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.
Resuehr
D
D
Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL 32306-4300, USA.
Wildemann
U
U
Sikes
H
H
Olcese
J
J
D056930:ARNTL Transcription Factors; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D056926:CLOCK Proteins; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D024721:E-Box Elements / Q000235:genetics / Q000502:physiology*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D052681:Gonadotrophs / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D056950:Period Circadian Proteins; D034622:RNA Interference; D011966:Receptors, LHRH / Q000037:antagonists & inhibitors / Q000235:genetics*; D017384:Sequence Deletion; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D056930:ARNTL Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D005136:Eye Proteins; D008156:Luciferases; D056950:Period Circadian Proteins; D011966:Receptors, LHRH; D015534:Trans-Activators; C538961:Bmal1 protein, mouse; C539023:Per1 protein, mouse; C538944:Clock protein, mouse