Background and methods: In our previous analyses, we found significantly lower levels of growth hormone receptor (GHR) mRNA in adipose tissues of obese than in those of lean individuals, suggesting that idiopathic obesity involves GH resistance due to decreased GHR availability. To understand the mechanism(s) behind this downregulation, we performed an in silico analysis of the three most relevant GHR gene promoters, which revealed putative response elements (REs) for a number of obesity adipose-associated factors, including tumor necrosis factor-alpha (TNFα), hypoxia-inducible factor-1-alpha (HIF-1α) and glucocorticoids. We then characterized the dose-dependent effects of these factors on GHR expression in HEK293 cells and in mature human SGBS (Simpson-Golabi-Behmel syndrome) adipocytes using quantitative reverse transcriptase-PCR and assessed the function of their putative REs by luciferase-reporter assays, site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays.
Results: TNFα treatments significantly reduced GHR mRNA levels and GHR promoter activities at doses ≥ 10 ng ml(-1) in both cell lines. Transient overexpression of HIF-1α or exposure to the hypoxia mimetic CoCl(2) significantly increased GHR mRNA levels and promoter activities. Dexamethasone had biphasic effects: there was a significant increase in GHR mRNA levels at 10(-10) M and in promoter activities at 10(-10) and 10(-8) M, whereas a significant decrease in both mRNA levels and promoter activities occurred at 10(-6) M. Site-directed mutagenesis of the putative nuclear factor-κB, HIF-1α and glucocorticoid REs resulted in the loss of these effects, whereas ChIP analysis confirmed specific transcription factor-promoter interactions.
Conclusions: Our results suggest that the increased activity of TNFα, HIF-1α and glucocorticoids in obese adipose tissues could alter GHR gene transcription through specific REs and that TNFα may be involved in the development of GH resistance.