Oxidative stress-induced downregulation of glycogen synthase kinase 3 beta in fetal membranes promotes cellular senescence

Objective: Oxidative stress (OS)-induced stress signaler p38 mitogen-activated protein kinase (p38MAPK) activation and fetal membrane senescence are associated with parturition. This study determined changes in glycogen synthase kinase 3 beta (GSK3 beta) and its regulation by p38MAPK in effecting senescence to further delineate the molecular mechanism involved in senescence. Methods: Primary human amnion epithelial cells and amnion mesenchymal cells were treated with cigarette smoke extract (CSE, OS inducer). Expression of total and phosphorylated GSK3 beta and p38MAPK, and that of GSK3 beta's downstream targets: beta-catenin (beta-Cat) and nuclear factor erythroid 2-related factor 2 (Nrf2) (western blot analysis), cell cycle regulation and senescence (flow cytometry) were determined. The specificity of GSK3 beta and p38MAPK's mechanistic role was tested by co-treating cells with their respective inhibitors, CHIR99021 and SB203580. Exosomal secretion of beta-Cat from OS-induced cells was confirmed by immunofluorescence confocal microscopy and western blot. Results: OS induced by CSE resulted in phosphorylation of GSK3 beta (inactivation) and p38MAPK (activation) that was associated with cell cycle arrest and senescence. Inhibitors to GSK3 beta and p38MAPK verified their roles. Glycogen synthase kinase 3 beta inactivation was associated with nuclear translocation of antioxidant Nrf2 and exosomal secretion of beta-Cat. Conclusions: OS-induced P-p38MAPK activation is associated with functional downregulation of GSK3 beta and arrest of cell cycle progression and senescence of amnion cells. Lack of nuclear translocation of beta-Cat and its excretion via exosomes further supports the postulation that GSK3 beta down-regulation by p38MAPK may stop cell proliferation preceding cell senescence. A better understanding of molecular mechanisms of senescence will help develop therapeutic strategies to prevent preterm birth.