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Gonadotropins induce rapid phosphorylation of the 3',5'-cyclic adenosine monophosphate response element binding
A Mukherjee1, O K Park-Sarge, K E Mayo
1Department of Biochemistry, Northwestern University, Evanston, Illinois 60208, USA.
Abstract:
The pituitary gonadotropins FSH and LH exert their effects on gonadal target cells, at least in part, through the activation of adenylyl cyclase and the production of the second messenger cAMP. To elucidate further the signal transduction pathways regulating gonadotropin-responsive genes in ovarian granulosa cells, we have investigated the expression 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 analysis 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 stimulation. Similar results were obtained using immunohistochemistry and Western 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 granulosa cells after gonadotropin treatment. In contrast, immunocytochemistry and Western protein blotting using an antipeptide antibody specific to CREB phosphorylated at serine 133 (P-CREB), which is the activated from of the CREB protein, revealed a dramatic increase in the phosphorylated form of CREB within 20 min of gonadotropin treatment of granulosa cells that was transient and was decreased by 60 min after gonadotropin treatment. Stimulation of P-CREB was observed using granulosa cells isolated from immature animals and treated with recombinant human FSH in vitro, or using granulosa cells isolated from immature animals primed with PMSG in vivo and treated with human CG (hCG) in vitro. Stimulation of P-CREB was also observed in ovarian granulosa cells isolated from animals treated with PMSG in vivo. These results indicate that 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.
Insights
Pituitary gonadotropins rapidly and temporarily increase phosphorylated cAMP response element binding protein (P-CREB) in ovarian granulosa cells, suggesting its crucial role in regulating gene transcription.
Area of Science:
- Reproductive Endocrinology
- Molecular Biology
- Cell Signaling
Background:
- Pituitary gonadotropins (FSH and LH) signal through cAMP in gonadal cells.
- The cAMP response element binding protein (CREB) mediates cAMP effects on gene transcription.
- Understanding CREB's role in ovarian granulosa cells is key to gonadotropin action.
Purpose of the Study:
- To investigate CREB expression and activation in ovarian granulosa cells following gonadotropin stimulation.
- To elucidate the role of CREB phosphorylation in gonadotropin-regulated gene expression.
Main Methods:
- In situ hybridization, RNA blot analysis, and RT-PCR for CREB mRNA.
- Immunohistochemistry and Western blotting for CREB and phosphorylated CREB (P-CREB) protein.
- In vitro and in vivo gonadotropin treatments of granulosa cells and ovarian tissues.
Main Results:
- CREB mRNA and total CREB protein levels remained unchanged after gonadotropin stimulation.
- Gonadotropin treatment induced a rapid (20 min), transient increase in P-CREB in granulosa cells.
- Both FSH and LH/hCG stimulated P-CREB, indicating rapid activation of CREB.
Conclusions:
- Gonadotropins rapidly and transiently phosphorylate CREB in ovarian granulosa cells.
- Activated P-CREB is likely a critical transcription factor for gonadotropin-regulated ovarian genes.
- This pathway highlights a key mechanism in ovarian follicle development and function.