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A4620 - In Hypoxia-Exposed Human Pulmonary Artery Smooth Muscle Cells, Loss of PTEN-Induced Kinase 1 (PINK1) Occurs Through Posttranscriptional Mechanisms and Alters the Cellular Glycolytic Phenotype
Author Block: D. E. Green1, T. C. Murphy1, C. Hart2; 1Medicine, Emory Univ School of Med, Atlanta, GA, United States, 2Atlanta VA Med Ctr/Emory Univ, Decatur, GA, United States.
Rationale: Pulmonary hypertension (PH) is a progressive vasculopathy characterized by dysregulated proliferation of pulmonary artery smooth muscle cells (PASMC). We recently demonstrated that hypoxia reduced the expression of PINK1, an essential mitochondrial outer membrane protein that regulates mitochondrial quality through mitophagy. Loss of PINK1 reduced mitochondrial respiration and enhanced PASMC proliferation. However, the mechanisms by which PINK1 was downregulated by hypoxia and the effects of reduced PINK1 signaling on the glycolytic phenotype of PASMC remain to be defined and will be investigated in the current study.
Methods: C57BL/6J mice and Sprague Dawley rats were exposed to normoxic (21% O2) or hypoxic (10% O2) conditions for 3-5 weeks. Protein was isolated from mouse and rat lungs, and rat pulmonary artery (PA) tissue in vivo, and protein and miRNA was isolated from human PASMC lysates. In parallel, glycolytic function in PINK1-depleted human PASMC was measured using the Seahorse Glycolysis Stress Test. Strategies to rescue PINK1 expression were explored in human PASMC by administering proteasome and PI3Kinase inhibitors (MG132 and LY 29004), and transfecting PASMC with miRNA-27a antisense oligonucleotides.
Results: Expression of PINK1 and its target, the E3 ubiquitin ligase, Parkin was reduced in lung and PA tissues isolated from hypoxia-exposed rats and mice, respectively. Hypoxia-induced reductions in lung and PASMC PINK1 levels were associated with reciprocal increases in miRNA-27a which putatively targets seed sequences in the PINK1 3’ UTR. Hypoxia, or siRNA-induced PINK1 depletion reduced Parkin protein and augmented the glycolytic reserve capacity in human PASMC. Basal PINK1 and Parkin levels were increased by administration of proteasome and lysosomal inhibitors or by transfection PASMC with miRNA-27a inhibitors.
Conclusions: In concert with our previous data that demonstrated that loss of PINK1 enhanced proliferation and apoptosis resistance in PASMC, our current findings demonstrate that hypoxic reductions in PINK1 may be mediated by posttranscriptional mechanisms that involve miRNA-27a, and that loss of PINK1 alters the glycolytic phenotype of PASMC. Inhibitory effects of hypoxia and stimulatory effects of MG132 and LY 29004 on PINK similarly affected Parkin expression, however further studies are needed to determine if alterations in PINK1 modulate Parkin phosphorylation and activity. Future studies are designed to investigate the contribution of other non-coding RNA species to the regulation of PINK1 and to examine the role of PINK1 on the regulation of metabolic programming and mitophagy in proliferating PASMC.