Stimulation of the Canonical WNT-Signaling Pathway Inhibits Follicle-Stimulating Hormone Responsive Genes in Rat Granulosa Cells.

Abstract

Beta-catenin, a key component of WNT signaling, participates in FSH mediated regulation of estrogen production. The purpose of these studies was to determine if β-catenin'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 recombinant mouse WNT3A (500 ng/ml) for 24 h before treatment with vehicle or FSH (100 ng/ml) for an additional 24 h (n = 5). Steroidogenic enzymes and ovarian differentiation factor mRNAs were quantified using real-time PCR. Expression of steroidogenic enzyme mRNAs aromatase (Cyp19a1), P450 side chain cleavage (Cyp11a1), and steroidogenic acute regulatory protein (Star) were increased (P < 0.05) following FSH treatment. Co-incubation of FSH and WNT3A reduced the ability of FSH to stimulate Cyp19a1 mRNA expression (P < 0.01) and Cyp11a1 and Star expression (P = 0.07). Subsequent steroid production demonstrated a parallel effect as FSH-induced estradiol was reduced from 559 pg/ml to 75.5 (± 131) pg/ml (P = 0.06) and progesterone was reduced from 1.51 ng/ml to 0.12 ng/ml (± 0.22) ng/ml (P < 0.01) with addition of WNT3A. Concomitant activation of FSH and WNT pathways results in marked reduction of LH receptor (P = 0.02) but not inhibin-alpha (P = 0.28). Additionally, WNT3A alone down regulated FSH receptor mRNA expression compared to controls (P < 0.01). To ascertain the contribution of WNT3A to suppression of the steroidogenic enzymes and differentiation factors, we used a known inhibitor of the WNT pathway, Dickkopf (DKK). Primary cultures of rat granulosa cells were pretreated with vehicle or recombinant human DKK4 (5μg/ml) before treatment with WNT3A (n = 2). Pretreatment of DKK4 preceding activation of the WNT signaling pathway with WNT3A, resulted in a restored ability of FSH to stimulate expression of the steroidogenic enzymes. Consequent production of estradiol and progesterone concentrations in cell culture media were also reestablished from 27.5 pg/ml to 430 (± 113) pg/ml (P < 0.01) and 0.02 ng/ml to 1 (± 0.26) ng/ml (P < 0.05), respectively following disruption of the WNT pathway by DKK4 when WNT3A and FSH were co-incubated when compared to vehicle treated controls. Expression of ovarian derived differentiation factor target genes, LH receptor, and inhibin-alpha returned to levels greater than controls in granulosa cells exposed to DKK before co-incubation with WNT3A and FSH (P = 0.01). FSH receptor mRNA expression returned to control levels when DKK disrupted WNT signaling before treatment of WNT3A. Forkhead box protein O1 (FoxO1) mRNA expression was analyzed as a potential mechanism for suppressing steroidogenic enzymes and differentiation factor mRNAs. FoxO1 mRNA expression was increased by WNT3A alone (P < 0.05) and in combination with FSH (P = 0.07). Disruption of WNT signaling resulted in a reduction of FoxO1 mRNA. Data suggest WNT signaling inhibits FSH target genes associated with maturation and differentiation of the ovarian follicle. A likely mechanism for WNT3A inhibition of FSH signaling is reduction of FSH receptor occurring through increased FOXO1, a repressor of FSH target genes. Supported in part by NIH (R15065668 to JAHG) and Oklahoma Center for the Advancement of Science and Technology (HR10-030S to JAHG).