GSK3β Inhibition Is the Molecular Pivot That Underlies the Mir-210-Induced Attenuation of Intrinsic Apoptosis Cascade during Hypoxia

Abstract

Apoptotic cell death is a deleterious consequence of hypoxia-induced cellular stress. The master hypoxamiR, microRNA-210 (miR-210), is considered the primary driver of the cellular response to hypoxia stress. We have recently demonstrated that miR-210 attenuates hypoxia-induced apoptotic cell death. In this paper, we unveil that the miR-210-induced inhibition of the serine/threonine kinase Glycogen Synthase Kinase 3 beta (GSK3β) in AC-16 cardiomyocytes subjected to hypoxia stress underlies the salutary protective response of miR-210 in mitigating the hypoxia-induced apoptotic cell death. Using transient overexpression vectors to augment miR-210 expression concomitant with the ectopic expression of the constitutive active GSK3β S9A mutant (ca-GSK3β S9A), we exhaustively performed biochemical and molecular assays to determine the status of the hypoxia-induced intrinsic apoptosis cascade. Caspase-3 activity analysis coupled with DNA fragmentation assays cogently demonstrate that the inhibition of GSK3β kinase activity underlies the miR-210-induced attenuation in the hypoxia-driven apoptotic cell death. Further elucidation and delineation of the upstream cellular events unveiled an indispensable role of the inhibition of GSK3β kinase activity in mediating the miR-210-induced mitigation of the hypoxia-driven BAX and BAK insertion into the outer mitochondria membrane (OMM) and the ensuing Cytochrome C release into the cytosol. Our study is the first to unveil that the inhibition of GSK3β kinase activity is indispensable in mediating the miR-210-orchestrated protective cellular response to hypoxia-induced apoptotic cell death.

NF-κB Transcriptional Activity Indispensably Mediates Hypoxia–Reoxygenation Stress-Induced microRNA-210 Expression

“…The quantitative abundance of the miR-210 promoter pull-down fragment in the experimental groups was determined by adopting a novel ELOHA approach [ 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 ]. Briefly, the purified pulled-down miR-210 promoter ( Section 4.6 ) was affinity captured on streptavidin beads using hybridization with biotin-labeled miR-210 promoter capture probe (Eurofins Genomics, Ebersberg, Germany).…”

“…The levels of miR-210 in the experimental cell lysates were determined by adopting a novel microRNA immunoassay approach [ 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 ], as previously described [ 16 , 104 ]. This miR-210 immunoassay approach allowed the direct quantitative determination of miR-210 in the same experimental lysates being subjected to specific downstream assays.…”