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.
Problem: Senescence of the fetal membranes and senescence-associated inflammation have been associated with parturition at term and pre-term in both mice and humans. Using a pregnant mouse model, we determined changes in multiple molecular signalers contributing to senescence and inflammation associated with parturition.
Method of study:Fetal membranes were collected from timed-pregnant CD-1 mice on gestation days (E) 13, 15, 17, 18, and 19. Immunohistochemistry (IHC) localized pro-cell growth factors glycogen synthase kinase 3β (GSK3β) and β-catenin. Gestational age-associated changes in pro-cell growth vs senescence mediators (p38 mitogen-activated protein kinase [p38MAPK]), prooxidants (heme oxygenase-1 [HO-1], peroxisome proliferator-activated receptor γ [PPARγ]), and pro-and anti-inflammatory cytokines (IL-6, IL-8, IL-10, and IL-1β) were determined by Western blots and Luminex assays. Results: Fetal membrane expressions of phosphorylated forms of GSK3β (inactivation) and p38MAPK (activation) increased, while β-catenin expression decreased, as gestation progressed. Antioxidant HO-1 expression decreased while PPARγ increased toward term gestation. IL-6 and IL-8 concentrations were highest on E19 (day of delivery), while IL-10 and IL-1β concentrations were highest on E15. Conclusion: Mouse fetal membranes showed a progressive senescence marker increase coincided with downregulation of cell growth factors. Development of senescence is associated with inflammation. Senescence-associated changes are natural and physiologic and indicative of fetal membranes' readiness for parturition.
K E Y W O R D Sfetal membranes, GSK3β, oxidative stress, p38MAPK, pre-term labor, senescence
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.