Metformin inhibits StAR expression in human endometriotic stromal cells via AMPK-mediated disruption of CREB-CRTC2 complex formation.

Endometriosis is an estrogen-dependent disease affecting reproductive women. Metformin could have a therapeutic effect on endometriosis through regulation of local estrogen production. The aim of this study was to investigate the molecular and cellular mechanism by which metformin regulates StAR expression in human endometriotic stromal cells (ESCs). ESCs derived from ovarian endometriomas were cultured with metformin and prostaglandin E2 (PGE2). StAR mRNA was measured by quantitative PCR; pregnenolone, progesterone, and estrogen production were measured by ELISA kits; steroidogenic acute regulatory protein (StAR), AMP-activated protein kinase, cAMP response element binding protein (CREB), and CREB-regulated transcription coactivator 2 (CRTC2) protein expression were measured by Western blot assay; and CRTC2 translocation and its association with CREB were assessed by coimmunoprecipitation assay and CRTC2-CREB complex binding by a chromatin immunoprecipitation assay. 1) StAR mRNA levels in ESCs are 264 times higher than those in endometrial cells. 2) Metformin downregulates the StAR mRNA expression (maximum 31.7%) stimulated by PGE2 (2.4-fold) in ESCs. 3) PGE2 induces CRTC2 translocation and enhances its association with CREB to form a transcription complex that binds to the StAR promoter region. 4) Metformin prevents the nuclear translocation of CRTC2 by increasing AMP-activated protein kinase phosphorylation. This inhibits transcription of StAR by disrupting formation of the CREB-CRTC2 complex, involved in activation of the StAR promoter cAMP response element. We have demonstrated a detailed mechanistic analysis of StAR expression regulated by metformin in ESCs. Our data highlight a role for CRTC2 in the mechanism by which metformin inhibits StAR expression.