Steroidogenesis in endometriosis: the importance of GATA6

Abstract

The production of steroids from cholesterol is initiated by the concerted effort of multiple factors coordinated in a tissue specific pattern. The same steroidogenic factors are also essential for the development of endometriosis. The PKA pathway and signaling from steroidogenic factor 1 (NR5A1) critically regulate steroidogenic gene expression, and these along with GATA6 have been implicated in the pathogenesis of endometriosis. This study evaluated the combinatorial impact of GATA6 and NR5A1 on steroidogenesis in diseased cells, and expanded these findings to demonstrate how these pathways permit healthy cells to acquire the steroidogenic potential that drives endometriosis. Basic science, in-vitro. Ectopic endometriotic fibroblasts (OSIS), obtained from the cyst walls of endometriomas, and normal eutopic endometrial fibroblasts (NoEM), obtained from the endometrium of subjects without endometriosis, were isolated in accordance with IRB-approved protocols. Human foreskin fibroblasts (BJ) were purchased commercially. NR5A1 and GATA6 were depleted in OSIS by the reverse transfection of gene-specific silencer select siRNAs in comparison to a scrambled siRNA. NoEM and BJ cells, which express little endogenous NR5A1 or GATA6, were transduced with adenoviruses bearing CMV-driven NR5A1, GATA6, or an empty CMV-null control. Expression of steroidogenic genes including steroidogenic acute regulatory protein (StAR), cholesterol side chain cleavage enzyme (CYP11A1), 3-beta-hydroxysteroid dehydrogenase type 2 (HSD3B2), 17α-hydroxylase (CYP17A1), and aromatase (CYP19A1), were assessed using RT-qPCR and immunoblotting. Depleting NR5A1 in OSIS significantly reduced the expression of StAR and CYP11A1, and attenuated HSD3B2, CYP17A1 and CYP19A1 as compared to controls. Depleting GATA6 in OSIS attenuated the PKA-dependent induction of CYP17A1 and CYP19A1 compared to untreated controls. In both NoEM and BJ cells, the expression of StAR and CYP11A1 was induced by NR5A1 alone; however, expression was significantly higher in NoEM than in BJ cells. In contrast, HSD3B2, CYP17A1, and CYP19A1 were synergistically increased by NR5A1 and GATA6 compared to either gene alone in NoEM, while in BJ cells expression of HSD3B2, CYP17A1 and CYP19A1 was minimal. Silencing of NR5A1 and GATA6 in endometriotic tissue leads to decreased expression of key steroidogenic enzymes. In non-endometriotic cells the ability of NR5A1 and GATA6 to work synergistically to increase steroid production depends on a tissue’s inherent potential for steroidogenesis. In conclusion, GATA6 appears to play an important role in the regulation of steroid production and ultimately in the pathogenesis of endometriosis.