Cheng Taksum, Pavlos Nathan J, Wang Cathy, Tan Jamie We-Yin, Lin Jian Ming, Cornish Jillian, Zheng Ming-Hao, Xu Jiake
Molecular Orthopaedics Laboratory, Centre for Orthopaedic Research, School of Surgery and Pathology, University of Western Australia, Queen Elizabeth II Medical Centre, Second Floor M Block, Nedlands, Western Australia 6009, Australia.
Mol Endocrinol. 2009 Jan;23(1):35-46. doi: 10.1210/me.2007-0465. Epub 2008 Nov 13.
Receptor activator of nuclear factor-kappaB ligand (RANKL) is a key factor necessary for osteoclast differentiation and activation. Mutations within the TNF-like core domain of RANKL have been recently reported in patients with osteoclast-poor autosomal recessive osteopetrosis. However, the functional consequence owing to RANKL mutations has not been well characterized. Here we describe the functional propensity of RANKL mutants in osteoclast differentiation and their impact on RANKL-mediated signaling cascades. Recombinant RANKL (rRANKL) mutants within the TNF-like core domain exhibited diminished osteoclastogenic potential as compared with wild-type rRANKL1 encoding the full TNF-like core domain [amino acids (aa) 160-318]. Consistent with the insufficient activities on osteoclastogenesis, rRANKL mutants showed reduced activation of nuclear factor-kappaB, IkappaBalpha degradation, and ERK phosphorylation. In addition, we found that rRANKL mutants interfered with wild-type rRANKL-induced osteoclastogenesis with deletion mutant rRANKL5 (aa 246-318) exhibiting the greatest inhibitory effect. The same mutant also significantly reduced wild-type rRANKL1 (aa 160-318)-induced osteoclastic bone resorption in vitro. BIAcore assays demonstrated that rRANKL5 alone, lacking the AA'' and CD loops, weakly binds to receptor activator of nuclear factor-kappaB (RANK). Intriguingly, preincubation of mutant rRANKL5 with rRANKL1 before exposure to RANK enhanced the maximal binding level to RANK, indicating that rRANKL5 forms hybrid trimeric complexes with rRANKL1. Furthermore, RANKL mutant mimicking human RANKL V277 mutation in patients, impairs osteoclast differentiation and signaling. Taken together, these data lend support to the notion that the TNF-like core domain of RANKL contains structural determinants that are crucial for osteoclast differentiation and activation, thus providing a possible mechanistic explanation for the observed phenotype in osteopetrotic patients harboring RANKL mutations.
核因子κB受体激活剂配体（RANKL）是破骨细胞分化和激活所必需的关键因子。最近在破骨细胞缺乏的常染色体隐性骨硬化症患者中报道了RANKL肿瘤坏死因子样核心结构域内的突变。然而，RANKL突变所导致的功能后果尚未得到充分表征。在此，我们描述了RANKL突变体在破骨细胞分化中的功能倾向及其对RANKL介导的信号级联反应的影响。与编码完整肿瘤坏死因子样核心结构域[氨基酸（aa）160 - 318]的野生型rRANKL1相比，肿瘤坏死因子样核心结构域内的重组RANKL（rRANKL）突变体表现出破骨细胞生成潜能降低。与破骨细胞生成活性不足一致，rRANKL突变体显示出核因子κB激活减少、IκBα降解减少以及ERK磷酸化减少。此外，我们发现rRANKL突变体干扰野生型rRANKL诱导的破骨细胞生成，其中缺失突变体rRANKL5（aa 246 - 318）表现出最大抑制作用。同一突变体在体外也显著降低了野生型rRANKL1（aa 160 - 318）诱导的破骨细胞骨吸收。BIAcore分析表明，单独的rRANKL5缺乏AA''和CD环，与核因子κB受体激活剂（RANK）的结合较弱。有趣的是，在暴露于RANK之前，将突变体rRANKL5与rRANKL1预孵育可提高与RANK的最大结合水平，表明rRANKL5与rRANKL1形成杂合三聚体复合物。此外，模仿患者中人类RANKL V277突变的RANKL突变体损害破骨细胞分化和信号传导。综上所述，这些数据支持了这样一种观点，即RANKL的肿瘤坏死因子样核心结构域包含对破骨细胞分化和激活至关重要性的结构决定因素，从而为携带RANKL突变的骨硬化症患者中观察到的表型提供了一种可能的机制解释。
