GnRH induces miR-132 and miR-212 and reduces p250RhoGAP and SirT-1 in pituitary LbetaT2 gonadotropes

Gonadotropin releasing hormone (GnRH) is vital to the proper pituitary gonadotrope function as it regulates LH and FSH synthesis and secretion. GnRH causes cell-cycle arrest in pituitary LbetaT2 gonadotrophs, leading to apoptosis. Microarray and q-PCR analyses show that the mouse EST, AK006051, encoding two intronic micro-RNAs, miR -132 and miR-212, is highly induced under GnRH treatment. As miR-132/212 have been linked to neuronal development, we hypothesized that these miRNAs might be linked to cell- cycle arrest in LbetaT2 gonadotrophs. GnRH treatment for increasing times causes a dose-dependent increase in AK006051 promoter activity and a concomitant increase in miR-132/212 expression which is abolished by pretreatment with the adenylate cyclase inhibitor SQ22536 and the MEK inhibitor U0126. Marked increases in both the number and morphology of neurites protruding from LbetaT2 cells are observed after 24h of GnRH treatment. As well, GnRH causes a pulse frequency-dependent increase in AK006051. GnRH inhibits p250RhoGAP expression through a miR-132/212 response element within the 3' UTR of p250RhoGAP. Knockdown of p250RhoGAP by siRNA induces the same morphology observed with GnRH treatment. Since p250RhoGAP stimulates proliferation in fibroblasts and suppresses neurite outgrowth in neurons, our findings suggest that miR-132/212 induction by GnRH is required for cell cycle arrest. We also suggest that apoptosis is due to degradation of SirT-1, another miR-132/212 target, leading to unchecked activation of p53. In conclusion, these data suggest a mechanism by which gonadotrophs synchronize their responses to GnRH leading to coordinated LH and FSH secretion by modulating synaptic-like contacts between gonadotrophs thereby regulating reproductive function