GnRH is released from the hypothalamus in a pulsatile manner and binds to specific receptors (GnRHR) in the anterior pituitary gland to stimulate follicle-stimulating hormone (FSH) and luteinizing hormone (LH) subunit gene expression and subsequent FSHβ and LHsecretion. The FSH and LH subunits are preferentially stimulated by pulsatile GnRH at low and high pulse frequencies, respectively. The transcription factors, cAMP response element binding protein (CREB) and inducible cAMP early repressor (ICER), have been implicated in the regulation of rat FSHβ gene expression. We hypothesized that these transcription factors are activated by distinct signaling pathways in response to pulsatile GnRH. GnRH stimulation of the gonadotrope-derived LTβ2 cell line resulted in an increase in CREB phosphorylation within 10 min. This response was attenuated by a PKA inhibitor (H89) in a dose-dependent fashion, whilst PKC (GF109203X), CamKII (KN93) and MEKI/II (U0126) inhibitors had minimal effects. Conversely, GnRH induction of ICER expression was significantly attenuated only by MEKI/II inhbitors U0126 and PD0325901 (highly specific). Experiments using a dominant negative PKA expression vector (DNPKA) demonstrated a significant reduction in GnRH-stimulated CREB phosphorylation, FSHβ mRNA levels, and rat FSHLβUC activity compared to empty vector controls, confirming a role for PKA in pCREB-mediated FSH β transcription by GnRH. In perifusion studies, CREB phosphorylation, FSH β mRNA levels and FSHβLUC activity were increased by pulsatile GnRH, with significantly greater increases at low (every 120 min) compared to high (every 30 min) pulse frequencies. Overexpression of DNPKA reduced GnRH-stimulated pCREB induction at low and high pulse frequencies, in parallel with both FSH βmRNA levels and FSHLβUC activity. We conclude that the signaling pathways mediating GnRH activation of CREB and ICER are distinct. The PKA pathway mediates GnRH-stimulated CREB phosphorylation and FSHβ transcription in response to continuous and pulsatile GnRH at both pulse frequencies, whereas GnRH induction of ICER occurs via MAPK pathways.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.