REGULATORY MECHANISM OF RANKL GENE EXPRESSION

破骨细胞分化因子（RANKL）是造血前体破骨细胞形成和维持所必需的。为了阐明RANKL基因表达和破骨细胞形成的机制，我们对小鼠和人的RANKL基因启动子进行了表征。人和小鼠RANKL基因启动子具有共同的结构、倒置tata和CAAT盒子、Runx2/Cbfa-1结合位点和维生素D响应元件（VDRE）。为了阐明溶骨转移的分子机制，我们对小鼠实验模型和尸检人骨标本的溶骨病变中RANKL基因的表达和破骨细胞的发生进行了研究。在小鼠和人肿瘤转移所致的溶骨病变中，RANKL在靠近癌细胞巢的间质/成骨细胞上表达。在小鼠模型中，产生pthrp的肿瘤产生溶骨损伤，而不产生pthrp的肿瘤很少引起RANKL表达和诱导破骨细胞。我们进一步分析了PTHrP对RANKL基因转录的影响。通过小鼠和人RANKL基因启动子缺失构建体的瞬时转染研究，PTHrP通过位于VDRE附近的c-AMP响应元件（CRE）上调小鼠和人RANKL基因的转录活性。EMSA显示特异性蛋白DNA结合，并与抗creb1和-ATF2抗体超移。因此，PTHrP通过间质/成骨细胞上RANKL的表达诱导破骨细胞骨吸收，提供一个有利于产生PTHrP的癌细胞存活的骨微环境。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 the formation and maintenance of osteoclasts from hematopoietic precursors. To clarify the mechanism of RANKL gene expression and osteoclastogenesis, mouse and human RANKL gene promoters were characterized. Both human and mouse RANKL gene promoter shares the common structure, inverted-TATA and CAAT boxes, Runx2/Cbfa-1 binding sites and vitamin D responsive element (VDRE). To elucidate the molecular mechanism of osteolytic bone metastasis, we assessed RANKL gene expression and osteoclastogenesis in osteolytic lesions of mouse experimental model and human bone specimen taken at the autopsy. 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. In the mouse model, PTHrP-producing tumor generated osteolytic lesion, whereas non-producing tumor rarely caused RANKL expression and induction of osteoclasts. We further analyzed the effects of PTHrP on RANKL gene transcription. By transient transfection studies using deletion constructs of mouse and human RANKL gene promoter, PTHrP upregulated the transcriptional activity through c-AMP responsive element (CRE) located close to VDRE in both mouse and human. EMSA showed specific protein DNA binding, and the supershift with anti-CREB1 and -ATF2 antibodies. Thus PTHrP induces osteoclastic bone resorption through the RANKL expression on stromal/osteoblastic cells, affording a bone microenvironment conducive to the survival of PTHrP-producing cancer cells.We have presented our data at various international as well as domestic meeting, and published scientific papers for academic journals.