{"paper_id":"622852fa-c7f6-457d-aba8-41717f83af92","body_text":"Abstract\nActivin A can stimulate aromatase P450 (P450arom) expression in eutopic endometrial stromal cells (ESCs) of endometriosis by activin type I receptor-Smad pathway. In order to identify Smad3/4 binding to P450arom promoter II that mediates activin A response in ESCs, polymerase chain reaction (PCR) products of a serial truncated deletion of the P450arom promoter II region between —904 and +87 bp were inserted into the pGL3-basic vector to generate the promoter reporter plasmids. Luciferase reporter plasmids were cotransfected into cells with or without activin A (25 ng/mL). The pGL3 —705/+87 revealed a luciferase activity similar to pGL3 —904/+87, whereas progressive truncation to position —464/+87 and — 192/+87, the luciferase activity was significant variation. Chromatin immunoprecipitation assay and Smad4-small interfering RNA (siRNA) testify that Smad3/4 binds to the activin A-responsive aromatase promoter in ESCs. Chromatin immunoprecipitation assay-PCR assay demonstrated anti-Smad3 antibody complexes could interact with the amplified DNA of the activin A-responsive P450arom promoter. Mutations of the binding site (—141/— 138 bp, — 165/— 162 bp) in P450arom promoter II significantly reduced promoter activity of activin A fold-induction to 26% and 28%, respectively. We cotransfected pGL3 —705/+87 with control siRNA and Smad4-siRNA into ESCs in the presence of activin A. Luciferase analysis showed that Smad4-siRNA abolished increased promoter activity of activin A-induced P450arom expression. The effect of activin A on the p-Smad3 accumulation in the cytoplasm and nucleus was significantly abrogated following the pretreatment of ESCs with Smad4-siRNA. 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