Submission Number: MAR-4-99-139

Abstract Number: 1

PULSATILE GnRH MODULATION OF GONADOTROPIN GENE TRANSCRIPTION: DIVERGENT SIGNALS AND MOLECULAR TARGETS.

Margaret A Shupnik* ^1,2, Jennifer Weck ², Shannon Jenkins ¹ and Alice C Anderson ¹

Department of Medicine and Center for Research in Reproduction, University of Virginia, Charlottesville, VA ¹
Department of Physiology, University of Virginia, Charlottesville, VA ²

Abstract:
Hypothalamic GnRH is released in pulses of defined amplitude and frequency that vary during the reproductive cycle and are crucial for normal development and fertility. GnRH stimulates the secretion and synthesis of pituitary gonadotropins,with rapid pulses favoring LH and slower pulses favoring FSH. GnRH stimulates rat gonadotropin gene transcription in a frequency-dependent, subunit-specific manner. In vivo, the common -subunit gene transcription is stimulated by rapid pulses (8-30 min intervals), while the LH gene is stimulated by intermediate frequencies (30-60 min) and FSH gene transcription is increased only by slower pulse intervals ( 120 min). In isolated perifused pituitary cells the -subunit gene is stimulated by either pulsatile or constant GnRH while the LH gene strictly requires pulsatile GnRH, confirming the critical nature of the GnRH signal on the gonadotrope. FSH is only modestly stimulated in vitro, suggesting additional regulatory pathways are important in vivo. Given only one gonadotrope GnRH receptor, we postulated differential gene sensitivity to intracellular signaling pathways and divergent nuclear transcription factors contributed to differential GnRH responses. GnRH stimulates calcium influx through L-type voltage-gated channels and also activates phospholipase C, leading to increased protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activity. In rat pituitary cells GnRH stimulated transcription of the LH and -subunit genes, but PKC activation stimulated the -subunit gene preferentially. In contrast, the L-channel antagonist nimodipine eliminated GnRH stimulation of LH with only partial suppression of the -subunit gene, and treatment with an L-channel agonist stimulated only LH. Transgenic animal studies and transfection with gene promoters similarly showed that GnRH stimulation of the LH promoter required calcium, with only slight effects on the -subunit. Addition of a MAPK inhibitor obliterated the GnRH response of the -subunit, but not the LH promoter. Thus, GnRH stimulation of the -subunit gene is more dependent on the MAPK/PKC pathway while the LH gene is more dependent on calcium. These differences are reflected in the transcription factors conferring GnRH sensitivity. Deletion/mutation analysis showed GnRH stimulation of the -subunit gene required two non-equivalent binding sites for Ets-domain proteins, which are direct nuclear targets of MAPK. The LH promoter has two interacting GnRH-responsive regions. A calcium-sensitive upstream element contains multiple Sp1 binding sites and a CArG region critical for pulsatile GnRH stimulation. A downstream region binds SF-1 and Egr-1 and is critical for basal expression,with Egr-1 sites cooperating with the upstream element for GnRH stimulation. Thus, differential sensitivity to GnRH signaling and distinct nuclear transcription factor targets for these signals contribute to GnRH regulation of these genes. .

Keywords: gonadotropin subunits, GnRH pulses, gene transcription

This abstract is being presented at: 10:30 AM in session:
Minisymposium I: PULSES, PEPTIDES, AND PROMOTORS: DIFFERENTIAL GnRH ACTIONS ON THE PITUITARY