The Chair of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia.
Int J Mol Sci. 2021 Jan 21;22(3):1054. doi: 10.3390/ijms22031054.
Glucocorticoid osteoporosis is a serious side effect of long term glucocorticoid uptake and it is caused by osteoblast apoptosis and imbalance in the major bone remodeling pathway RANK/RANKL/OPG. The impact of glucocorticoid on the maintenance of RANK/RANKL/OPG is well explored; dexamethasone was shown to disturb the ratio between and level by decreasing the expression level of and increasing level of . Here, were aimed to decipher whether glucocorticoid receptor directly influences promoter activity and its transcriptional regulation. We demonstrate that overexpression of glucocorticoid receptor (GR) NR3C1 increased promoter activity in human osteosarcoma, cervical cancer (2-fold) and adenocarcinoma cells (4.5-fold). Mutational analysis revealed that +352 site in the promoter is functional glucocorticoid responsive element (GRE) since the effect of GR on promoter activity was diminished by mutation at this site. Overexpression of upregulated mRNA expression 1.5-fold in human A549 and HOS cells. On the other hand silencing of caused slight decrease in mRNA level, suggesting that NR3C1 directly accounts for transcriptional regulation. Using electrophoretic mobility shift assay we demonstrate that NR3C1 binds to the proximal promoter region. Our study provides evidences that NR3C1 directly upregulates transcription in human cell lines and connects the missing link in the mechanism of RANK/RANKL/OPG imbalance of glucocorticoid induced osteoporosis.
糖皮质激素性骨质疏松症是长期糖皮质激素摄入的严重副作用，它是由成骨细胞凋亡和主要骨重塑途径 RANK/RANKL/OPG 失衡引起的。糖皮质激素对 RANK/RANKL/OPG 维持的影响已经得到了充分的研究；地塞米松通过降低 和增加 的表达水平，显示出干扰 和 水平之间的比例，从而扰乱了两者之间的平衡。在这里，我们旨在破译糖皮质激素受体是否直接影响 启动子活性及其转录调控。我们证明，糖皮质激素受体（GR）NR3C1 的过表达增加了人骨肉瘤、宫颈癌（2 倍）和腺癌细胞（4.5 倍）中的 启动子活性。突变分析表明， 启动子中的+352 位点是功能性糖皮质激素反应元件（GRE），因为该位点的突变减弱了 GR 对 启动子活性的影响。过表达 在人 A549 和 HOS 细胞中上调 mRNA 表达 1.5 倍。另一方面，沉默 导致 mRNA 水平略有下降，表明 NR3C1 直接负责 转录调控。通过电泳迁移率变动分析，我们证明 NR3C1 与 启动子的近端区域结合。我们的研究提供了证据，表明 NR3C1 直接上调人细胞系中的 转录，并连接了糖皮质激素诱导骨质疏松症中 RANK/RANKL/OPG 失衡机制中的缺失环节。
