Lee Francis Young-In, Kim Dae-Won, Karmin Jaime A, Hong Daewha, Chang Seong-Sil, Fujisawa Motoyuki, Takayanagi Hiroshi, Bigliani Louis U, Blaine Theodore A, Lee Hahn-Jun
Center for Orthopaedic Research, Department of Orthopaedic Surgery, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.
J Biol Chem. 2005 Aug 19;280(33):29929-36. doi: 10.1074/jbc.M414600200. Epub 2005 Jun 13.
To clarify the role of calpain in the receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis, RANKL-induced calpain activation was examined by using murine RAW 264.7 cells and bone marrow-derived monocyte/macrophage progenitors. We found that calpain activity increased in response to RANKL in both cell types based on alpha-spectrinolysis and that mu-calpain, rather than m-calpain, was activated during RANKL-supported osteoclastogenesis in RAW 264.7 cells. Overexpression of mu-calpain clearly augmented RANKL-supported osteoclastogenesis in RAW 264.7 cells, thereby implicating its pivotal role in this process. Cell-permeable calpain inhibitors, including calpastatin and calpeptin, were sufficient to suppress RANKL-supported osteoclastogenesis based on decreased expression of the osteoclastogenic marker, matrix metalloproteinase 9, and the generation of tartrate-resistant acid phosphatase-positive multinucleated cells in both cell types. Calpain inhibitors suppressed NF-kappaB activation via inhibition of the cleavage of inhibitor of NF-kappaB(IkappaBalpha)in RAW 264.7 cells. Taken together, our findings suggest that mu-calpain is essential to the regulation of RANKL-supported osteoclastogenesis via NF-kappaB activation.
为阐明钙蛋白酶在核因子-κB 受体激活剂配体（RANKL）支持的破骨细胞生成中的作用，我们使用小鼠 RAW 264.7 细胞和骨髓来源的单核细胞/巨噬细胞祖细胞检测了 RANKL 诱导的钙蛋白酶激活情况。我们发现，基于α-血影蛋白水解，两种细胞类型中钙蛋白酶活性均随 RANKL 刺激而增加，且在 RAW 264.7 细胞的 RANKL 支持的破骨细胞生成过程中，μ-钙蛋白酶而非 m-钙蛋白酶被激活。μ-钙蛋白酶的过表达明显增强了 RAW 264.7 细胞中 RANKL 支持的破骨细胞生成，从而表明其在该过程中起关键作用。包括钙蛋白酶抑制蛋白和钙肽素在内的细胞可渗透钙蛋白酶抑制剂，基于破骨细胞生成标志物基质金属蛋白酶 9 表达的降低以及两种细胞类型中抗酒石酸酸性磷酸酶阳性多核细胞的生成减少，足以抑制 RANKL 支持的破骨细胞生成。钙蛋白酶抑制剂通过抑制 RAW 264.7 细胞中核因子-κB 抑制蛋白（IκBα）的裂解来抑制核因子-κB 的激活。综上所述，我们的研究结果表明，μ-钙蛋白酶通过激活核因子-κB 对 RANKL 支持的破骨细胞生成的调节至关重要。
