Metformin inhibits StAR expression in human endometriotic stromal cells via AMPK-mediated disruption of CREB-CRTC2 complex formation.
BACKGROUND
Endometriosis is an estrogen-dependent disease affecting reproductive women. Metformin could have a therapeutic effect on endometriosis through regulation of local estrogen production.


OBJECTIVE
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.
Department of Obstetrics and Gynecology, Peking University First Hospital, Beijing 100034, People's Republic of China.
Jia-Ning
JN
Xu
Cheng
C
Zeng
Yan
Y
Zhou
Chao
C
Peng
Ying-Fang
YF
Zhou
Qing
Q
Xue
D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000328:Adult; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004717:Endometrium / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D006801:Humans; D008687:Metformin / Q000494:pharmacology*; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D017154:Stromal Cells / Q000187:drug effects* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D000098682:Steroidogenic Acute Regulatory Protein
C115163:CREB1 protein, human; C493245:CRTC2 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D010750:Phosphoproteins; D014157:Transcription Factors; D000098682:Steroidogenic Acute Regulatory Protein; D008687:Metformin; D055372:AMP-Activated Protein Kinases