Kitazawa Sohei, Kajimoto Kazuyoshi, Kondo Takeshi, Kitazawa Riko
Division of Molecular Pathology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
J Cell Biochem. 2003 Jul 1;89(4):771-7. doi: 10.1002/jcb.10567.
Receptor activator of NF-kappaB ligand (RANKL) has been identified as requisite for osteoclastogenesis. To elucidate the molecular mechanism that conducts its catabolic action on bone, the effect of 1alpha,25 dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) on osteoclastogenesis and RANKL mRNA expression was examined by coculture, RT-PCR and nuclear run-on studies. By accelerating the transcription rate of the RANKL gene in SaOS2 osteoblastic cells, 1alpha,25(OH)(2)D(3) enhanced in vitro osteoclast formation from peripheral monocytes. Cloning and characterization of the 5'-flanking region of the human RANKL gene revealed that the basic promoter comprises inverted TATA- and CAAT-boxes flanked by RUNX2 binding sites. Both electrophoresis mobility shift assay (EMSA) and transfection studies demonstrated that 1alpha,25(OH)(2)D(3) activated human RANKL promoter through vitamin D responsive elements (VDRE) located at -1584/-1570 by binding VDR and RXRalpha heterodimers in a ligand-dependent manner. The results provide direct evidence that 1alpha,25(OH)(2)D(3) augments osteoclastogenesis by transactivating the human RANKL gene in osteoblastic cells through VDRE.
核因子κB受体活化因子配体（RANKL）已被确定为破骨细胞生成所必需的因子。为了阐明其对骨骼产生分解代谢作用的分子机制，通过共培养、逆转录-聚合酶链反应（RT-PCR）和核转录分析研究了1α,25-二羟基维生素D3（1α,25(OH)2D3）对破骨细胞生成和RANKL mRNA表达的影响。1α,25(OH)2D3通过加速SaOS2成骨细胞中RANKL基因的转录速率，增强了外周单核细胞在体外形成破骨细胞的能力。对人RANKL基因5'侧翼区域的克隆和特性分析表明，基本启动子包含倒置的TATA盒和CAAT盒，两侧为RUNX2结合位点。电泳迁移率变动分析（EMSA）和转染研究均表明，1α,25(OH)2D3通过位于-1584/-1570的维生素D反应元件（VDRE），以配体依赖的方式结合维生素D受体（VDR）和维甲酸X受体α（RXRα）异二聚体，从而激活人RANKL启动子。这些结果提供了直接证据，表明1α,25(OH)2D3通过VDRE在成骨细胞中转录激活人RANKL基因，从而增强破骨细胞生成。
