Neuroendocrinology

Luteinizing Hormone Beta Promoter Stimulation by Adenylyl Cyclase and Cooperation with Gonadotropin-Releasing Hormone 1 in Transgenic Mice and LBetaT2 Cells

Abstract
Rat luteinizing hormone beta (Lhb) gene transcription is stimulated by hypothalamic gonadotropin-releasing hormone 1 (GNRH1), and this response may be modulated by other signaling pathways such as cAMP. Here we characterize the ability of cAMP, alone or with GNRH1, to stimulate Lhb gene transcription in mouse pituitary and clonal gonadotrope cells. Both cAMP and pituitary adenylate-cyclase-activating peptide (ADCYAP1) increase GNRH1 stimulation of luciferase activity in pituitaries of mice expressing the rat Lhb-luciferase transgene, suggesting cAMP and GNRH1 pathways interact in vivo. cAMP stimulation of the Lhb-luciferase transgene was similar between females in metestrus and proestrus, but GNRH1 stimulation was greater at proestrus. Additive effects with combined treatments were observed at metestrus and proestrus. Elevated intracellular cAMP stimulated Lhb promoter activity in LbetaT2 clonal gonadotrope cells, alone and with GNRH1. In LbetaT2 cells, cAMP stimulation of the Lhb promoter was eliminated by inhibition of PKA; GNRH1 stimulation was partially suppressed by either PKA or PRKCC inhibitors. Only the proximal GNRH1-responsive region of the promoter was required for cAMP stimulation, and mutation of the 3'NR5A1 site diminished the response. Regulation of primary mRNA transcripts from the endogenous Lhb gene by cAMP and GNRH1 correlated with results from the Lhb-luciferase transgene or transfected promoter. Occupancy of the endogenous promoter by EGR1 was increased by GNRH1 with or without forskolin, but forskolin alone had little effect. Thus, cAMP stimulation of Lhb promoter activity, and enhancement of GNRH1 stimulation, occurs in multiple physiological states independent of steroid status, via a PKA-dependent mechanism.

Key words: Mechanisms of Hormone Action • Cyclic adenosine monophosphate • Gonadotropin-releasing hormone • Luteinizing hormone • Steroid hormones

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