Gonadotropin releasing hormone stimulation and androgen suppression of luteinizing hormone gene transcription.

摘要

Gonadotropin releasing hormone (GnRH) controls expression of the luteinizing hormone subunit genes, α and LHβ, and steroids can modify the response to GnRH. Two enhancer regions, distal and proximal, have been identified on the rat LHβ gene promoter and must cooperate for full GnRH stimulation. It has been hypothesized that the transcription factors binding to these regions, including Sp1, Egr-1 and SF-1, may interact directly or indirectly via a coactivator to enhance LHβ transcription. In transfection experiments in clonal gonadotrope LβT2 cells, the coactivator small nuclear RING finger protein (SNURF), enhanced basal and GnRH stimulated expression of LHβ. SNURF interacts with Sp1 and SF-1 and point mutations or deletions of SNURF functional domains demonstrated that these interactions are required for coactivation. We tested direct pituitary affects of androgen on modulation of the LHβ promoter in LβT2 cells and in pituitaries of transgenic mice expressing LHβ-luciferase. Stimulation of LHβ expression by GnRH was suppressed by nanomolar androgen concentrations in isolated pituitary cells from mice with functional nuclear androgen receptors (AR), but not in littermates with inactivating mutant AR. GST-pulldown studies demonstrated that the DNA binding domain of AR interacts with Sp1 and Egr-1 and increasing concentrations of AR reduce Sp1 binding to the LHβ promoter in EMSA experiments. SNURF binds to AR, and SNURF overexpression overcomes androgen suppression of GnRH-stimulated LHβ promoter activity. To test the effects of GnRH on transcription factor occupancy of the LHβ promoter, we used the chromatin immunoprecipitation (ChIP) assay. GnRH treatment increased the association of acetylated histone H3 with the LHβ promoter, and transcription factors Sp1, Egr-1 and SF-1 coordinately cycle on and off the promoter over time of GnRH exposure. Transcription factor occupancy of the promoter was stimulated after 15 minutes of GnRH treatment, cycling occurred over approximately 30-minute intervals and dissipated after 4 hours. These studies demonstrate that GnRH stimulation of LHβ involves a specific coactivator SNURF and is associated with dynamic occupancy of the LHβ promoter by transcription factors. Thus, there is a direct effect of androgen on the pituitary to suppress GnRH stimulation and this suppression may involve AR interaction with transcription factors such as Sp1.