Metabolic signalling to somatotrophs: Transcriptional and post-transcriptional mediators

Abstract

In normal individuals, pituitary somatotrophs optimise body composition by responding to metabolic signals from leptin. To identify mechanisms behind the regulation of somatotrophs by leptin, we used Cre-LoxP technology to delete leptin receptors (LEPR) selectively in somatotrophs and developed populations purified by fluorescence-activated cell sorting (FACS) that contained 99% somatotrophs. FACS-purified, Lepr-null somatotrophs showed reduced levels of growth hormone (GH), growth hormone-releasing hormone receptor (GHRHR), and Pou1f1 proteins and Gh (females) and Ghrhr (both sexes) mRNAs. Pure somatotrophs also expressed thyroid-stimulating hormone (TSH) and prolactin (PRL), both of which were reduced in pure somatotrophs lacking LEPR. This introduced five gene products that were targets of leptin. In the present study, we tested the hypothesis that leptin is both a transcriptional and a post-transcriptional regulator of these gene products. Our tests showed that Pou1f1 and/or the Janus kinase/signal transducer and activator of transcription 3 transcriptional regulatory pathways are implicated in the leptin regulation of Gh or Ghrhr mRNAs. We then focused on potential actions by candidate microRNAs (miRNAs) with consensus binding sites on the 3' UTR of Gh or Ghrhr mRNAs. Somatotroph Lepr-null deletion mutants expressed elevated levels of miRNAs including miR1197-3p (in females), miR103-3p and miR590-3p (both sexes), which bind Gh mRNA, or miRNA-325-3p (elevated in both sexes), which binds Ghrhr mRNA. This elevation indicates repression of translation in the absence of LEPR. In addition, after detecting binding sites for Musashi on Tshb and Prl 3' UTR, we determined that Musashi1 repressed translation of both mRNAs in in vitro fluc assays and that Prl mRNA was enriched in Musashi immunoprecipitation assays. Finally, we tested ghrelin actions to determine whether its nitric oxide-mediated signalling pathways would restore somatotroph functions in deletion mutants. Ghrelin did not restore either GHRH binding or GH secretion in vitro. These studies show an unexpectedly broad role for leptin with respect to maintaining somatotroph functions, including the regulation of PRL and TSH in subsets of somatotrophs that may be progenitor cells.

Keywords: Cre-LoxP; Musashi; ghrelin; growth hormone; growth hormone releasing hormone; leptin; leptin receptors; mRNAs; miRNAs; mouse; post-transcriptional regulation; prolactin; somatotroph; thyroid stimulating hormone; transcriptional activators; translational regulation.