Androgen suppression of GnRH-stimulated rat LH beta gene transcription occurs through Sp1 sites in the distal GnRH-responsive promoter region

Steroids may regulate LH subunit gene transcription by modulating hypothala mic GnRH pulse patterns or by acting at the pituitary gonadotrope to alter promoter activity. We tested direct pituitary effects of the androgen dihyd rotestosterone (DHT) to modulate the rat LH beta promoter in transfected L beta T2 clonal gonadotrope cells and in pituitaries of transgenic mice expr essing LH beta -luciferase. The LH beta promoter (-617 to +44 bp)-luciferas e construct was stimulated in L beta T2 cells 7- to 10-fold by GnRH. Androg en treatment had little effect on basal promoter activity but suppressed Gn RH stimulation by approximately 75%. GnRH stimulation of LH beta was also s uppressed by DHT in isolated pituitary cells from male or female mice with functional nuclear ARs, but not in male littermates with mutant AR. GnRH st imulation of the LH beta promoter requires interactions between a complex d istal response element containing two specificity protein-1 (Spl) binding s ites and a CArG box, and a proximal element with two bipartite binding site s for steroidogenic factor-1 and early growth response protein-1 (Egr-1). D HT effectively suppressed promoter constructs with an intact distal respons e element. The distal response element does not bind AR, but AIR reduces Sp l binding to this region. Glutathione-S-transferase pull-down studies demon strated direct interactions of AIR with Spl, which requires the DNA-binding domain of AR, and weaker interactions with Egr-1. We conclude that androge n suppression of the rat LH beta promoter occurs primarily through direct i nteraction of AR with Spl, with some possible role through binding to Egr-1 . These interactions result in interference with GnRH-stimulated gene trans cription by reducing cooperation between the distal and proximal GnRH respo nse elements.