Regulation of Expression of the Rat Orthologue of Mouse Double Minute 2 (MDM2) by H2O2-induced Oxidative Stress in Neonatal Rat Cardiac Myocytes

Abstract

The Mdm2 ubiquitin ligase is an important regulator of p53 abundance and p53-dependent apoptosis. Mdm2 expression is frequently regulated by a p53 Mdm2 autoregulatory loop whereby p53 stimulates Mdm2 expression and hence its own degradation. Although extensively studied in cell lines, relatively little is known about Mdm2 expression in heart where oxidative stress (exacerbated during ischemia-reperfusion) is an important pro-apoptotic stimulus. We demonstrate that Mdm2 transcript and protein expression are induced by oxidative stress (0.2 mM H 2 O 2 ) in neonatal rat cardiac myocytes. In other cells, constitutive Mdm2 expression is regulated by the P1 promoter (5 to exon 1), with inducible expression regulated by the P2 promoter (in intron 1). In myocytes, H 2 O 2 increased Mdm2 expression from the P2 promoter, which contains two p53-response elements (REs), one AP-1 RE, and two Ets REs. Exposure of cardiac myocytes to sufficiently high levels of reactive oxygen species (ROS) 6 such as H 2 O 2 leads to their death (1-3), and this probably involves a continuum from apoptosis to necrosis, depending on the severity of the oxidative stress (4). In aerobic tissues such as the heart, the mitochondria probably represent a significant source of ROS, and increased ROS production by these organelles during hypoxia and ischemia-reperfusion injury may be particularly important in myocardial injury (5, 6). However, at lower concentrations, ROS have been reported to promote either growth of the cardiac myocyte (7) or to induce "preconditioning" (8), either of which potentially increases the ability of the cardiac myocyte to survive cytotoxic stresses. H 2 O 2 -induced oxidative stress simultaneously stimulates a number of potentially pro-apoptotic and cytoprotective signaling pathways in the whole heart or cardiac myocytes (9), and the final outcome (cell death or survival) could depend on which signaling pathway(s) predominates and endures. As shown by our microarray studies, H 2 O 2 can positively and negatively regulate global gene expression in cardiac myocytes (3, 10). One gene consistently up-regulated by H 2 O 2 in rat cardiac myocytes at toxic and nontoxic concentrations is the orthologue of transformed mouse 3T3 cell double minute 2 (Mdm2) (3, 10), a proto-oncogene (11, 12), to which we will refer as Rdm2. 7 The human orthologue will be abbreviated as HDM2. The Mdm2 protein binds to the pro-apoptotic p53 tumor suppressor transcription factor to inhibit its transactivating activity (13). Perhaps more importantly, Mdm2 is a ubiquitin-protein isopeptide ligase that ubiquitinates p53 and other proteins (13), thus promoting their proteasomal degradation. Indeed, the stability of p53 protein appears to be of major importance in controlling its abundance (13). In addition, Mdm2 may autoubiquitinate to promote its own degradation (14, 15).Regulation of Mdm2 expression is complex and involves two alternative promoters. The P1 promoter lies 5Ј to exon 1 and the P2 promoter lies within intron 1 (16). P1 primarily regulates Th...

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