Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Department of Orthopedics, First Hospital of Wuhan, Wuhan 430022, China.
Bone. 2018 Sep;114:257-267. doi: 10.1016/j.bone.2018.06.015. Epub 2018 Jun 19.
Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an essential component of the signaling complex that mediates osteoclastogenesis. As an adaptor protein of E3 ligase function, TRAF6 regulates NF-κB signaling via TAK1 and I-κB kinase (IKK) activation. Here, we investigated novel mechanisms by which TRAF6 signaling is regulated under receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis.
A yeast two-hybrid screen system identified cellular factors that interact with TRAF6. The interactions were confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays, followed by immuno-blotting. The role of TRAF6 in bone growth and remodeling was determined by osteoclast differentiation and bone-resorption pit assays. Regulatory mechanisms were examined by co-immunoprecipitation, immuno-blotting, real-time polymerase chain reaction, and luciferase reporter assays.
We show that B-cell chronic lymphatic leukemia protein 3 (BCL3) interacts with TRAF6 through its ankyrin-repeat domain and inhibits osteoclastogenesis in bone marrow derived macrophages (BMDMs). Further, TRAF6 interacts with CYLD to mediate BCL3 deubiquitination, which facilitates the cytoplasmic accumulation of BCL3 and represses BCL3 and p50 complex-mediated cyclin D1 transcription.
TRAF6 promotes RANKL-induced osteoclastogenesis by regulating novel non-canonical NF-κB signaling via BCL3 deubiquitination, indicating that BCL3 provides valuable insights into bone loss-associated diseases.
肿瘤坏死因子受体相关因子 6（TRAF6）是介导破骨细胞形成的信号复合物中的一个必需组成部分。作为 E3 连接酶功能的衔接蛋白，TRAF6 通过 TAK1 和 I-κB 激酶（IKK）的激活来调节 NF-κB 信号。在这里，我们研究了 TRAF6 信号在核因子-κB 受体激活物配体（RANKL）诱导的破骨细胞形成下受到调节的新机制。
酵母双杂交筛选系统鉴定了与 TRAF6 相互作用的细胞因子。通过谷胱甘肽 S-转移酶下拉和共免疫沉淀测定，以及免疫印迹来确认相互作用。通过破骨细胞分化和骨吸收陷窝测定来确定 TRAF6 在骨生长和重塑中的作用。通过共免疫沉淀、免疫印迹、实时聚合酶链反应和荧光素酶报告基因测定来检查调节机制。
我们表明 B 细胞慢性淋巴细胞白血病蛋白 3（BCL3）通过其锚蛋白重复结构域与 TRAF6 相互作用，并抑制骨髓来源巨噬细胞（BMDMs）中的破骨细胞形成。此外，TRAF6 与 CYLD 相互作用以介导 BCL3 的去泛素化，这促进了 BCL3 的细胞质积累，并抑制了 BCL3 和 p50 复合物介导的细胞周期蛋白 D1 转录。
TRAF6 通过调节新的非经典 NF-κB 信号通路促进 RANKL 诱导的破骨细胞形成，通过 BCL3 去泛素化作用，表明 BCL3 为与骨丢失相关的疾病提供了有价值的见解。
