RESUMEN
Expression of pituitary FSH and LH, under the control of pulsatile GnRH, is essential for fertility. cAMP response element-binding protein (CREB) has been implicated in the regulation of FSHß gene expression, but the molecular mechanisms by which pulsatile GnRH regulates CREB activation remain poorly understood. We hypothesized that CREB is activated by a distinct signaling pathway in response to pulsatile GnRH in a frequency-dependent manner to dictate the FSHß transcriptional response. GnRH stimulation of CREB phosphorylation (pCREB) in the gonadotrope-derived LßT2 cell line was attenuated by a protein kinase A (PKA) inhibitor, H89. A dominant negative PKA (DNPKA) reduced GnRH-stimulated pCREB and markedly decreased GnRH stimulation of FSHß mRNA and FSHßLUC activity, but had little effect on LHßLUC activity, indicating relative specificity of this pathway. In perifusion studies, FSHß mRNA levels and FSHßLUC activities were increased by pulsatile GnRH, with significantly greater increases at low compared with high pulse frequencies. DNPKA markedly reduced these GnRH-stimulated FSHß responses at both low and high pulse frequencies. Correlating with FSHß activation, both PKA activity and levels of pCREB were increased to a greater extent by low compared with high GnRH pulse frequencies, and the induction of pCREB was also attenuated by overexpression of DNPKA at both low and high pulse frequencies. Taken together, these data indicate that a PKA-mediated signaling pathway mediates GnRH activation of CREB at low-pulse frequencies, playing a significant role in the decoding of the hypothalamic GnRH signal to result in frequency-dependent FSHß activation.