ANALYSIS OF THE GENE PROMOTERS OF MOUSE OSTEOCLAST DIFFERENTIATION FCTOR (RANKL)

核因子-kB受体激活剂配体(RANKL)是一种膜结合的信号转导分子，是诱导破骨细胞所必需的。我们鉴定了小鼠RANKL基因的启动子区域，确定了倒置的TATA和CAAT-box，推测的Runx2结合位点(-190，-205和-365)，以及推测的维生素D反应元件(VDRE，-935)。Northern印迹和核连续杂交分析表明维生素D3上调RANKL基因在转录水平的表达，瞬时转染法研究表明维生素D3的诱导作用被VDRE(-937/-922)突变和-723区缺失所抵消。在VD3处理的细胞中检测到VDRE/核蛋白超移位复合体与抗VDR和RXRβ_抗体结合。此外，当与小鼠骨髓巨噬细胞共培养时，传代后的ST2细胞由于RANKL mRNA表达减少而显示出破骨细胞生成减少。然而，转染RANKL启动子的早期和晚期ST2细胞显示出相同水平的荧光素酶活性和维生素D3诱导，这表明RANKL基因的表达依赖于传代次数，这可能涉及顺式调控机制。由于CpG基因座(-66/+246)在传代后期的ST2细胞中主要甲基化，转录和翻译起始点附近的CpG甲基化可能在一定程度上调节RANKL基因的表达。我们在各种国际和国内会议上公布了我们的数据，并在学术期刊上发表了科学论文。

Receptor activator of NF-kB ligand (RANKL) is a membrane-bound signal transducer necessary for the induction of osteoclasts. We characterized the promoter region of the mouse RANKL gene, identifying inverted TATA- and CAAT-boxes, putative Runx2 binding sites (-190, -205 and -365), and a putative vitamin D response element (VDRE, -935). Northern blot and nuclear run-on analyses showed that vitamin D_3 upregulates RANKL gene expression at the transcriptional level, and transient transfectikon studies revealed that the inductive effect of vitamin D_3 was abolished by mutation of the putative VDRE (-937/-922) and deletion of the region up to -723. An elecrophoretic motility shift assay demonstrated that the VDR-RXR_heterodimer binds to AGGTCAGCCTGGTTCA (-937/-922) ; VDRE/nuclear protein supershift complexes bound to anti-VDR and RXRβ_antibodies were detected in vitamin D_3-treated ST2 cells. Furthermore, when cocultured with mouse bone marrow macrophages, later-passage ST2 cells showed a decrease in osteoclastogenesis because of decreased RANKL mRNA expression. However, earlier- and later-passage ST2 cells transfected with a RANKL promoter construct showed the same level of luciferase activity and vitamin D_3 induction, suggesting that a cis-regulatory mechanism might be involved in the dependence of RANKL gene expression on passage number. Since CpG loci (-66/+246) were predominantly methylated in later-passage ST2 cells, CpG methylation around the transcription and translation start sites probably regulates, in part, RANKL gene expression.We have presented our data at various international as well as domestic meeting, and published scientific papers for academic journals.