Effects Of Oxidative Stress-induced Cleavage Of GSK-3β By Calpain On GSK-3β Activity Regulation And Its Underlying Mechanisms

Background:Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine protein kinase, its dysfunction may play an essential role in the pathogenesis of psychiatric, metabolic, neurodegenerative diseases, in which oxidative stress exists concurrently. GSK-3β activity is regulated by serine (inhibitory) and tyrosine (stimulatory) phosphorylation, by insulin and wnt signaling pathways, protein complex formation etc. Our previous studies reported a phenomenon that GSK-3β activity was up-regulated under oxidative stress situation. However, it is ambiguous how oxidative stress regulates GSK-3β activity. Previous studies have reported that N-terminal cleavage of GSK-3by calpain leads to the loss of its inhibitory domain, producing two fragments with kinase activity, which means calpain activation produces a truncation of activated GSK-3.Objective:To investigate the effects of hydrogen peroxide (H2O2) on GSK-3β activity in HEK293/Tau441cell lines, and explore the effects of oxidative stress-induced cleavage of GSK-3β by calpain on its activity regulation and its underlying mechanisms.Methods:After cell culture and drug treatment, we used CCK-8kit to measure cell viability, and measured the expression level of SOD and MDA to evaluate whether the oxidative stress model was successfully established, and then detected GSK-3β activity, used western blot to detect phosphorylation level of GSK-3β at different residues and expression level of associated active forms of kinases that might be involved in GSK-3β phosphorylation; we also used I×71confocal microscopy to detect changes of intracellular calcium concentration in Fura-2-AM labeled HEK293/Tau441cells after administration at different time points, and then used western blot to analysis the cleavages of GSK-3β; finally, we treated cells with hydrogen peroxide and calpain inhibitor IV simultaneously to explore whether the truncation of GSK-3β was calpain-specific.Results:1. GSK-3β activity was increased by twofold in H2O2treated HEK293/Tau441compared with the control group. Surprisingly, H2O2dramatically increased phosphorylation of GSK-3β at serine9, an inactive form of GSK-3β, while there were no obvious changes of phosphorylation of GSK-3β on tyrosine216;2. Fura-2-AM labeled cells detected by confocal microscopy showed that intracellular Ca2+of HEK293/Tau441cells was increased since1min after administration and peaked after3min exposure to H2O2, and western blot results showed GSK-3β was cleavaged into two fragments;3. We treated cells with H2O2and specific calpain inhibitor IV simultaneously, and found that the truncation of GSK-3β was significantly inhibited.Conclusions:These findings suggest that H2O2increases GSK-3β activity through calpain activation caused by calcium influx after administration, which counteracts the inhibitory effects of phosphorylaton of GSK-3β at serine9induced by hydrogen peroxide.