GnRH pulses regulate gonadotropin subunit gene transcription in a frequency-dependent, subunit-specific manner. The alpha-subunit gene is stimulated by constant GnRH and by rapid to intermediate pulse frequencies, while stimulation of LHbeta subunit gene transcription requires intermediate frequency pulses. We have defined the GnRH-responsive elements of the rat LH subunit gene promoters by deletion/mutation analysis and transfection studies in rat pituitary cells and two clonal gonadotrope cell lines. The alpha-subunit gene GnRH-responsive region lies between -411 and -375 bp. The region contains two Ets-domain protein binding sites, and mutating either site obliterates the response. DNA protein binding studies demonstrate the two sites are not equivalent, and that Ets-1 does not mediate this response. Studies of the LHbeta promoter reveal a major GnRH-responsive region between -456 and -342 bp. Within this region, two Sp1 binding sites contribute to the GnRH response, and the 3'Sp1 site is also critical for basal expression. The 5'Sp1 site partially overlaps a CArG box, and mutating the CArG element specifically eliminates the response to pulsatile GnRH. DNA containing this mutation cannot form intermediate mobility complexes with nuclear proteins, but retains Sp1 binding. Mutation of the 3'Sp1 site and either the 5'Sp1 or CArG element partially restores GnRH stimulation, suggesting a downstream element contributes to the full GnRH response. These studies demonstrate that unique composite elements and transcription factors are responsible for GnRH stimulation of the LH subunit genes and may contribute to their differential responses to GnRH pulses.

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 LHbeta promoter in transfected LbetaT2 clonal gonadotrope cells and in pituitaries of transgenic mice expressing LHbeta-luciferase. The LHbeta promoter (-617 to +44 bp)-luciferase construct was stimulated in LbetaT2 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 LHbeta 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 LHbeta 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 LHbeta 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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