Molecular mechanisms underlying activin regulation of the gonadotropin releasing hormone receptor gene

Abstract

Activin regulation of the mouse GnRHR promoter was thought to occur solely through the GRAS element (1;2). However, the mouse GnRHR promoter is activin regulated while the rat promoter is not. To investigate this divergence in activin regulation between the two promoters the 1 bp difference in the GRAS element was exchanged between the two promoters. The rat promoter with the mouse GRAS homolog did not gain activin regulation, while the mouse promoter with the rat GRAS element did lose activin regulation in accordance with the importance of GRAS. This was the first evidence that while necessary GRAS alone is not sufficient for activin regulation of the mouse GnRHR promoter (3). Using a series of chimeric exchanges and block replacements in the mouse promoter, I identified a new element I termed the Downstream Activin Response Element or DARE (4). I suggest that GRAS and DARE together comprise a unique activin responsive unit (ARU) in the proximal promoter of the murine GnRHR gene. Although activin responsiveness of the murine GnRHR gene promoter has been extensively studied using the gonadotrope derived αT3-1 cell line, whether this response is evident in a more physiological context was unknown. Thus, I sought to determine if the activin responsive phenotype of the GnRHR gene promoter defined in vitro is recapitulated in transgenic mice. Transgenic mice harboring the wild type mouse GnRHR gene promoter fused to the cDNA for luciferase (-1900wt) (7) were infected with an adenoviral construct that expresses follistatin (Ad-follistatin). I find that adenoviral mediated over-expression of follistatin leads to an approximately 50% decrease in pituitary luciferase expression in the -1900wt mice. These data are the first to demonstrate activin responsiveness of a GnRHR promoter construct in vivo. Finally, I repeated this experimental paradigm in a separate line of transgenic mice harboring the 1900 bp promoter fragment containing a loss of function mutation in GRAS (- 1900μGRAS). Consistent with the critical role of GRAS in mediating activin responsiveness in vitro, I find that pituitary expression of luciferase in the -1900μGRAS line is not affected by follistatin over-expression.