Abstract 1778: Estrogen Protects Cardiomyocytes from Apoptosis by Suppressing p38alpha-mediated p53 activation and by Downregulating a Negative Feedback from p53 on p38beta
To investigate the molecular mechanisms of estrogen-mediated protection of cardiomyocytes in hypoxic stress. Estrogen is anti-apoptotic and promotes survival of cardiomyocytes under ischemia-related stress. We have previously shown that estrogen protects cardiomyocytes exposed to simulated ischemia-reperfusion (I/R) by differentially regulating pro-apoptotic p38alpha and pro-survival p38beta. However, little is known about how E2 modulation of the kinases alters the apoptotic signaling in cardiomyocytes. An attractive downstream target is p53, a well-known mediator of apoptosis and a substrate of p38alpha. We propose that the cytoprotective actions of estrogen involve regulation of p53 via p38. Cultured neonatal rat cardiomyocytes underwent hypoxia followed by reoxygenation (H/R) to simulate I/R. The extent of apoptosis was determined by examining annexin V-positive cells under fluorescent microscopy. The whole cell lysate was collected after H/R, and the protein of interest was immunoprecipitated and immunoblotted. The p38 activity was determined by immunoprecipitating the protein and performing in-vitro kinase assays on the substrate, ATF2. In our model, p53 played a significant role in H/R-induced myocyte death: Inhibition of p53 (by pifithrin-alpha or by siRNA) significantly reduced the number of apoptotic cells by half. Phosphorylation of p53 (p-p53) at serine 15 increased after H/R, while E2 effectively inhibited this form of p53 activation. Use of a specific agonist for each estrogen receptor isoform (ERalpha or ERbeta) demonstrated that both isoforms participate in inhibition of p-p53. E2 attenuated transcription of apoptosis-specific targets of p53, puma and noxa, as determined by RT PCR. In addition, inhibition of p53 augmented the p38beta activity, suggesting a baseline repression of this pro-survival kinase by p53 that may be reversed by E2. Our results demonstrate that during H/R stress, cardiomyocytes undergo p53-dependent apoptosis following phosphorylation of p53 by p38alpha. E2 protects cardiomyocytes by inhibiting p38alpha-p53 signaling, leading to pro-survival p38beta activation.