E-box Regulation of Gonadotropin-releasing Hormone (GnRH) Receptor Expression in Immortalized Gonadotrope Cells

The pituitary gland's ability to respond to the hypothalamic hormone GnRH (gonadotropin-releasing hormone) depends directly on the gonadotrope-specific expression of the GnRH receptor (GnRHR), a G-protein coupled transmembrane protein coded by the GnRHR gene. In the present study, we have investigated the potential regulatory role of seven noncanonical E-box enhancer sequences within the 856bp proximal 5'-flanking region of the mGnRHR gene in regulating transcription. These sequences are known to mediate the action of clock gene proteins on the expression of a diverse array of genes both central and peripheral. In the present studies the expression of all of the cognate clock genes was identified in the alphaT3-1 gonadotrope cell line. Additionally, luteinizing hormone-immunoreactive cells in the adult rodent pituitary gland were also shown to co-express the PERIOD-1 protein. By means of chromatin immunoprecipitation of alphaT3-1 nuclear extracts we were able to capture promoter fragments of the GnRHR and Period-1 genes, indicating that E-boxes in these promoters bind the CLOCK protein. RNA interference experiments with alphaT3-1 cells in which Bmal1 expression was attenuated also confirmed the involvement of E-boxes in transcriptional regulation of the mGnRHR gene. Subsequent luciferase reporter assay experiments with GnRHR constructs possessing intact or mutated E-boxes confirmed the use of these sequences for the regulation of mGnRH-R/luc expression. Transient overexpression of the dominant negative E-box-binding factor CLOCK-Delta19, or the inhibitory clock protein mPER1, markedly reduced CLOCK/BMAL1-driven mGnRH-R/luc expression in a dose-dependent fashion. Our data implicate the clock genes as important factors controlling GnRHR expression in murine gonadotrope cells.