Gonadotropin-releasing hormone activates the equine luteinizing hormone beta promoter through a protein kinase C/mitogen-activated protein kinase pathway

GnRH regulation of LH secretion is well understood and involves Ca2+ mobili zation. However, the mechanism by which GnRH activates transcription of the LH beta gene is controversial. GnRH is known to elevate intracellular calc ium and activate the protein kinase C (PKC) pathway. The present study eval uated the pathway(s) involved in GnRH induction of LH beta transcription. W e have previously reported that the equine LH beta (eLH beta -448/+60) prom oter is active in alpha T3-1 cells. Therefore, we created a clonal, stably transfected alpha T3-1 gonadotroph cell line harboring the eLH beta promote r (-448/+60) fused to the luciferase reporter gene. Administration of a GnR H agonist resulted in induction of promoter activity that was completely in hibited by the antagonist antide, Various calcium-affecting drugs had no ef fect on the promoter. Administration of phorbol 12-myristate 13-acetate (PM A) elicited an activation similar to, albeit lower than, that with GnRH. Do wn-regulation or pharmacological inhibition of PKC completely blocked PMA's induction of the promoter, while GnRH induction was only partly attenuated . Treatment with the mitogen-activated protein kinase (MAPK) kinase inhibit or, PD98059, completely inhibited the activation of eLH beta by PMA but onl y partly diminished GnRH's induction. Expression of the transcription facto r, early growth response protein 1 (Egr1), correlated completely with activ ation of MAPK, suggesting that Egr1 is the factor through which PKC/MAPK ac ts. Our data suggest that GnRH induces activity of the eLH beta promoter by activating a signal transduction cascade involving PKC-MAPK-Egr1 but that has no significant requirement for calcium.