BMP6 Increases TGF-Beta1 Production by Up-Regulating Furin Expression in Human Granulosa-Lutein Cells

Abstract

Background: Bone morphogenetic protein 6 (BMP6) and transforming growth factor--β1 (TGF--β1) are key intraovarian regulators that play essential roles in regulating mammalian follicular function and promoting oocyte maturation. Dysregulated BMP6 or TGF-β1 signaling is associated with polycystic ovary syndrome. Furin is a proprotein convertase that promotes the activation of diverse functional proteins by cleaving protein precursors. Methods: Primary and immortalized (SVOG) human granulosa-lutein (hGL) cells were used as study models. RT-qPCR, western blot analyses, and enzyme immunoassay were used to examine the effects of recombinant human BMP6 on the expression of furin and production of TGF-β1. Dual inhibition approaches (kinase receptor inhibitors and small interfering RNAs targeted knockdown) were used to investigate the underlying molecular mechanisms by which BMP6 regulated the expression of furin. Findings: BMP6 significantly up-regulated the expression of furin and increased the production of TGF-β1. Additionally, both activin receptor-like (ALK)2 and ALK3 were involved in BMP6-induced up-regulation of furin. Furthermore, knockdown of furin abolished BMP6-induced increases in TGF--β1 production, and knockdown of endogenous SMAD4 reversed the increase in furin expression induced by BMP6. Interpretation: The ALK2/3-mediated canonical SMAD signaling pathway is required for the stimulatory effect of BMP6 on furin expression, which in turn increases the production of TGF-β1 in hGL cells. Our findings shed insights into the molecular interactions and mechanisms of two intrafollicular growth factors in hGL cells, which could be potentially applied as therapeutic targets for patients with polycystic ovary syndrome. Funding Statement: Canadian Institutes of Health Research Foundation Scheme Grant #143317. Declaration of Interests: The authors have no conflict of interests to declare. Ethics Approval Statement: All participants signed a written informed consent. The study was approved by Research Ethics Board from the University of British Columbia.