Androgen Receptor Drives Transcription of Rat PACAP in Gonadotrope Cells

Abstract

Gonadotropin expression is precisely regulated within the hypothalamic-pituitary-gonadal axis through the complex interaction of neuropeptides, gonadal steroids. and both gonadal- and pituitary-derived peptides. In the anterior pituitary gland, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) modulates gonadotropin biosynthesis and secretion, acting both alone and in conjunction with GnRH. Steroid hormone feedback also influences gonadotropin expression via both direct and indirect mechanisms. Evidence from nonpituitary tissues suggests that PACAP may be a target for gonadal steroid regulation. In the present study, we show that androgen markedly stimulates rat (r) PACAP promoter-reporter activity in the LβT2 mature mouse gonadotrope cell line. 5'-Serial deletion analysis of reporter constructs identifies 2 regions of androgen responsiveness located at (-915 to -818) and (-308 to -242) of the rPACAP promoter. Androgen receptor (AR) binds directly to DNA cis-elements in each of these regions in vitro. Site-directed mutagenesis of 3 conserved hormone response element half-sites straddling the (-308 to -242) region dramatically blunts androgen-dependent PACAP promoter activity and prevents AR binding at the mutated promoter element. Chromatin immunoprecipitation demonstrates that endogenous AR binds the homologous region on mouse chromatin in LβT2 cells in both the presence and absence of androgen. These data demonstrate that androgen stimulates PACAP gene expression in the pituitary gonadotrope via direct binding of AR to a specific cluster of evolutionarily conserved hormone response elements in the proximal rPACAP gene promoter. Thus, androgen regulation of pituitary PACAP expression may provide an additional layer of control over gonadotropin expression within the hypothalamic-pituitary-gonadal axis.

Androgen-dependent sexual dimorphism in pituitary tryptophan hydroxylase expression: relevance to sex differences in pituitary hormones

“…Notably, the two AREs identified can act in a synergistic fashion to activate tph1 transcription, as seen by a comparison of data between the construct with wild-type sequence and that with mutations introduced into both half-sites of the AREs in figure 2j. Considering that the canonical AREs identified are only 263 bp apart and that multiple non-canonical ARE half-sites in close proximity can act synergistically [38,39], the half-sites of these canonical AREs may interact to some extent with one another.…”

Gonadotropin-Releasing Hormone and Gonadal Steroids Regulate Transcription Factor mRNA Expression in Primary Pituitary and Immortalized Gonadotrope Cells

“…We and others have demonstrated expression of ERα, PR-B, and AR in this cell line. 14,59 –62…”

“…We and others have demonstrated expression of ERa, PR-B, and AR in this cell line. 14,[59][60][61][62] The LbT2 cells were treated with 100 nmol/L GnRH for 6 or 24 hours (Figure 6). A substantially greater number of transcripts were modulated by GnRH in the LbT2 cells compared with the response in the mixed cell population.…”