Transcriptional Regulation of The Porcine GnRH Receptor Gene by Glucocorticoids

Authors

Chanho Lee, University of Nebraska-LincolnFollow

Date of this Version

12-2013

Citation

Lee, C. 2013. Transcriptional Regulation of the Porcine GnRH Receptor Gene by Glucocorticoids. M.S. Thesis, University of Nebraska-Lincoln, Lincoln, NE.

Comments

A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Animal Science, Under the Supervision of Professor Brett R. White. Lincoln, Nebraska: December, 2013

Copyright (c) 2013 Chanho Lee

Abstract

Binding of GnRH to its receptor (GnRHR) stimulates the synthesis and secretion of the gonadotropins, as well as up-regulation of GnRHR. Thus, the interaction between GnRH and GnRHR represents a central point for regulation of reproduction. Glucocorticoids alter reproduction by reducing GnRH responsiveness of gonadotropes within the anterior pituitary gland, potentially via transcriptional regulation of the GnRHR gene. Investigation into this mechanism, however, revealed that the murine GnRHR gene was stimulated by glucocorticoids. To determine the effect of glucocorticoids on porcine GnRHR gene expression, gonadotrope-derived αT3-1 cells were transiently transfected with a vector containing 5118 bp of 5’ flanking sequence for the porcine GnRHR gene fused to luciferase for 12 h and treated with increasing concentrations of the glucocorticoid agonist, dexamethasone (0, 1, 10, 100 and 1,000 nM) for an additional 12 h prior to harvest. Maximal induction of luciferase activity was detected at 100 nM of dexamethasone (2-fold over vehicle; P < 0.05). Deletion from 274 to 323 bp of proximal promoter eliminated glucocorticoid responsiveness, suggestive of a glucocorticoid response element (GRE). Electrophoretic mobility shift assays (EMSAs) using a radiolabeled oligonucleotide spanning -290/-270 bp of proximal promoter revealed increased binding of nuclear extracts from αT3-1 cells treated with 100 nM dexamethasone compared to vehicle. Mass spectrometry analysis of isolated proteins from a pull-down using a biotinylated oligonucleotide (-290/-270 bp) identified PARP-1 as the binding component. EMSAs with either GR or PARP-1 antibodies resulted in a supershift of the specific binding complex, whereas addition of both antibodies abolished the supershift. Inhibition of p38 and ERK1/2 mitogen-activated protein kinase (MAPK) pathways decreased dexamethasone-induced promoter activity (P < 0.05), indicating their involvement in glucocorticoid stimulation of the promoter. Thus, our working model for glucocorticoid responsiveness of the porcine GnRHR gene suggests that binding of glucocorticoid to its receptor (GR), triggers GR phosophorylation by p38 and ERK1/2 MAPK pathways, resulting in the recruitment of PARP-1 by phosphorylated, ligand-bound GR to a GRE located within -290/-270 bp of the porcine GnRHR promoter.

Adviser: Brett R. White