title: Smad3/4 Binding to Promoter II of P450arom So As to Regulate Aromatase Expression in Endometriosis.
abstract: Activin 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. In conclusion, activated Smad3 proteins can bind to P450arom promoter -705/+87 bp region, responsive to activin A in ESCs, which can promote P450arom transcription.
authors.affiliation: 1 State Key Laboratory of Reproductive Medicine, Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China.|2 Department of Obstetrics and Gynecology, Taian Central Hospital, Taian, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Qu
authors.affiliation: 1 State Key Laboratory of Reproductive Medicine, Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: 1 State Key Laboratory of Reproductive Medicine, Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China.
authors.forename: Xiadi
authors.initials: X
authors.lastname: Wu
authors.affiliation: 1 State Key Laboratory of Reproductive Medicine, Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Zheng
authors.affiliation: 1 State Key Laboratory of Reproductive Medicine, Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Hou
authors.affiliation: 1 State Key Laboratory of Reproductive Medicine, Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China.
authors.forename: Yugui
authors.initials: Y
authors.lastname: Cui
authors.affiliation: 1 State Key Laboratory of Reproductive Medicine, Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China.
authors.forename: Yundong
authors.initials: Y
authors.lastname: Mao
authors.affiliation: 1 State Key Laboratory of Reproductive Medicine, Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, Nanjing, China.
authors.forename: Jiayin
authors.initials: J
authors.lastname: Liu
mesh_terms: D028341:Activins / Q000235:genetics / Q000378:metabolism; D001141:Aromatase / Q000235:genetics / Q000378:metabolism*; D004715:Endometriosis / Q000235:genetics / Q000378:metabolism*; D004717:Endometrium / Q000378:metabolism; D005260:Female; D006801:Humans; D011401:Promoter Regions, Genetic; D051900:Smad3 Protein / Q000378:metabolism*; D051901:Smad4 Protein / Q000378:metabolism*; D017154:Stromal Cells / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D051900:Smad3 Protein; D051901:Smad4 Protein; C422405:activin A; D028341:Activins; D001141:Aromatase
keywords: Smad3; activin A; aromatase; endometriosis; transcription