Murakami H, Takahashi N, Tanaka S, Nakamura I, Udagawa N, Nakajo S, Nakaya K, Abe M, Yuda Y, Konno F, Barbier A, Suda T
Department of Biochemistry, School of Dentistry, Showa University, Tokyo, Japan.
Bone. 1997 May;20(5):399-404. doi: 10.1016/s8756-3282(97)00025-2.
Signaling pathways mediated by tyrosine phosphorylation and dephosphorylation have been reported to be involved in the regulation of cytoskeletal organization in osteoclasts, the principal cells responsible for bone resorption. We examined the effects of tiludronate [(4-chlorophenyl)thiomethylene bisphosphonate] on the cytoskeleton and the balance of phosphotyrosine levels in osteoclast-like multinucleated cells (OCLs) formed in cocultures of mouse osteoblastic cells and bone marrow cells. When OCLs were placed on plastic dishes in the presence of 10% fetal bovine serum, they formed a ringed structure of F-actin dots (actin ring) within 2 h. Tiludronate did not inhibit the process of actin ring formation, but it disrupted preformed actin rings in a time- and a dose-dependent manner. Western blot analysis using an antiphosphotyrosine antibody revealed that tyrosine phosphorylation of certain proteins in OCLs was stimulated by tiludronate added to the purified OCLs. Tyrosine kinase activity of the p60c-src immunoprecipitated from cell lysates of the purified OCLs was not affected by tiludronate directly added to the kinase assay. OCL lysates stimulated dephosphorylation of tyrosine-phosphorylated substrates such as phosphoneuroprotein 14 and epidermal growth factor receptors. Like sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, tiludronate dose-dependently inhibited tyrosine dephosphorylation of those substrates induced by OCL lysates. These findings suggest that tiludronate disrupts the preformed actin rings and suppresses bone-resorbing activity by inhibiting protein tyrosine phosphatases in osteoclasts.
据报道，由酪氨酸磷酸化和去磷酸化介导的信号通路参与破骨细胞（负责骨吸收的主要细胞）中细胞骨架组织的调节。我们研究了替鲁膦酸盐[（4-氯苯基）硫亚甲基双膦酸盐]对在小鼠成骨细胞和骨髓细胞共培养中形成的破骨细胞样多核细胞（OCLs）的细胞骨架和磷酸酪氨酸水平平衡的影响。当OCLs在10%胎牛血清存在下置于塑料培养皿中时，它们在2小时内形成了F-肌动蛋白点的环状结构（肌动蛋白环）。替鲁膦酸盐不抑制肌动蛋白环的形成过程，但它以时间和剂量依赖的方式破坏预先形成的肌动蛋白环。使用抗磷酸酪氨酸抗体的蛋白质免疫印迹分析显示，添加到纯化的OCLs中的替鲁膦酸盐刺激了OCLs中某些蛋白质的酪氨酸磷酸化。从纯化的OCLs细胞裂解物中免疫沉淀的p60c-src的酪氨酸激酶活性不受直接添加到激酶测定中的替鲁膦酸盐的影响。OCLs裂解物刺激了酪氨酸磷酸化底物如神经磷酸蛋白14和表皮生长因子受体的去磷酸化。与蛋白质酪氨酸磷酸酶抑制剂原钒酸钠一样，替鲁膦酸盐剂量依赖性地抑制了由OCLs裂解物诱导的这些底物的酪氨酸去磷酸化。这些发现表明，替鲁膦酸盐通过抑制破骨细胞中的蛋白质酪氨酸磷酸酶来破坏预先形成的肌动蛋白环并抑制骨吸收活性。
