The role of PINK1 in the hypoxic stress response through the regulation of Hif-1alpha translation.

摘要

Several studies have demonstrated the role of mitochondrial dysfunction in the loss of dopaminergic neurons in Parkinson's disease (PD). PTEN-induced putative kinase -- 1 (PINK1) is a gene associated with an autosomal recessive form of familial PD. PINK1 is important in maintaining mitochondrial integrity and cell viability under oxidative stress, but it remains to be determined whether these functions require direct interaction with mitochondrial targets or work through indirect means. Given the commonality between the cellular dysfunction in neurodegenerative diseases and the protective effects of PINK1 reported in ischemic stroke models, we determined whether PINK1 could be part of a general stress regulatory response.;Utilizing hypoxic stress as a model, we discovered that PINK1 deficiency attenuated induction of Hypoxia inducible factor-1 alpha (Hif-1alpha) in three different cell types including PINK1 deficient murine embryonic fibroblasts (MEFs), primary cortical neuronal cultures, and HeLa cells with reduced level of PINK1 by siRNA compared to wild type (WT) controls. Since Hif-1alpha is a master transcriptional regulator of metabolic and survival genes under hypoxic stress, the effect of PINK1 deficiency on hypoxic response was also examined. Assessment of mRNA levels of three Hif-1alpha target hypoxic response element (HRE) genes, Glucose transporter -- 1 (GLUT-1), Vascular endothelial growth factor a (VEGFa), and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), as well as measurement of luciferase activity of a Hif-1alpha HRE-luciferase construct, showed reduction of HRE promoter activity in PINK1 deficient cells. The data demonstrates that PINK1, through translational regulation of Hif-1alpha HRE-luciferase construct, showed reduction of HRE promoter ac, mediates HRE gene response needed for the hypoxic stress response.;Since PINK1 could have a great impact on the hypoxic stress response, it was imperative to understand the mechanism by which PINK1 regulates Hif-1alpha levels during hypoxic stress. We determined that this attenuation was not attributable to decreased Hif-1alpha mRNA, Hif-1alpha hydroxylation, or proteasome degradation. The degradation rate of Hif-1alpha during hypoxia in the presence of cyclohexamide was independent of PINK1, suggesting that these changes seen in Hif-1alpha were due to alternations in translation. Radiolabeling de novo Hif-1alpha protein synthesis confirmed the reduced Hif-1alpha translation in PINK1 deficient cells compared to WT cells under hypoxic conditions. The cap-dependent translation inhibitor, eukaryotic initiation factor 4E binding protein 1 (4E-BP1) showed a shift towards hyper-phosphorylated forms in PINK1 KO MEFs compared to WT. This data suggests that stress induced cap-independent translation of Hif-1alpha may be hindered in the absence of PINK1. Thus, PINK1 may be necessary for the regulation of stress responses in general that could both have direct and indirect impact on mitochondrial homeostasis and cell survival in PD through Hif-1alpha.