Kim Sungtae, Yamazaki Miwa, Zella Lee A, Shevde Nirupama K, Pike J Wesley
Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Dr., Madison, WI 53706, USA.
Mol Cell Biol. 2006 Sep;26(17):6469-86. doi: 10.1128/MCB.00353-06.
RANKL is a tumor necrosis factor (TNF)-like factor secreted by mesenchymal cells, osteoblast derivatives, and T cells that is essential for osteoclastogenesis. In osteoblasts, RANKL expression is regulated by two major calcemic hormones, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and parathyroid hormone (PTH), as well as by several inflammatory/osteoclastogenic cytokines; the molecular mechanisms for this regulation are unclear. To identify such mechanisms, we screened a DNA microarray which tiled across the entire mouse RankL gene locus at a 50-bp resolution using chromatin immunoprecipitation (ChIP)-derived DNA precipitated with antibodies to the vitamin D receptor (VDR) and the retinoid X receptor (RXR). Five sites of dimer interaction were observed on the RankL gene centered at 16, 22, 60, 69, and 76 kb upstream of the TSS. These regions contained binding sites for not only VDR and RXR, but also the glucocorticoid receptor (GR). The most distant of these regions, termed the distal control region (RL-DCR), conferred both VDR-dependent 1,25(OH)(2)D(3) and GR-dependent glucocorticoid (GC) responses. We mapped these activities to an unusual but functionally active vitamin D response element and to several potential GC response elements located over a more extensive region within the RL-DCR. An evolutionarily conserved region within the human RANKL gene contained a similar vitamin D response element and exhibited an equivalent behavior. Importantly, hormonal activation of the RankL gene was also associated with chromatin modification and RNA polymerase II recruitment. Our studies demonstrate that regulation of RankL gene expression by 1,25(OH)(2)D(3) is complex and mediated by at least five distal regions, one of which contains a specific element capable of mediating direct transcriptional activation.
核因子κB受体活化因子配体（RANKL）是一种由间充质细胞、成骨细胞衍生物和T细胞分泌的肿瘤坏死因子（TNF）样因子，对破骨细胞生成至关重要。在成骨细胞中，RANKL的表达受两种主要的钙调节激素，即1,25-二羟维生素D3 [1,25(OH)2D3] 和甲状旁腺激素（PTH），以及几种炎症/破骨细胞生成细胞因子的调节；这种调节的分子机制尚不清楚。为了确定这些机制，我们使用与维生素D受体（VDR）和视黄酸X受体（RXR）抗体沉淀的染色质免疫沉淀（ChIP）衍生DNA，以50 bp的分辨率筛选了覆盖整个小鼠RankL基因座的DNA微阵列。在RankL基因上观察到五个二聚体相互作用位点，位于转录起始位点（TSS）上游16、22、60、69和76 kb处。这些区域不仅包含VDR和RXR的结合位点，还包含糖皮质激素受体（GR）的结合位点。这些区域中最远的区域，称为远端控制区（RL-DCR），赋予了VDR依赖性的1,25(OH)2D3反应和GR依赖性的糖皮质激素（GC）反应。我们将这些活性定位到一个不寻常但功能活跃的维生素D反应元件以及位于RL-DCR内更广泛区域的几个潜在GC反应元件上。人类RANKL基因内的一个进化保守区域包含一个类似的维生素D反应元件，并表现出相同的行为。重要的是，RankL基因的激素激活也与染色质修饰和RNA聚合酶II募集有关。我们的研究表明，1,25(OH)2D3对RankL基因表达的调节是复杂的，由至少五个远端区域介导，其中一个区域包含一个能够介导直接转录激活的特定元件。
