Negative Regulation of Human Growth Hormone Gene Expression by Insulin Is Dependent on Hypoxia-inducible Factor Binding in Primary Non-tumor Pituitary Cells

Insulin controls growth hormone (GH) production at multiple levels, including via a direct effect on pituitary somatotrophs. There are no data, however, on the regulation of the intact human (h) GHgene (hGH1) by insulin in non-tumor pituitary cells, but the proximal promoter region (nucleotides -496/+1) responds negatively to insulin in transfected pituitary tumor cells. A DNA-protein interaction was also induced by insulin at nucleotides -308/-235. Here, we confirmed the presence of a hypoxia-inducible factor 1 (HIF-1) binding site within these sequences (-264/-259) and investigated whether HIF-1 is associated with insulin regulation of endogenous hGH1. In the absence of primary human pituitary cells, transgenic mice expressing the intact hGH locus in a somatotroph-specific manner were generated. A significant and dose-dependent decrease in hGH and mouse GH RNA levels was detected in primary pituitary cell cultures from these mice with insulin treatment. Increasing HIF-1 alpha availability with a hypoxia mimetic significantly decreased hGH RNA levels and was accompanied by recruitment of HIF-1 alpha to the hGH1 promoter in situ as seen with insulin. Both inhibition of HIF-1 DNA binding by echinomycin and RNA interference of HIF-1 alpha synthesis blunted the negative effect of insulin on hGH1 but not mGH. The insulin response is also sensitive to histone deacetylase inhibition/trichostatin A and associated with a decrease in H3/H4 hyperacetylation in the proximal hGH1 promoter region. These data are consistent with HIF-1-dependent down-regulation of hGH1 by insulin via chromatin remodeling specifically in the proximal promoter region.