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EGR-1/ASPP1 inter-regulatory loop promotes apoptosis by inhibiting cyto-protective autophagy.

ABSTRACT: The decrease of ASPP1 (Apoptosis-Stimulating Protein of p53 1), a known p53 activator, has been linked to carcinogenesis and the cytotoxic resistance in various cancers, yet the underlying mechanisms of ASPP1 expression and its complex functions are not yet clear. Here, we report that ASPP1 forms an inter-regulatory loop with Early Growth Response 1 (EGR-1), and promotes apoptosis via inhibiting cyto-protective autophagy, independent of the well-documented p53-dependent mechanisms. We show that ASPP1 mRNA and protein were remarkably elevated by ectopic EGR-1 expression or endogenous EGR-1 activation, in cells with different tissue origins and p53 status. Conversely, RNAi-mediated EGR-1 knockdown suppressed ASPP1. The further mechanism studies revealed that ASPP1 promoter, mapped to -283/+88, which contained three conserved EGR-1 binding sites, was required for both binding and transactivity of EGR-1. In addition, we demonstrate that ASPP1 promoted EGR-1 in a positive feedback loop by preventing proteasome-mediated EGR-1 degradation or promoting EGR-1 nuclear import in response to anticancer natural compound Quercetin. Furthermore, albeit activating p53 in the nucleus is the well-studied function of ASPP1, we found that ASPP1 was predominately localized in the cytoplasm. Interestingly, the cytoplasmic ASPP1 retained its pro-apoptosis capability. Mechanistically, ASPP1 suppressed Atg5-Atg12 and also bound with Atg5-Atg12 to prevent its further complex formation with Atg16, resulting in the inhibition of cyto-protective autophagy. In conclusion, our results provide new insights into EGR-1/ASPP1 regulatory loop in sensitizing Quercetin-induced apoptosis. EGR-1/ASPP1, therefore, may be potentially used as therapeutic targets to improve cancer's response to pro-apoptosis treatments.

SUBMITTER: Zhao K

PROVIDER: S-EPMC5520923 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Microvascular obstruction (MVO) can result in coronary microcirculation embolism and myocardial microinfarction. Myocardial injury induced by MVO is characterized by continuous ischemia and hypoxia of cardiomyocytes. Autophagy and apoptosis are closely associated with various cardiovascular diseases. Based on our previous study, we observed a decrease in miR-30e-3p expression and an increase in Egr-1 expression in a rat coronary microembolization model. However, the specific function of miR-30e-3p in regulating autophagy and apoptosis in an ischemia/hypoxia (IH) environment remains to be deciphered. We exposed cardiomyocytes to an IH environment and then determined whether miR-30e-3p was involved in promoting cardiomyocyte autophagy and inhibiting apoptosis by regulating Egr-1. Methods:Cardiomyocytes were isolated from rats for our in vitro study. miR-30e-3p was either overexpressed or inhibited by transfection with lentiviral vectors into cardiomyocytes. 3-Methyladenine (3-MA) was used to inhibit autophagy. RT-qPCR and western blotting were used to determine the expression levels of miR-30e-3p, Egr-1, and proteins related to the autophagy and apoptosis process. Autophagic vacuoles and autophagic flux were evaluated using transmission electron microscopy (TEM) and confocal microscopy, respectively. Cardiomyocyte viability was evaluated using the MTS assay. Cell injury was assessed by lactate dehydrogenase (LDH) leakage, and apoptosis was determined by flow cytometry. Results:Both miR-30e-3p expression and autophagy were significantly inhibited, and apoptosis was increased in cardiomyocytes after 9 hours of IH exposure. Overexpression of miR-30e-3p increased autophagy and inhibited apoptosis, as well as suppressed Egr-1 expression and decreased cell injury. In addition, inhibition of miR-30e-3p reduced autophagy and increased apoptosis and cell injury. Conclusions:miR-30e-3p may be involved in promoting cardiomyocyte autophagy and inhibiting apoptosis by indirectly regulating Egr-1 expression in an IH environment.

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EGR-1/ASPP1 inter-regulatory loop promotes apoptosis by inhibiting cyto-protective autophagy.

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