Abstract 567 Endocrinology & Diabetes II Platform, Saturday, 5/1

The precise coordinated expression and release of gonadotropin releasing hormone (GnRH) is essential for the onset and progression of puberty and a functioning mammalian reproductive system. However, the specific factors that control GnRH gene expression at puberty remain largely unknown. Regulation of GnRH neuronal activity has been difficult to study due to the paucity of cell number and their scattered distribution. Immortalized neuronal cell lines created by targeted tumorigenesis in the mouse (NLT and Gn11) have been shown to secrete variable amounts of GnRH, with NLT cells producing about ten times higher levels of GnRH than Gn11 cells. Comparison of these cell lines, therefore, provides a means of identifying the factor or factors that may be responsible for cell-specific expression of GnRH.

A subtraction hybridization technique using NLT and Gn11 mRNA has been used to isolate a cDNA clone that is differentially expressed in NLT cells, designated NLT expression factor 1 (NLT-EF1). Northern blot analysis confirms differential expression, revealing three-fold higher levels of NLT-EF1 mRNA present in NLT cells when compared to Gn11 cells, and identifies NLT-EF1 in hypothalamus, cortex, cerebellum, lung, and ovary and testis. A full-length clone of EF1 has been isolated from a mouse brain library that encodes a protein product of MW 67 kilodaltons.

Transient transfections studies were performed on Gn11 cells to investigate the ability of NLT EF-1 to transactivate the hGnRH promoter. Cells were transfected with the hGnRH promoter region from D1131/+5 fused to a luciferase reporter gene using lipofectamine. Co-transfection with NLT EF-1 stimulated -1131/+5 LUC reporter 2.5 fold when compared with co-transfection with NLT EF-1 alone. This data indicates that NLT EF-1 is able to transactivate a construct containing elements of the hGnRH promoter, and, therefore, may regulate GnRH gene expression by interacting with binding sites located within the region between D1131 and +5 of the hGnRH promoter.

Due to previously published data characterizing the protein kinase C pathway in the GnRH neuron, Northern blot analysis was used to examine the effects of treatment with the phorbol ester, TPA, on the expression of NLT EF-1. NLT EF-1 expression increased two fold after stimulation by TPA in both NLT and GN11 cells. Transient transfection studies on hGnRH promoter constructs co-transfected with NLT EF-1 reveal that treatment with TPA in the presence of EF-1 increases luciferase expression 15-fold with a hGnRH promoter construct containing the region D412/+5, containing an AP-1 site, but not with a mutant D412/+5 LUC construct in which the AP-1 site is abolished. This suggests that the action of EF-1 may be through stimulation of the protein kinase C pathway and AP-1 activation. Interestingly, chemical blockade of tyrosine kinase activity prevents advancement of puberty in rats with precocious puberty induced by hypothalamic lesions.