Roles of metalloprotease-disintegrins in osteoclastogenesis

金属蛋白酶-去整合素是一个跨膜糖蛋白家族，在细胞-细胞相互作用和蛋白质如肿瘤坏死因子α（TNFα）的胞外域加工中发挥作用。最近，一种膜结合的TNF家族蛋白被命名为破骨细胞分化因子（ODF，也称为RANKL、OPGL和TRANCE），已被鉴定为正常破骨细胞生成的必需因子。ODF在成骨基质细胞中表达，并通过细胞-细胞直接接触支持破骨细胞生成。在一定条件下，ODF的胞外结构域脱落并释放到培养基中。这种脱落消除了基质细胞和骨髓破骨细胞前体之间的细胞-细胞相互作用，因此，可能显着改变破骨细胞生成的效率。然而，控制ODF脱落的机制仍有待阐明。我们已经开发了一种检测可溶性ODF（sODF）的检测系统，其中sODF被6 ×组氨酸标记的骨保护素（OPG）从培养基或组织液中捕获，ODF的诱饵受体，并使用抗ODF抗体通过免疫印迹检测。当小鼠颅骨来源的成骨细胞/基质细胞在维生素D3的存在下培养时，膜结合ODF的表达显著升高。在这些条件下，未检测到sODF。然而，当细胞同时用佛波醇12-肉豆蔻酸酯-13-乙酸酯（PMA）或促炎细胞因子（如IL-1、TNFα、IL-17或IL-11）处理时，在培养基中检测到大量sODF。金属蛋白酶抑制剂KB 8301能有效地抑制ODF的脱落。在金属蛋白酶-去整合素家族蛋白中，我们在小鼠颅骨衍生的成骨细胞/基质细胞中鉴定了TACE（ADAM 17）和Kuzbanian（ADAM 10）的mRNA。需要进一步研究以确定哪些酶参与脱落过程。

Metalloprotease-disintegrins are a family of transmembrane glycoproteins that play roles in cell-cell interaction and in the processing of the ectodomains of proteins such as tumor necrosis factor α (TNFα). Recently, a membrane-bound TNF family protein designated as osteoclast differentiation factor (ODF, also called RANKL, OPGL and TRANCE) has been identified as an essential factor for normal osteoclastogenesis. ODF is expressed in osteoblastic stromal cells and support osteoclastogenesis by direct cell-cell contact. Under certain condition, the ectodomain of ODF is shed and released into the media. This shedding abolishes cell-cell interaction between stromal cells and bone marrow osteoclast precursors, therefore, may significantly alter the efficiency of osteoclastogenesis. However, the mechanism which controls the shedding of ODF remains to be elucidated. We have developed an assay system to detect soluble ODF (sODF), in which the sODF is captured from culture media or tissue fluid by 6xhistidine-tagged osteoprotegerin (OPG), a decoy receptor of ODF, and detected by immunoblotting using an anti-ODF antibody. When murine calvaria-derived osteoblast/stromal cells were cultured in the presence of vitamin D3, the expression of membrane-bound ODF was significantly elevated. Under these conditions, sODF was not detected. However, when cells were simultaneously treated with phorbol 12-myristate-13-acetate (PMA) or proinflammatory cytokines, such as IL-1, TNFα, IL-17 or IL-11, a significant amount of sODF was detected in the culture media. The shedding of ODF was potently inhibited by KB8301, a metalloprotease inhibitor. Of the metalloprotease-disintegrin family proteins, we identified mRNAs for TACE (ADAM17) and Kuzbanian (ADAM10) in murine calvaria-derived osteoblast/atromal cells. Further studies are necessary to determine which enzymes are involved in the shedding process.