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

Abstract

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 (GSK3β) 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 GSK3β and p38MAPK, and that of GSK3β’s downstream targets: beta-catenin (β-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 GSK3β and p38MAPK’s mechanistic role was tested by co-treating cells with their respective inhibitors, CHIR99021 and SB203580. Exosomal secretion of β-Cat from OS-induced cells was confirmed by immunofluorescence confocal microscopy and western blot. Results OS induced by CSE resulted in phosphorylation of GSK3β (inactivation) and p38MAPK (activation) that was associated with cell cycle arrest and senescence. Inhibitors to GSK3β and p38MAPK verified their roles. Glycogen synthase kinase 3 beta inactivation was associated with nuclear translocation of antioxidant Nrf2 and exosomal secretion of β-Cat. Conclusions OS-induced P-p38MAPK activation is associated with functional downregulation of GSK3β and arrest of cell cycle progression and senescence of amnion cells. Lack of nuclear translocation of β-Cat and its excretion via exosomes further supports the postulation that GSK3β 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.

Changes in mediators of pro‐cell growth, senescence, and inflammation during murine gestation

“…The IHC findings in CD‐1 mice were similar to those seen in humans where GSK3β (phosphorylated and total forms) and β‐catenin were localized to all layers (amnion and chorion) of laboring as well as non‐laboring fetal membranes …”

“…To localize proteins of interest in fetal membrane tissues, IHC was performed as previously described . Briefly, fetal membranes collected from CD‐1 mice were fixed in 4% paraformaldehyde at 4°C overnight.…”

“…Fetal membranes collected on different gestational days were lysed with RIPA lysis buffer (50 mmol/L Tris pH 8.0, 150 mmol/L NaCl, 1% Triton X‐100, and 1.0 mmol/L EDTA pH 8.0, 0.1% SDS) supplemented with protease and phosphatase inhibitor cocktail and phenylmethylsulfonyl fluoride as described previously by our laboratory . A Pierce BCA Protein Assay Kit (Thermo Scientific) was utilized to determine protein concentration of samples, and 45 µg of tissue lysate was used for loading the gels for Western blotting, which was performed as previously described by our laboratory .…”

“…Senescence of fetal membranes and a buildup of inflammation within the uterine cavity are plausible factors associated with both term and pre‐term labor . Recently, we reported that glycogen synthase kinase 3β (GSK3β) can also contribute to p38MAPK‐mediated senescence in human fetal membranes . GSK3β is a multifunctional kinase that plays an important role in cell growth and maintaining cellular homeostasis .…”

“…β‐catenin is an important downstream target of GSK3β, and as an integral part of the Wnt pathway, it is also an effecter of cell growth. We have recently demonstrated that activation of p38MAPK in response to OS, in amnion cells, causes phosphorylation leading to inactivation of GSK3β, which may ultimately contribute to cell senescence . In response to OS, GSK3β in activation coincides with the shipment of β‐catenin out of amnion cells packaged within extracellular vesicles, which may indirectly promote cellular senescence …”