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Androgen Suppression of GnRH-Stimulated Rat LHß Gene Transcription Occurs Through Sp1 Sites in the Distal GnRH-Responsive Promoter Region

Departments of Pharmacology (D.C.), Internal Medicine (S.J., N.F., A.C.A., D.J.H., M.A.S.), and Biology (E.R.), University of Virginia, Charlottesville, Virginia 22908; and Department of Pediatrics and Department of Biochemistry and Biophysics (E.M.W.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Steroids may regulate LH subunit gene transcription by modulating hypothalamic GnRH pulse patterns or by acting at the pituitary gonadotrope to alter promoter activity. We tested direct pituitary effects of the androgen dihydrotestosterone (DHT) to modulate the rat LHß promoter in transfected LßT2 clonal gonadotrope cells and in pituitaries of transgenic mice expressing LHß-luciferase. The LHß promoter (-617 to +44 bp)-luciferase construct was stimulated in LßT2 cells 7- to 10-fold by GnRH. Androgen treatment had little effect on basal promoter activity but suppressed GnRH stimulation by approximately 75%. GnRH stimulation of LHß was also suppressed by DHT in isolated pituitary cells from male or female mice with functional nuclear ARs, but not in male littermates with mutant AR. GnRH stimulation of the LHß promoter requires interactions between a complex distal response element containing two specificity protein-1 (Sp1) binding sites and a CArG box, and a proximal element with two bipartite binding sites for steroidogenic factor-1 and early growth response protein-1 (Egr-1). DHT effectively suppressed promoter constructs with an intact distal response element. The distal response element does not bind AR, but AR reduces Sp1 binding to this region. Glutathione-S-transferase pull-down studies demonstrated direct interactions of AR with Sp1, which requires the DNA-binding domain of AR, and weaker interactions with Egr-1. We conclude that androgen suppression of the rat LHß promoter occurs primarily through direct interaction of AR with Sp1, with some possible role through binding to Egr-1. These interactions result in interference with GnRH-stimulated gene transcription by reducing cooperation between the distal and proximal GnRH response elements.

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