Is gonadotrope expression of the gonadotropin releasing hormone receptor gene mediated by autocrine/paracrine stimulation of an activin response element?

Expression of the FSH beta subunit and GnRH receptor (GnRHR) genes in gonadotropes is stimulated by activin. We sought to identify the cis-acting element(s) in the murine GnRHR gene promoter which confer activin responsiveness. We established that 600 bp of 5' flanking sequence from the murine GnRHR gene were sufficient to confer activin responsiveness in the gonadotrope-derived alpha T3-1 cell line. Since alpha T3-1 cells, like gonadotropes, secrete activin, we examined the ability of follistatin, an activin binding protein, to block the activin response. Increasing concentrations of follistatin from 0 to 100 ng/ml resulted in a dose dependent decrease in activity of the -600 promoter. Contained within this region are three elements important for expression in alpha T3-1 cells: a Steroidogenic Factor-1 binding site (SF-1), an Activator Protein-1 (AP-1) element, and an element termed the GnRH receptor activating sequence or GRAS. A block mutation of GRAS inhibited the ability of the promoter to respond to follistatin. A more refined analysis using a series of two-bp mutations which scan GRAS and flanking sequence revealed exact convergence of GRAS with activin/follistatin responsiveness. Finally, a construct consisting of 3 copies of GRAS placed upstream of a heterologous minimal promoter (3xGRAS-PRL-LUC) was responsive to both activin stimulation and follistatin inhibition in alpha T3-1 cells. Thus, autocrine/paracrine stimulation of gonadotropes by activin illustrates a unique mechanism for cell-specific gene expression.