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.

Involvement of Transcription Factor 21 in the Pathogenesis of Fibrosis in Endometriosis

“…Endometriosis is a benign disease going through tumor-like processes to form endometriotic lesions. Tumor-like processes include aggression, 5 evasion [epithelial-mesenchymal transition (EMT)], 6,7 adhesion (CD10 and integrins), 8 invasion (matrix metalloproteinases), 9 angiogenesis (vascular endothelial growth factor), 10,11 surviving (hormones and aromatase), 12 fibroproliferation, 13 and inflammation (cytokines). 14,15 Although these mechanisms have been studied for a long time, more research is still needed as the current medical therapies have many adverse effects and are not plausible because of the high recurrence rate after performing conservative surgery.…”

Platelets induce increased estrogen production through NF-κB and TGF-β1 signaling pathways in endometriotic stromal cells

“…The result of 4.5 fold increase in estrogen production in endometriotic stromal cells stimulated with activated platelets vs. those unstimulated is remarkably close to the reported fold increase found between testosterone-added endometriotic and endometrial stromal cells (4.1 for follicular phase and 4.5 for luteal phase) 41 . Previous studies have reported that the TGF-β1/Smad3 signaling pathway is involved in the transcriptional regulation of aromatase in several cell types [56][57][58] , and particularly in endometriotic stromal cells 59 . www.nature.com/scientificreports www.nature.com/scientificreports/ Therefore, our finding is also consistent with these reports.…”

Tumor necrosis factor alfa and interleukin 1 alfa induced phosphorylation and degradation of inhibitory kappa B alpha are regulated by estradiol in endometrial cells

“…On the other hand, persistently low levels of IkBα immunoreactivity in ectopic endometrial cells are likely to be related to the increased local inflammation observed in endometriosis and may contribute to the increased inflammatory cytokine levels in the peritoneal cavity of women with endometriosis ( 60 , 61 ) . Endometriosis is an estrogen-dependent disease and implants of endometriosis have sufficient enzymes for the local production of estrogen ( 54 , 59 , 62 , 63 , 64 ) . The low levels of IkBα in ectopic endometrial cells suggest that the signaling effects of estrogen on IkBα may function similarly to those observed in eutopic endometrium.…”