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Smad3/4 Binding to Promoter II of P450arom So As to Regulate Aromatase Expression in Endometriosis

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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.

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Authors and Affiliations

  1. State Key Laboratory of Reproductive Medicine, Center of Clinical Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University, 300 Guangzhou Road, Gulou District, Nanjing, 210029, China

    Juan Qu MD, Yuanyuan Zhu MD, Xiadi Wu MD, Juan Zheng MD, Zhen Hou PhD, MD, Yugui Cui PhD, Yundong Mao MD, PhD & Jiayin Liu MD, PhD

  2. Department of Obstetrics and Gynecology, Taian Central Hospital, Taian, China

    Juan Qu MD

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  1. Juan Qu MD
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Correspondence to Zhen Hou PhD, MD.

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Qu, J., Zhu, Y., Wu, X. et al. Smad3/4 Binding to Promoter II of P450arom So As to Regulate Aromatase Expression in Endometriosis. Reprod. Sci. 24, 1187–1194 (2017). https://doi.org/10.1177/1933719116681517

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