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Regulatory mechanism of bone metabolism by multimers of RANKL

RANKL多聚体对骨代谢的调控机制

基本信息

批准号：
15591963
负责人：
IKEDA Tohru
金额：
$ 2.24万
依托单位：
Nagasaki University (2004)Tokyo Medical and Dental University (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

We identified three RANKL isoforms. RANKL1 was identical to the originally reported RANKL. RANKL2 had a shorter intracellular domain. RANKL3 did not have the intracellular or transmembrane domains. The three RANKL isoforms was cloned into expression vectors, respectively, and transfected into NIH3T3 cells. In addition, multiple RANKL isoforms were cotransfected into NIH3T3 cells. NIH3T3 cells expressing RANKL isoform(s) were cocultured with bone marrow macrophages, and osteoclastogeneisis was analyzed. NIH3T3 cells expressing RANKL1 or RANKL2 formed tartrate-resistant acid phosphatase-positive cells. Coexpression of RANKL1 and RANKL2 induced fusion of perosteoclasts. NIH3T3 cells expressing RANKL3 had no effect on the formation of tartrate-resistant acid phosphatase-positive cells, but significantly inhibited fusion of preosteoclasts when coexpressed with RANKL1 and RANKL2. These findings suggest that multiple multimeric RANKL structures regulate bone metabolism.We also developed new sandwich ELISA system to detect RANKL protein, and analyzed the concentration of soluble RANKL using serum taken from 50 postmenopausal women. Serum concentration of RANKL was positively correlated with age and concentration of urinary deoxypiridinoline. These findings suggest that serum concentration of RANKL reflects bone metabolism.We also analyzed the expression of RANKL in human peripheral T lymphocytes. The expression of RANKL was very low in the T lymphocytes, but higher expression was detected in the stimulated T lymphocytes and T lymphocytes derived from inflammatory lesions. These findings suggest that RANKL is important to the function of T lymphocytes in addition to osteoclastogenesis.

我们鉴定了三种RANKL亚型。RANKL 1与最初报告的RANKL相同。RANKL 2具有较短的胞内结构域。RANKL 3不具有胞内或跨膜结构域。将RANKL的3种亚型分别克隆到表达载体中，转染NIH3T3细胞。此外，将多种RANKL同种型共转染至NIH 3T3细胞中。将表达RANKL同种型的NIH 3T3细胞与骨髓巨噬细胞共培养，并分析破骨细胞生成。表达RANKL 1或RANKL 2的NIH3T3细胞形成抗酒石酸酸性磷酸酶阳性细胞。RANKL 1和RANKL 2共表达诱导破骨细胞融合。表达RANKL 3的NIH3T3细胞对抗酒石酸酸性磷酸酶阳性细胞的形成没有影响，但当与RANKL 1和RANKL 2共表达时，显着抑制破骨细胞前体的融合。我们还建立了一种新的检测RANKL蛋白的夹心ELISA方法，并对50例绝经后妇女血清中可溶性RANKL的浓度进行了分析。血清RANKL浓度与年龄、尿脱氧吡啶啉浓度呈正相关。本研究还分析了RANKL在人外周血T淋巴细胞中的表达。RANKL在T淋巴细胞中的表达很低，但在刺激的T淋巴细胞和炎性病变来源的T淋巴细胞中检测到较高的表达。这些结果表明，RANKL是重要的T淋巴细胞的功能，除了破骨细胞生成。