Metformin Inhibits StAR Expression in Human Endometriotic Stromal Cells via AMPK-mediated Disruption of CREB-CRTC2 Complex Formation

J Clin Endocrinol Metab. 2014 Aug;99(8):2795-803. doi: 10.1210/jc.2014-1593. Epub 2014 May 13.

Abstract

Context: Endometriosis is an estrogen-dependent disease affecting reproductive women. Metformin could have a therapeutic effect on endometriosis through regulation of local estrogen production.

Objects: 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).

Methods: 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.

Results: 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.

Conclusions: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / metabolism*
  • Adult
  • Cells, Cultured
  • Cyclic AMP Response Element-Binding Protein / metabolism
  • Down-Regulation / drug effects
  • Down-Regulation / genetics
  • Endometrium / drug effects*
  • Endometrium / metabolism
  • Female
  • Humans
  • Metformin / pharmacology*
  • Phosphoproteins / genetics*
  • Phosphoproteins / metabolism
  • Phosphorylation / drug effects
  • Protein Binding / drug effects
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Stromal Cells / drug effects*
  • Stromal Cells / metabolism
  • Transcription Factors / metabolism

Substances

  • CREB1 protein, human
  • CRTC2 protein, human
  • Cyclic AMP Response Element-Binding Protein
  • Phosphoproteins
  • Transcription Factors
  • steroidogenic acute regulatory protein
  • Metformin
  • AMP-Activated Protein Kinases