ROLE OF RANKL IN OSTEOLYTIC BONE METASTASIS

破骨细胞分化因子（RANKL）是造血前体细胞向破骨细胞分化所必需的。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中评估了RANKL基因表达和破骨细胞生成。在小鼠和人肿瘤转移所致溶骨性病变中，RANKL表达于癌细胞巢附近的基质/成骨细胞，分析DNA甲基化对其mRNA表达和破骨细胞生成的影响。使用了ST 2细胞亚群：表达RANKL的P9和不表达RANKL的P16。RANKL基因启动子-65/+350区域的CpG甲基化，尤其是TATA盒上游3个碱基，在P16中高于P9。启动子甲基化降低了P16细胞的转录活性，5-aza-dC处理恢复了P16细胞RANKL的表达。RANKL基因启动子的甲基化抑制RANKL基因表达。成骨细胞对骨吸收刺激反应的异质性可能归因于RANKL基因启动子的甲基化状态，我们分析了成骨细胞分化所必需的转录因子Runx 2对RANKL基因的调控。我们通过RT-PCR、ChIP测定和siRNA沉默分析了ST 2细胞和Runx 2缺失小鼠衍生的C6细胞。Runx 2通过浓缩染色质抑制稳态RANKL基因表达，同时对RANKL基本启动子显示出轻微的正作用。我们在各种国际和国内会议上展示了我们的数据，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.