Date Published: January 17, 2014
Publisher: Public Library of Science
Author(s): Andrea D. Stapp, Belinda I. Gómez, Craig A. Gifford, Dennis M. Hallford, Jennifer A. Hernandez Gifford, John Davis.
Beta-catenin (CTNNB1), a key component of wingless-type mouse mammary tumor virus integration site family (WNT) signaling, participates in follicle stimulated hormone-mediated regulation of estrogen (E2) production. The purpose of these studies was to determine if CTNNB1’s contribution to FSH-mediated steroidogenesis in primary rat granulosa cells was due in part to extracellular stimulation of the canonical WNT signaling pathway. To achieve this purpose, primary cultures of rat granulosa cells were exposed to vehicle or a canonical member of the WNT signaling pathway, WNT3A, before co-culture and in the presence or absence of FSH for 24 h. Activation of the canonical WNT signaling pathway was determined by dose-dependent induction of Axin2 mRNA expression and stimulation of the CTNNB1/T cell factor promoter-reporter TOPflash. WNT pathway induction was demonstrated at doses of 50 and 500 ng/mL of WNT3A. Granulosa cells treated with WNT3A in combination with FSH had enhanced CTNNB1/T cell factor transcriptional activity above cells treated with WNT3A alone. Steroidogenic enzymes and ovarian differentiation factor mRNAs were quantified via quantitative PCR. Expression of steroidogenic enzyme mRNAs aromatase (Cyp19a1), P450 side chain cleavage (Cyp11a1), and steroidogenic acute regulatory protein (Star) were increased following FSH treatment. Co-incubation of WNT3A and FSH reduced the ability of FSH to stimulate steroidogenic enzymes and subsequent E2 and progesterone (P4) production. Concomitant activation of FSH and WNT pathways results in marked reduction of ovarian differentiation factors, LH receptor (Lhcgr) and inhibin-alpha (Inha). Therefore, WNT inhibits FSH target genes and steroid production associated with maturation and differentiation of the ovarian follicle.
The wingless-type mouse mammary tumor virus integration site (WNT) family of secreted glycoproteins participates in a variety of cellular processes including embryonic induction, axis specification, cell fate determination and differentiation , . WNT ligands can activate three distinct intracellular signaling pathways which result in different biological activities. However, the most well studied WNT pathway is the canonical WNT signaling cascade which signals through the transcriptional co-factor, β-catenin (CTNNB1) to regulate target gene expression , . Members of the canonical WNT signaling pathway are generally classified by their ability to transform mammary epithelial cell lines and include WNT-1,-2,-3A and -8 , . Canonical WNTs are important in tissue homeostasis and recognized for their role in controlling cellular decisions to proliferate and differentiate –. However, mis-regulation of WNT/CTNNB1 signaling is linked to a range of pathologies including cancers of the breast, colon, and skin .
The ability of FSH to facilitate maturation of ovarian follicles and synthesis of follicular E2 relies on the input from a variety of signaling molecules , . Secreted WNT glycoproteins have been identified as regulators of ovarian cell function and follicular organization. The Wnt family of genes are hormonally regulated in rodent and bovine ovaries. In rodent ovaries, Wnt4 expression is elevated in response to human chorionic gonadotropin and highly expressed in terminally differentiated luteal cells . Likewise, we reported that bovine granulosa cells demonstrate an upregulation of Wnt2 mRNA expression following FSH stimulation . The canonical WNT pathway relies on activation of the downstream effector, CTNNB1 to transduce a signal. A requirement for CTNNB1 in FSH regulation of key steroidogenic enzymes has been identified in primary culture of rat granulosa cells , and reduction of CTNNB1 resulted in a compromised ability of FSH to stimulate Cyp19a1 mRNA and subsequent E2 production . Additionally, granulosa cells of large bovine antral follicles producing high amounts of E2 demonstrated an increase in CTNNB1 accumulation compared to low E2 producing follicles . Collectively, these data suggest FSH and WNT signaling pathways may work together to impact steroid production in the postnatal ovary. Surprisingly, our data provide novel evidence indicating activation of canonical WNT signaling inhibits expression of FSH target genes associated with regulation of follicle maturation and steroid hormone production. Specifically, exogenous stimulation of primary granulosa cells with recombinant WNT3A effectively mutes the ability of FSH to regulate transcription of the steroidogenic enzymes Cyp19a1, Star and Cyp11a1 and decreases production of both E2 and P4. WNT3A is a member of the canonical WNT family of secreted molecules and is recognized for its’ ability to stabilize CTNNB1 protein which accumulates and enters the nucleus to activate transcription of TCF/LEF target genes. WNT3A is expressed in postnatal ovaries of mice  and cattle  and therefore may be involved in regulating ovarian gene expression. An interaction between WNT signaling and G-protein coupled gonadotropin receptors is evident and appears to be dependent on stage-specific development of the ovarian follicle. The negative regulation of WNT signaling on gonadotropin stimulation of steroidogenic enzymes and ovarian differentiating factors is consistent with previous studies in which granulosa cells from mice expressing dominant stable CTNNB1 had reduced expression of Lhcgr, Star, and Cyp11a1 following forskolin-induced cAMP activation and PMA-activated PKC signaling . The suggestion by Fan et al.  that overactivation of CTNNB1 is responsible for the negative effects on LH-induced events is probable given our findings that the greatest observed increase in transcriptional activity of the CTNNB1/TCF responsive promoter (TOPflash) occurred in cells stimulated by WNT and FSH at doses capable of muting steroid synthesis. Our data show that WNT3A and FSH act synergistically to activate TOPflash, whereas WNT3A blunts the FSH response on other gene targets. These data highlight the importance of promoter context as TOPflash is an artificial minimal promoter composed of several TCF response elements. It is not likely that a minimal promoter will mimic native promoters that are composed of many different response elements. TOPflash is simply a positive control demonstrating WNT3A activity, especially when added with FSH.