Department of Experimental Medicine, McGill University, Montreal, QC, Canada.
Int J Obes (Lond). 2011 Dec;35(12):1520-9. doi: 10.1038/ijo.2011.10. Epub 2011 Mar 8.
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.
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.
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.
在我们之前的分析中，我们发现肥胖个体的脂肪组织中生长激素受体 (GHR) mRNA 的水平明显低于瘦个体，这表明特发性肥胖涉及由于 GHR 可用性降低而导致的 GH 抵抗。为了了解这种下调的机制，我们对三个最相关的 GHR 基因启动子进行了计算机分析，发现了一些肥胖脂肪相关因子的潜在反应元件 (RE)，包括肿瘤坏死因子-α (TNFα)、缺氧诱导因子-1-α (HIF-1α) 和糖皮质激素。然后，我们使用定量逆转录聚合酶链反应 (qRT-PCR) 研究了这些因素对 HEK293 细胞和成熟人 SGBS（辛普森-高拉比-贝姆综合征）脂肪细胞中 GHR 表达的剂量依赖性影响，并通过荧光素酶报告基因检测、定点突变和染色质免疫沉淀 (ChIP) 检测评估了它们潜在 RE 的功能。
TNFα 处理在两种细胞系中以≥ 10 ng ml(-1)的剂量显著降低 GHR mRNA 水平和 GHR 启动子活性。瞬时过表达 HIF-1α 或暴露于缺氧模拟物 CoCl(2)显著增加 GHR mRNA 水平和启动子活性。地塞米松具有双相作用：在 10(-10) M 时 GHR mRNA 水平显著增加，在 10(-10) 和 10(-8) M 时启动子活性显著增加，而在 10(-6) M 时 GHR mRNA 水平和启动子活性均显著降低。潜在的核因子-κB、HIF-1α 和糖皮质激素 RE 的定点突变导致这些作用丧失，而 ChIP 分析证实了特定转录因子-启动子相互作用。
我们的结果表明，肥胖脂肪组织中 TNFα、HIF-1α 和糖皮质激素活性的增加可能通过特定的 RE 改变 GHR 基因转录，并且 TNFα 可能参与 GH 抵抗的发生。
