Regulation of Pituitary MT 1 Melatonin Receptor Expression by Gonadotrophin-Releasing Hormone ( GnRH ) and Early Growth Response Factor-1 ( Egr-1 ) : In Vivo and In Vitro Studies

Melatonin receptor expression exhibits profound developmental changes through poorly understood mechanisms. In mammals, a current model suggests that pubertal reactivation of gonadotrophin-releasing hormone (GnRH) secretion down-regulates MT1 melatonin receptors in pituitary gonadotroph cells, via the induction of early growth response factor-1 (EGR-1). Here we have examined this model by testing the hypotheses that inhibition of Mt1 expression by GnRH occurs directly in gonadotroph cells, can be reversed in adulthood by blockade of GnRH receptors, and requires EGR-1. We first confirmed the endogenous expression of Mt1 mRNA in the aT3-1 gonadotroph cell line. Stimulation of these cells with a GnRH agonist resulted in a rapid increase of Egr-1 mRNA expression, which peaked after 30–60 minutes, and a more prolonged elevation of nuclear EGR-1 immunoreactivity. Moreover, the GnRH agonist significantly decreased Mt1 mRNA. We then treated adult male rats with the GnRH antagonist cetrorelix or saline. After 4 weeks of daily injections, cetrorelix significantly reduced serum LH concentration and testis weight, with histological analysis confirming absence of spermatogenesis. Despite the successful inhibition of GnRH signalling, pituitary Mt1 expression was unchanged. Next we studied the proximal region of the rat Mt1 promoter. Consistent with previous work, over-expression of the transcription factor PITX-1 increased Mt1-luciferase reporter activity; this effect was dependent on the presence of consensus PITX-1 promoter binding regions. Over-expression of EGR-1 inhibited PITX-1-stimulated activity, even following mutation of the consensus EGR-1 binding site. Finally, we studied Egr1 mice and observed no difference in pituitary Mt1 expression between Egr1 and wild-type litter mates. This work demonstrates that GnRH receptor activation directly down-regulates Mt1 expression in gonadotroph cells. However, pituitary Mt1 expression in adults is unaltered by blockade of GnRH signalling or absence of EGR-1. Our data therefore suggest that melatonin receptor regulation by GnRH is not reversible in adulthood and doesn’t require EGR-1. Citation: Bae S-E, Wright IK, Wyse C, Samson-Desvignes N, Le Blanc P, et al. (2014) Regulation of Pituitary MT1 Melatonin Receptor Expression by GonadotrophinReleasing Hormone (GnRH) and Early Growth Response Factor-1 (Egr-1): In Vivo and In Vitro Studies. PLoS ONE 9(3): e90056. doi:10.1371/journal.pone.0090056 Editor: Martin Young, University of Alabama at Birmingham, United States of America Received November 28, 2013; Accepted January 27, 2014; Published March 21, 2014 Copyright: 2014 Bae et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC; grant BB/F020309/1; http://www.bbsrc.ac.uk/home/ home.aspx). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: j.johnston@surrey.ac.uk