ROLE OF RANKL IN OSTEOLYTIC BONE METASTASIS

破骨细胞分化因子(RANKL)是破骨细胞向造血祖细胞分化所必需的。为了阐明溶骨性骨转移的机制，我们在小鼠实验模型和人骨标本上检测了RANKL基因的表达和破骨细胞的形成。在小鼠和人肿瘤转移的溶骨性病变中，RANKL在靠近癌巢的基质/成骨细胞上均有表达，分析了DNA甲基化对其mRNA表达和破骨细胞生成的影响。ST2细胞亚群：表达RANKL的P9和不表达RANKL的P16。P16组RANKL基因启动子-65/+350区CpG甲基化高于P9组，尤其是TATA-box上游3个碱基。在体外，启动子结构的甲基化降低了P16细胞的转录活性，5-aza-DC处理恢复了P16细胞RANKL的表达。RANKL基因启动子甲基化抑制RANKL基因表达。成骨细胞对骨吸收刺激的异质性可能与RANKL基因启动子的甲基化状态有关。然后，我们分析了成骨细胞分化所必需的转录因子Runx2对RANKL基因的调控。我们通过RT-PCR、芯片分析和siRNA沉默分析了ST2细胞和Runx2基因缺失的小鼠来源的C6细胞。Runx2通过浓缩染色质抑制RANKL基因的稳定表达，同时对RANKL基本启动子显示出轻微的积极作用。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。

Osteoclast differentiation factor (RANKL) is requisite for osteoclasts differentiation from hematopoietic precursors. To elucidate the mechanism of osteolytic bone metastasis, we assessed RANKL gene expression and osteoclastogenesis in mouse experimental model and human bone specimen. In both mouse and human osteolytic lesion due to cancer metastasis, RANKL expression was observed on the stromal/osteoblastic cells close to the cancer cell nests.The effect of DNA methylation on its mRNA expression and osteoclastogenesis was analyzed. Subpopulations of ST2 cells were used : P9 which expresses RANKL and P16 which does not. CpG methylation of the -65/+350 region of the RANKL gene promoter, especially 3 bases upstream of TATA-box, was higher in P16 than in P9. In vitro methylation of the promoter construct reduced the transcriptional activity,5-aza-dC treatment recovered RANKL expression of P16 cells. Methylation of the RANKL gene promoter suppresses RANKL gene expression. The heterogeneity of osteoblastic cells in response to bone-resorbing stimuli may be attributed to the methylation status of the RANKL gene promoter.We then analyzed the RANKL gene regulation by Runx2,transcrition factor essential for osteoblastic differentiation. We analyzed ST2 cells and Runx2 null mouse-derived C6 cells by RT-PCR,ChIP assay, and siRNA silencing. Runx2 suppresses the steady-state RANKL gene expression by condensing chromatin, while showing a slightly positive effect on RANKL basic promoter.We have presented our data at various international as well as domestic meetings, and published scientific papers for academic journals.