High glucose-induced p66Shc mitochondrial translocation regulates autophagy initiation and autophagosome formation in syncytiotrophoblast and extravillous trophoblast.

BACKGROUND: p66Shc, as a redox enzyme, regulates reactive oxygen species (ROS) production in mitochondria and autophagy. However, the mechanisms by which p66Shc affects autophagosome formation are not fully understood. METHODS: p66Shc expression and its location in the trophoblast cells were detected in vivo and in vitro. Small hairpin RNAs or CRISPR/Cas9, RNA sequencing, and confocal laser scanning microscope were used to clarify p66Shc's role in regulating autophagic flux and STING activation. In addition, p66Shc affects mitochondrial-associated endoplasmic reticulum membranes (MAMs) formation were observed by transmission electron microscopy (TEM). Mitochondrial function was evaluated by detected cytoplastic mitochondrial DNA (mtDNA) and mitochondrial membrane potential (MMP). RESULTS: High glucose induces the expression and mitochondrial translocation of p66Shc, which promotes MAMs formation and stimulates PINK1-PRKN-mediated mitophagy. Moreover, mitochondrial localized p66Shc reduces MMP and triggers cytosolic mtDNA release, thus activates cGAS/STING signaling and ultimately leads to enhanced autophagy and cellular senescence. Specially, we found p66Shc is required for the interaction between STING and LC3II, as well as between STING and ATG5, thereby regulates cGAS/STING-mediated autophagy. We also identified hundreds of genes associated several biological processes including aging are co-regulated by p66Shc and ATG5, deletion either of which results in diminished cellular senescence. CONCLUSION: p66Shc is not only implicated in the initiation of autophagy by promoting MAMs formation, but also helps stabilizing active autophagic flux by activating cGAS/STING pathway in trophoblast.

ARID1A recruits GATA2 to regulate the senescence of trophoblast cells under high-glucose condition.

INTRODUCTION: Gestational diabetes mellitus (GDM) is a common complication during pregnancy. The hyperglycemic stimulation of gestational diabetes inhibits the invasion of the placental trophoblast cells. Some studies have indicated that the senescence of trophoblast cells weakens their invasive capacity, while the mechanism of trophoblast cells senescence in GDM remain elusive. METHODS: We performed western blotting and Immunohistochemical staining to investigate AT-Rich Interaction Domain 1A (ARID1A) expression in GDM placental tissues. 5 mM and 30 mM glucose treated HTR-8/SVneo cells to simulate normal glucose (NG) stress and high glucose (HG) stress. Cell proliferation capacity was investigated by CCK8 assay and cell cycle assay. SA-ß-gal was used to detect cellular senescence. Chip-seq characterized the binding site of ARID1A to CDKN1A. In conjunction with bioinformatics analysis, co-immunoprecipitation assays, Chip-qPCR and luciferase reporter assays were performed to prove ARID1A recruits GATA2 to CDKN1A. RESULTS: We found that ARID1A has a higher expression levels in GDM placental tissues compared to the control. ARID1A overexpression suppressed cell proliferation, induced cell cycle arrest and promoted cell senescence. Conversely the inhibition of ARID1A significantly rescues HG induced senescence of trophoblast cells. To further characterize the mechanism by which ARID1A regulate the transcription of CDKN1A, co-immunoprecipitation assays, Chip-qPCR and luciferase reporter assay indicate that ARID1A recruits GATA2 to regulate the transcriptional activity of CDKN1A. DISCUSSION: Our study uncovers a ARID1A mediated regulatory mechanism in GDM trophoblast cell senescence and suggests that targeting the placental ARID1A might provide new diagnostic and therapeutic strategies for GDM.