Glucocorticoid Responsiveness of the Porcine GnRH Receptor (GnRHR) Gene Is Conferred by an Element(s) Located Between -290/-270 bp of Proximal Promoter.

Abstract

The binding of GnRH to its receptor results in the synthesis and secretion of the gonadotropins, as well as stimulation of the gene encoding its own receptor. Thus, the interaction between GnRH and GnRHR represents a central point for regulation of reproductive function. Glucocorticoids can alter reproduction by reducing GnRH responsiveness of gonadotropes within the anterior pituitary gland, potentially via transcriptional regulation of the GnRHR gene. In addition, transcription of the murine GnRHR gene is stimulated by glucocorticoids. To determine the effect of glucocorticoids on porcine GnRHR gene expression, we isolated 5118 bp of 5' flanking sequence for the porcine GnRHR gene and produced reporter constructs containing the GnRHR promoter fused to the cDNA encoding luciferase (-5118LUC). The gonadotrope-derived αT3-1 cell line was transiently transfected with -5118LUC for 12 h and treated with increasing concentrations of the glucocorticoid agonist, dexamethasone (1, 10, 100 and 1,000 nM) for an additional 12 h prior to harvest. A dose-dependent increase in luciferase activity was observed with maximal induction noted at 100 nM dexamethasone (2-fold over vehicle; P < 0.05). The dexamethasone induction of the -5118 promoter was blocked by the glucocorticoid antagonist, mifepristone (100 pM). To determine the location of the glucocorticoid response element(s) within the GnRHR promoter, we performed transient transfection assays with luciferase reporter constructs containing progressively less 5' flanking region for the porcine GnRHR gene. Dexamethasone-stimulated luciferase activity was maintained following reduction of the full length GnRHR promoter to 323 bp upstream of the translational start site. However, further deletion to 274 bp of proximal promoter eliminated glucocorticoid responsiveness, suggesting the presence of a glucocorticoid response element(s) within this region. Electrophoretic mobility shift assays using 32P-labeled oligomers spanning this region revealed increased binding of nuclear extracts from αT3-1 cells treated with 100 nM dexamethasone compared to vehicle treated cells to the -290/-270 bp oligonucleotide. Sequence analysis of this region revealed putative binding sites for PR, ER, GR, COUP-TF and GATA, as well as RXR α, β, and γ. Inhibition of p38 MAPK and ERK 1/2 pathways significantly decreased dexamethasone-induced promoter activity, indicating the involvement of p38 MAPK and ERK 1/2 pathways for glucocorticoid responsiveness of the promoter. Further, our Western blot data revealed that the JNK pathway is also involved in dexamethasone-stimulated promoter activity. In summary, glucocorticoid responsiveness of the porcine GnRHR gene is conferred by an element(s) located between 270 and 290 bp of proximal promoter that is activated by p38 MAPK, ERK 1/2, and JNK pathways. (platform)