Poly [ADP-Ribose] Polymerase-1 (PARP-1) Confers Glucocorticoid Responsiveness of the Porcine GnRH Receptor (GnRHR) Gene.

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. Investigation of this possible mechanism, however, revealed that transcription of the murine GnRHR gene is stimulated by glucocorticoids. To determine the effect of glucocorticoids on porcine GnRHR gene expression, gonadotrope-derived alphaT3-1 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), and this response was blocked by the glucocorticoid antagonist, mifepristone (100 pM). Deletion from 274 to 323 bp upstream of the translational start site eliminated glucocorticoid responsiveness, suggesting the presence of a GRE(s) within this region. Electrophoretic mobility shift assays (EMSA) using a 32P-labeled oligonucleotide spanning -290/-270 bp of proximal promoter revealed increased binding of nuclear extracts from alphaT3-1 cells treated with 100 nM dexamethasone compared to vehicle. Sequence analysis of this region indicated putative binding sites for PR, ER, GR, COUP-TF and GATA, as well as RXR alpha, beta, and gamma. However, competitive oligonucleotides for each of these transcription factors, including GR, were unable to compete for binding to the radiolabeled -290/-270 bp probe in EMSAs. Mass spectrometry analysis of isolated proteins from a pulldown using a biotinylated oligonucleotide (-290/-270 bp) indentified PARP-1 as the key binding partner. To confirm the mass spectrometry result, we performed EMSAs with antibodies specific for either GR (sc-1004X; Santa Cruz Biotechnology, Santa Cruz, CA) or PARP-1 (614301; BioLegend, San Diego, CA). Both the GR and PARP-1 antibodies generated a supershift of the specific binding complex. Interestingly, addition of the PARP-1 and GR antibodies together abolished the supershift. Since the competitive oligonucleotide for GR was unable to abrogate the DNA/protein complex whereas inclusion of antibodies confirmed that GR was a member of the specific binding complex, this suggests that GR must recruit PARP-1 in order to bind with the GRE. Inhibition of p38 and ERK1/2 mitogen-activated protein kinase (MAPK) pathways significantly decreased dexamethasone-induced promoter activity (P < 0.05), indicating the involvement of these signaling pathways in glucocorticoid stimulation of the promoter. Thus, our working model for glucocorticoid responsiveness of the porcine GnRHR gene suggests that, upon binding to its receptor, glucocorticoid triggers phosophorylation of GR 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. (platform)