MECHANISMS OF REGULATION OF GNRH GENE EXPRESSION

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The overall goal of this program is to identify and characterize the cellular and molecular mechanisms which regulate GnRH gene expression. Initial studies will be conducted in an immortalized mouse GnRH neuronal cell model, the GT1-7 cell. We will focus on the PKC and Ca++ signal transduction systems since they have already been shown to elicit significant effects on GnRH transcription, mRNA turnover and secretion. Moreover, Ca++ is a major intracellular path way activated by the excitatory amino acid (EAA) glutamate and we have previously shown that NMDA, has extremely rapid effects on GnRH gene expression at the cytoplasmic level. These findings will be extended to investigations in animals in order to verify that the observed mechanisms are of primary importance in vivo. There are four specific aims: Aim 1:Characterize the elements in the mouse GnRH promoter and the trans- acting factors which are responsible for mediating the negative regulation by the PKC and calcium pathways in the GT1-7 cells. The function of two mouse specific promoter elements will also be identified. Aim 2:Previous studies in cultures of GT1-7 cells have shown that phorbol esters cause a decrease in the stability of GnRH mRNA concomitant with a decrease in poly (A) tail length and a decrease in the number of ribosomes associated with GnRH mRNA. Ca++ ionophores have similar effects on GnRH mRNA stability. Our hypothesis is that GnRH mRNA turnover plays an important role in setting the level of GnRH gene expression. We will determine the mechanism by which activation of the PKC and Ca++ pathways decreases the stability of the GnRH mRNA in GT1-7 cells. Aim 3: Elucidate the mechanism(s) by which rapid changes in GnRH mRNA levels are elicited in the hypothalamus. We will use an EAA paradigm previously shown to significantly modulate GnRH gene expression in vivo and analyze changes in the relative level of polyA and polysome loading of GnRH mRNA in rat hypothalamic neurons. To determine if the post- transcriptional regulatory elements present in GnRH mRNA function in vivo, transgenic mice expressing mutant GnRH mRNA constructs will be created and the effects of EAA treatment analyzed. Aim 4: Using perifusion of GT1 cells, it was reported that different modes of addition of EAAs elicit different responses in GnRH release or in Ca++ activation. We have also seen that secreted GnRH peptide is cleaved to GnRH(1-5) which subsequently antagonizes the NMDA receptor, possibly a mechanism by which GnRH exerts inhibitory ultra-short loop feedback on GnRH neurons. In this study, we will determine if different modes of treatment of GT1-7 cells with EAAs will differentially affect GnRH gene transcription and/or GnRH mRNA stability in a perifusion system.