Regulatory mechanism of bone metabolism by multimers of RANKL

我们确定了三个RANKL亚型。RANKL1与最初报道的rank1相同。RANKL2的胞内结构域较短。RANKL3不具有胞内或跨膜结构域。将三个RANKL亚型分别克隆到表达载体中，转染NIH3T3细胞。此外，将多个RANKL亚型共转染到NIH3T3细胞中。将表达RANKL亚型的NIH3T3细胞与骨髓巨噬细胞共培养，分析其破骨细胞发生情况。表达RANKL1或RANKL2的NIH3T3细胞形成抗酒石酸酸性磷酸酶阳性细胞。RANKL1和RANKL2的共表达诱导破骨细胞融合。表达RANKL3的NIH3T3细胞对酒石酸盐抗性酸性磷酸酶阳性细胞的形成没有影响，但与RANKL1和RANKL2共表达时显著抑制破骨前细胞的融合。这些发现表明多种多聚RANKL结构调节骨代谢。我们还开发了新的夹心ELISA检测RANKL蛋白，并利用50例绝经后妇女血清分析了可溶性RANKL的浓度。血清RANKL浓度与年龄、尿脱氧吡啶啉浓度呈正相关。这些结果提示血清RANKL浓度反映骨代谢。我们还分析了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，但在受刺激的T淋巴细胞和炎性病变的T淋巴细胞中表达较高。这些发现表明RANKL除了对破骨细胞的发生外，对T淋巴细胞的功能也很重要。

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.