E-box regulation of gonadotropin-releasing hormone (GnRH) receptor expression in immortalized gonadotrope cells

Oct 12, 2007Molecular and cellular endocrinology

Control of Reproductive Hormone Receptor Levels by E-box Sequences in Immortalized Gonad Hormone Cells

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Abstract

Seven noncanonical E-box enhancer sequences are identified as potential regulators of the GnRH receptor gene expression.

The GnRH receptor gene is controlled by specific enhancer sequences that may respond to clock gene proteins.
Expression of clock genes was detected in gonadotrope cells, linking these genes to GnRH receptor regulation.
Luteinizing hormone-producing cells in the rodent pituitary co-express the PERIOD-1 protein, suggesting a role in hormone regulation.
Chromatin immunoprecipitation confirmed that E-boxes in the promoters of GnRHR and Period-1 genes bind to the CLOCK protein.
RNA interference targeting Bmal1 supported the role of E-boxes in regulating GnRHR gene transcription.
Overexpression of certain inhibitory clock proteins significantly decreased the expression levels of the GnRH receptor.

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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.

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E-box regulation of gonadotropin-releasing hormone (GnRH) receptor expression in immortalized gonadotrope cells

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