GONADOTROPINS INDUCE RAPID PHOSPHORYLATION OF THE 3',5'-CYCLIC ADENOSINE-MONOPHOSPHATE RESPONSE ELEMENT-BINDING PROTEIN IN OVARIAN GRANULOSA-CELLS

The pituitary gonadotropins FSH and LH exert their effects on gonadal target cells, at least in part, through the activation of adenylyl cyc lase and the production of the second messenger cAMP. To elucidate fur ther the signal transduction pathways regulating gonadotropin-responsi ve genes in ovarian granulosa cells, we have investigated the expressi on of the cAMP response element binding protein (CREB), which mediates many of the effects of cAMP by modulating the transcription of target genes in a cAMP-dependent fashion. In situ hybridization, RNA blot an alysis and RT-PCR RNA, quantification demonstrated that CREB messenger RNA (mRNA) is expressed at low levels throughout the ovary, and that CREB mRNA levels do not change appreciably after gonadotropin stimulat ion. Similar results were obtained using immunohistochemistry and West ern protein blotting to examine CREB protein in ovaries isolated from immature animals treated with gonadotropins or immunocytochemistry and Western protein blotting to examine the CREB protein in cultured gran ulosa cells after gonadotropin treatment. In contrast, immunocytochemi stry and Western protein blotting using an antipeptide antibody specif ic to CREB phosphorylated at serine 133 (P-CREB), which is the activat ed form of the CREB protein, revealed a dramatic increase in the phosp horylated form of CREB within 20 min of gonadotropin treatment of gran ulosa cells that was transient and was decreased by 60 min after gonad otropin treatment. Stimulation of P-CREB was observed using granulosa cells isolated from immature animals and treated with recombinant huma n FSH in vitro, or using granulosa cells isolated from immature animal s primed with PMSG in vivo and treated with human CG (hCG) in vitro. S timulation of P-CREB was also observed in ovarian granulosa cells isol ated from animals treated with PMSG in vivo. These results indicate th at both gonadotropins can induce a rapid and transient phosphorylation of the CREB protein in granulosa cells, leading to the activation of a factor likely to play an important role in the transcription of many gonadotropin-regulated ovarian genes.