2025-04-09 東京科学大学
<関連情報>
- https://www.isct.ac.jp/ja/news/6l04f1v3ac7r
- https://www.isct.ac.jp/plugins/cms/component_download_file.php?type=2&pageId=&contentsId=1&contentsDataId=1261&prevId=&key=348c627c855901471f90d1497a924527.pdf
- https://www.sciencedirect.com/science/article/abs/pii/S8756328225000857
RANK-RANKL結合の可視化と定量化による疾患研究および創薬への応用 Visualization and quantification of RANK-RANKL binding for application to disease investigations and drug discovery
Ken-ichi Nakahama, Shiho Hidaka, Kanako Goto, Mayu Tada, Tomoya Doi, Hiroyuki Nakamura, Masako Akiyama, Masahiro Shinohara
Bone Available online: 25 March 2025
DOI:https://doi.org/10.1016/j.bone.2025.117473
Graphical abstract
Highlights
- Visualization of RANK-RANKL binding by a new method using NanoBiT technology
- Quantification of RANK-RANKL binding in living cells revealed ARO mechanisms.
- A new lead compound for osteoporotic patients is found by this system.
Abstract
Receptor activator of NFκB (RANK)-receptor activator of NFκB ligand (RANKL) binding triggers the differentiation of osteoclasts, bone-resorbing cells. The imbalance between bone resorption by osteoclasts and bone formation by osteoblasts causes bone diseases. We herein report the real-time detection of RANK-RANKL binding using the NanoLuc method. Large-BiT-RANK and RANKL-Small-BiT fusion proteins were expressed in HeLa cells, and their co-culture exhibited chemiluminescence in the presence of luciferase substrates. This luminescence was inhibited by the treatment of cells with an anti-RANKL neutralization antibody, indicating that luminescence is dependent on RANK-RANKL binding. Moreover, mutations in RANKL (M198K or G278R) and RANK (G54R or K171G), based on mutations in autosomal recessive osteopetrosis (ARO) patients, did not exhibit the luminescence in the presence of their wild-type counterparts. HeLa cells expressing RANKL mutants did not support osteoclastogenesis. These results clearly indicate that the loss of binding by RANK-RANKL mutants is responsible for osteoclast-poor osteopetrosis in ARO patients. A nuclear factor kappa B reporter gene assay showed the impaired signal transduction of RANK (G54R) by RANKL. Therefore, our method successfully detected and quantified RANK-RANKL binding in living cells. Furthermore, our method is not only useful for investigating the mechanisms underlying osteoclast-poor ARO, but also for the screening of lead compounds that inhibit RANK-RANKL binding in osteoporosis patients. We identified a new compound with a three-dimensional structure that inhibits RANK-RANKL binding using our method. Our detection system for RANK-RANKL binding will contribute to both the development of anti-osteopetrosis drugs and a more detailed understanding of bone cell biology.
