was considered as one arbitrary unit. *p <0.05 versus mock, †p <0.05 versus no treatment in each group. Download figure Download PowerPoint To characterize the roles of p42/44 MAPK in AT2 receptor-induced apoptosis, we measured the effect of PD98059, a specific inhibitor of MEK, on the AT2 receptor-stimulated apoptosis (Figure 5). PD98059 at 30 μM, a concentration that blocked accumulation of phospho-p42/44 MAPK, alone did not induce apoptosis, nor did it potentiate an apoptotic response to AT2 receptor expression (not shown). MEK activates p42/44 MAPK by phosphorylation and the AT2 receptor-coupled MAPK-phosphatase 1 dephosphorylates p42/44 MAPK (Yamada et al., 1996). Down-regulation of p42/p44 MAPK activity in VSMCs, neuroblastoma cells, cultured primary neurons and rat fibroblasts has been suggested to contribute to the anti-growth effect associated with the AT2 receptor (Duff et al., 1995; Nakajima et al., 1995; Huang et al., 1996; Yamada et al., 1996). However, this regulatory loop does not seem to be critical in the apoptotic pathway, but may have a role in exerting an anti-growth effect. We did not investigate the activation of the c-jun-kinase, because an increase in its activity is slower and specific inhibitors do not yet exist. Furthermore, nearly complete blockade of apoptosis by SB203580 is indicative of an exclusive role of p38 MAPK in AT2 receptor-induced apoptosis. We examined activation of caspases since the activation of class II caspases, a process essential for the execution of catastrophic proteolysis of regulatory proteins in a cell destined to die, is considered a hallmark of programmed cell death (Ashkenazi and Dixit, 1998). The caspase-3 inhibitor Ac-DEVD-cmk (1 μM) inhibited ∼30% of the AT2 receptor-induced apoptosis (Figure 5). The partial blockade might be because this class of inhibitors are not very potent. The measured caspase-3-like activity in cell-free extracts prepared from WT AT2 receptor-transfected cells was significantly higher than that in pcDNA3-transfected (mock) and N127G mutant-transfected cells (Figure 7). The increase in caspase-3-like activity in the WT AT2 receptor-transfected cells was not suppressed by the antagonist PD123319. The observations that N127G mutant receptor does not induce apoptosis or activate caspase-3-like activity and that the WT receptor activates caspase-3-like activity suggest that this activity is critical for induction of apoptosis. The involvement of other caspases in the process can not be ruled out. Thus, the activation and involvement of the caspase cascade in the AT2 receptor-induced apoptotic process are indicated. Apoptosis induced by AT2 receptor expression is not enhanced by Ang II nor inhibited by the AT2 receptor antagonist Several lines of evidence suggest that the activation of programmed cell death may be a constitutive function of the AT2 receptor. In AT2 receptor-transfected CHO and A7r5 cell models, induction of apoptosis was not enhanced by the agonist [Sar1]Ang II and was not inhibited by the antagonist PD123319 (Figure 3B). A similar mechanism may be involved in apoptosis in R3T3 fibroblasts as discussed earlier. Since Ang II dependence of apoptosis induction by the AT2 receptor has been indicated in several studies earlier, we systematically examined the influence of [Sar1]Ang II and PD123319 at several different levels of expression in A7r5 cells (Figure 4B). Measured apoptosis was neither enhanced by [Sar1]Ang II nor inhibited by PD123319. Treatment of cells with 0.1–10 μM [Sar1]Ang II did not increase the apoptotic index (calculated from the slope). A seeming decrease in the apoptotic index at receptor densities ≤300 fmol/mg, by treatment with the AT2 receptor antagonist PD123319, was not statistically significant, indicating a lack of inhibition by the compound. The drug had no effect on apoptosis at receptor densities ≥300 fmol/mg. In another experiment, we varied the AT2 receptor number from 10 to 720 fmol/mg protein, with and without [Sar1]Ang II or PD123319, to measure the time of onset of apoptosis. The differences among the three conditions were small and not significant. In the CHO cells, treatment with 0.1–10 μM [Sar1]Ang II did not increase the percentage of apoptosis and also had no influence on the apoptosis index (not shown). To examine Ang II independence of various AT2 receptor signals that contribute to apoptosis, we evaluated the potency of various pharmacological inhibitors on apoptosis with and without [Sar1]Ang II treatment (Figure 5). These experiments were carried out at two different levels of AT2 receptor expression, 265 ± 25 and 585 ± 30 fmol/mg protein, as shown in Figure 5. Combined treatment with [Sar1]Ang II + PD98059 did not have a synergistic effect on apoptosis, as would be anticipated, at both levels of expression (Figure 5). Furthermore, [Sar1]Ang II treatment did not antagonize the inhibition of apoptosis by SB203580, and the caspase-3 inhibitor DEVD-cmk. The most unexpected observation from these studies is the lack of an apoptosis-activating effect by [Sar1]Ang II. These findings are comparable to the observations reported in earlier studies that indicate that Ang II does not induce the cascade of events that are hallmarks of receptor regulation by agonists. For instance, Pucell et al. (1991) and Hein et al. (1997) reported that Ang II does not stimulate endocytosis of the AT2 receptor, implying that signals responsible for endocytosis are not generated. Similarly, AT2 receptor–Gi immune complex is insensitive to dissociation by GTPγS, unlike what would be expected (Zhang and Pratt, 1996). Blockade of AT2 receptor-induced apoptosis by SB203580 suggests that members of the p38 MAPK family are recruited for mediating this process (Figure 5). Accumulation of phospho-p38 MAPK in AT2-transfected cells is blocked by SB203580 but not affected by the agonist [Sar1]Ang II and the antagonist PD123319 (Figure 6). This observation indicates that the p38 MAPK activation is linked to the level of expression of AT2 receptor protein and not to activation of the AT2 receptor by the ligand. The SB series inhibitors are proposed to be specific inhibitors of α and β isoforms of p38 MAPK. In some cell types (such as Jurkat lymphoma and HeLa cells), p38β is involved in attenuation of apoptosis and p38α in augmentation of apoptosis (Wang et al., 1998). Assuming a similar mode of action in AT2 receptor-transfected A7r5 and CHO cells, inhibition of p38α may shift the balance in favor of anti-apoptotic effects. Ectopic expression of p38 MAPK is known to induce cell death in PC12 cells (Wang et al., 1998). Activated p38 MAPK is cap
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