YB1: A New Player in FSH Regulation of Gene Expression in Granulosa Cells
Donaubauer, Elyse Marie
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Within the ovarian follicle, immature oocytes are surrounded and supported by granulosa cells (GCs). Stimulation of GCs by follicle stimulating hormone (FSH) promotes their proliferation and differentiation, events that are necessary for fertility. FSH activates multiple signaling pathways to regulate genes necessary for follicular maturation. This research focuses on the regulation by FSH of extracellular signal-regulated kinase (ERK) and identifies Y-box binding protein-1 (YB-1) as a downstream component of ERK signaling. FSH-dependent ERK(Thr202/Tyr204) phosphorylation is protein kinase A (PKA) dependent, yet requires the constitutively active upstream ERK signaling pathway proteins. Treatment with EGFR inhibitor AG1478, a dominant-negative RAS, and the MEK inhibitor PD98059 all blocked FSH-dependent ERK(Thr202/Tyr204) phosphorylation. We hypothesized that FSH via PKA regulates ERK phosphorylation by inhibiting the activity of a protein phosphatase that dephosphorylates ERK in the absence of FSH. Results show that treatment with MAP kinase phosphatase 3 (dual specificity phosphatase 6) (MKP3(DUSP6)) inhibitors increase ERK(Thr202/Tyr204) phosphorylation in the absence of FSH to levels comparable to ERK phosphorylated in the presence of FSH. Further, ERK coimmunoprecipitated with MKP3(DUSP6), and treatment with MKP3(DUSP6) inhibitors blocked dephosphorylation of recombinant phospho-ERK2-GST. Together these results indicate that MKP3(DUSP6) is the phosphatase that dephosphorylates ERK in the absence of FSH. We further identified YB-1 as a downstream target of ERK. YB-1 is a nucleic acid binding protein that regulates transcription and translation. Our results show that FSH promotes an increase in the phosphorylation of YB-1 on Ser102 within 15 min that is maintained until ~8 h post treatment. FSH-stimulated phosphorylation of YB-1(Ser102) is prevented by pretreatment of GCs with the PKA-selective inhibitor PKI, the MEK inhibitor PD98059, and the ribosomal S6 kinase-2 (RSK-2) inhibitor BI-D1870. Transduction of GCs with the dephospho-adenoviral-YB-1(S102A) mutant prevented the FSH-dependent induction of Egfr, Cyp19a1, and Inha mRNAs. Collectively, these results demonstrate novel regulation of ERK(Thr202/Tyr204) phosphorylation by FSH and identify ERK-dependent gene targets that are required for follicular maturation. These results reveal that the ERK signaling pathways contributes to GC maturation by promoting the phosphorylation of the transcriptional activator YB-1 that is required for expression of at least three crucial FSH target genes.