Purchase this article with an account.
Morgan M. Brooks, Sudha Neelam, Rafal Fudala, Ignacy Gryczynski, Patrick R. Cammarata; Lenticular Mitochondrial Protection, Part 2: Elevated BAX Overrides GSK-3β and Induces Mitochondrial Depolarization in Atmospheric Oxygen. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1059.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Cardioprotection literature suggests that glycogen synthase kinase-3β (GSK-3β) is a crucial enzyme regulating mitochondrial permeability membrane transition (mPMT). Using direct inhibition with the GSK-3β inhibitor, SB216763, and indirect inhibition of GSK-3β with the MEK inhibitor, U0126, the circumstances by which GSK-3β dictates mPMT with human lens epithelial (HLE-B3) cells were determined.
Specific antisera and western blot analyses was used to detect BAX, ERK1/2, GSK-3β and glycogen synthase, GS (a substrate of GSK-3β) and pGS (useful in monitoring GSK-3β’s activity). JC-1 fluorescent dye analysis was employed to determine the extent of mitochondrial depolarization.
SB216763 (12µM) inactivated GSK-3β as verified by the lack of pGS. The BAX levels were unchanged for SB-treated and control cells. SB-treated cells, when challenged by oxidative stress, failed to depolarize relative to control cells. Such mitochondrial protection is consistent with the inactivity of GSK-3β blocking mPMT by preventing the opening of the mitochondrial permeability transition pore. Treatment of HLE-B3 cells with UO126 (10µM) inhibited ERK and GS phosphorylation, indicating that GSK-3β is inactivated in the absence of pERK. Unlike SB treatment, UO126 profoundly increased BAX levels over time relative to control cells. JC-1 analysis showed that UO-treated cells displayed substantially increased depolarization relative to control cells.
Addition of either SB216763 or U0126 to HLE-B3 cells, maintained in atmospheric oxygen, elicited the inactivation of GSK-3β. However, whereas SB-treatment affords mitochondrial protection upon oxidative stress, UO-treatment under similar conditions displays significant mPMT, indicating that despite the fact that inactivation of GSK-3β should have prevented the opening of the transition pore, profound mitochondrial membrane depolarization nonetheless occurred. The answer lies in the fact that BAX levels substantially increased with UO-treatment but not with SB-treatment. BAX is a pro-apoptotic member of Bcl-2 family and its translocation to the mitochondrial outer membrane prompts mPMT by opening of the mitochondrial permeability transition pore. In summary, an elevation in BAX levels induces mitochondrial depolarization because it supersedes the lenticular mitoprotection normally afforded by the inactivation of GSK-3β activity.
This PDF is available to Subscribers Only