Abstract 33

Truncation mutants of RANKL within the TNF-like core domain inhibit RANKL-induced osteoclast differentiation and activation

Cathy Wang¹, Jamie We-Yin Tan¹, Nathan Pavlos, Jian-Ming Li² Jill Cornish1, Ming-Hao Zheng, Jiake Xu¹

^1. Department of Orthopaedic Surgery, QEII Medical Centre, University of Western Australia, Perth, WA, 6009 Australia Department of Medicine

^2. University of Auckland, New Zealand

Receptor activator of NF-kB ligand (RANKL) is a crucial factor necessary for osteoclast differentiation and activation. In this study we have examined the role of the TNF-like core domain of RANKL in osteoclast differentiation and activation. To this end, a series of truncation mutants of the TNF-like core domain of RANKL were expressed as GST-fusion proteins, and their biological activities assessed using a number of pro-osteoclastogenic systems. Osteoclastogenesis assays revealed that while GST-rRANKL (aa160-318) containing the full TNF-like core region strongly induced osteoclast formation, RANKL truncation mutants GST-rRANKL (aa239- 318), (aa160-268), (aa160-291), (aa246-318) display significantly decreased osteoclastogenic activity. Consistently, the decrease in osteoclast number correlates with decreased TRACP activity and reduced calcitonin receptor and cathapsin K gene expression. Furthermore, competition studies revealed that all RANKL truncation mutants were capable of inhibiting RANKL (aa160-318)-induced osteoclast formation but with different efficacy, RANKL mutant (aa246-318) being the most potent. RANKL mutant (aa246-318) was also found to competitively decrease RANKL (aa160-318)-induced osteoclastic bone resorption in vitro. Interestingly, GST pull down studies reveal that all RANKL mutants have reduced binding affinity to RANK. In addition, all RANKL mutants display significant reduction in the activation of crucial osteoclastic signalling pathways including NF-kB, ERK, JNKs and have decreased IKBá degradation as compared to the full-length protein. Together, our data indicate that RANKL mutants within the TNF-like core domain may act as competitive inhibitors of RANKL-induced osteoclast differentiation and activation and thus may offer potential therapeutic approaches to combat bone lytic disorders.

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