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Follicle-stimulating hormone stimulates beta-catenin via protein kinase B in granulosa cells
Follicle-stimulating hormone regulation of ovarian estradiol production requires involvement of beta-catenin (CTNNB1), a transcriptional co-factor. In cultured bovine granulosa cells, FSH treatment increases protein abundance of CTNNB1 as well as protein kinase B (AKT), a molecule known to regulate components of the CTNNB1 degradation complex. However, whether FSH induction of CTNNB1 is through direct modulation of AKT remains to be determined. To elucidate the effects of AKT signaling on CTNNB1 induction and subsequent estradiol production, bovine granulosa cells were cultured in the presence or absence of known AKT activators and inhibitors. Total protein was collected for analysis by Western blot and culture medium for estradiol quantification by RIA. Values were analyzed using one-way ANOVA procedure of SAS. To investigate specific contributions of AKT to CTNNB1 accumulation, granulosa cells were treated with IGF-1, a well-established AKT activator, in the presence or absence of FSH. Granulosa cells treated with FSH, IGF-1, and FSH+IGF-1 increased (0.68 fold) CTNNB1 accumulation compared to controls (P = 0.09; n = 6). Estradiol medium concentrations increased (P = 0.001; n = 4) in cells treated with FSH, IGF-1, and FSH+IGF-1 (166, 379 and 397%, respectively) compared to non-treated controls. A subsequent study utilizing lithium chloride (LiCl), another activator of the AKT pathway, demonstrated similar results. Granulosa cells were cultured in the presence or absence of LiCl with and without FSH. Consistent with data from IGF-1 treated cells, LiCl, FSH, and FSH+LiCl increased CTNNB1 accumulation (0.79 fold) compared to non-treated controls (P = 0.03; n = 3). In contrast, inhibition of AKT signaling with LY294002 suppressed (P = 0.02; n = 3) CTNNB1 by 1.93 fold compared to controls. Co-treatment of FSH and LY294002 reduced the ability of FSH to increase CTNNB1 (P = 0.03). LY294002 treatment reduced estradiol medium concentrations 1.14 fold when compared to control levels, while co-incubation of FSH+LY294002 and FSH treatment induced estradiol to similar levels above controls (P = 0.0001; n = 4). Results demonstrate activation of AKT is required for CTNNB1 accumulation and estradiol production in bovine granulosa cells. These data suggest that induced CTNNB1 accumulation by FSH and IGF1 activation of AKT may be the lynch pin molecule responsible for FSH and IGF1 synergistic steroidogenic activity.
Keywords:
protein kinase B, Beta-catenin, granulosa cells