MicroRNA-21 Is a Downstream Effector of AKT That Mediates Its Antiapoptotic Effects via Suppression of Fas Ligand

Abstract

MicroRNA-21 (miR-21) is highly up-regulated during hypertrophic and cancerous cell growth. In contrast, we found that it declines in cardiac myocytes upon exposure to hypoxia. Thus, the objective was to explore its role during hypoxia. We show that miR-21 not only regulates phosphatase and tensin homologue deleted on chromosome 10 (PTEN), but also targets Fas ligand (FasL). During prolonged hypoxia, down-regulation of miR-21 proved necessary and sufficient for enhancing expression of both proteins. We demonstrate here for the first time that miR-21 is positively regulated via an AKT-dependent pathway, which is depressed during prolonged hypoxia. Accordingly, hypoxia-induced down-regulation of miR-21 and up-regulation of FasL and PTEN were reversed by activated AKT and reproduced by a dominant negative mutant, wortmannin, or PTEN. Moreover, the antiapoptotic function of AKT partly required miR-21, which was sufficient for inhibition of caspase-8 activity and mitochondrial damage. In consensus, overexpression of miR-21 in a transgenic mouse heart resulted in suppression of ischemia-induced up-regulation of PTEN and FasL expression, an increase in phospho-AKT, a smaller infarct size, and ameliorated heart failure. Thus, we have identified a unique aspect of the function of AKT by which it inhibits apoptosis through miR-21-dependent suppression of FasL. MicroRNA (miRNA)3 are molecules approximately twenty ribonucleotides long that specifically target mRNA through partial complementarity and, thereby, inhibit translation and/or induce their degradation. miR-21 is one of the most commonly and dramatically up-regulated miRNA in many cancers (1, 2) and has been implicated in the inhibition of programmed cell death (2). Some of its validated targets include tropomyosin 1 (3), PTEN (2, 4, 5), programmed cell death 4 (Pdcd4) (6, 7), TAp63 isoform of p53 family, and LRRFIP1, an inhibitor of NFB signaling (8). Similarly, miR-21 is one of the most highly and consistently up-regulated miRNA during cardiac hypertrophy (9 -12). Thum et al. (13) show that miR-21 is predominantly up-regulated in the myofibroblasts where it targets sprouty1 and enhances their survival and, thereby, fibrosis in the heart. Similarly, Roy et al. (14) show that miR-21 is elevated in the myofibroblast-infiltrated area 7 days after ischemia/ reperfusion and suppresses metalloprotease-2 via targeting PTEN. More recently, studies have shown that miR-21 exerts an antiapoptotic function in cardiac myocytes via inhibiting PDCD4 (15) and reduces infarct size via local viral delivery to the heart (16). However, the signaling pathway that regulates miR-21 has not been identified.Two of the molecules that play a major role in ischemic injury of the heart include PTEN and FasL. PTEN is a major negative regulator of AKT (17) whose activity is modulated by its abundance, oxidation, or phosphorylation (18). It is also targeted by miR-21, which provides a specific post-transcriptional mechanism for regulating its expression (2, 4, 5). PTEN has been regarded as the A...

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