A Peptide Mimicking a TNF Receptor Loop Blocks RANKL-induced Signaling

Objective: RANK ligand (RANKL) and TNFα both promote osteoclast differentiation by binding to their cognate receptors RANK (receptor activator of NF-κB) and TNF receptor, respectively. A critical ligand contact site on the TNF receptor is partly conserved in RANK. We therefore tested the effect of a peptide (WP9QY) that mimics this TNF receptor contact site on RANKL-induced osteoclast formation and activation. Methods: Osteoclastogenesis assay was performed using 30ng/ml RANKL and 20ng/ml M-CSF together with 0-100μM WP9QY. Bone resorption assay was done using authentic mature osteoclasts. They were isolated from 4 day-old C57BL/6J mice, then stimulated by 50ng/ml RANKL or together with WP9QY. EMSA, immunoprecipitation, and western blotting were used for showing the inhibitory effects of a peptide downstream of RANK. BIAcore was used for a binding study. C57BL/6J mice were used for both ovariectomy and low calcium feeding studies. Results: WP9QY dose-dependently inhibited osteoclastogenesis in RANKL-treated bone marrow cells, even when TNF receptors were missing or blocked, prevented RANKL-induced increased bone resorption by isolated osteoclasts, and blocked RANKL-induced signaling downstream of RANK in RAW 264.7 cells. In vivo, WP9QY prevented the increased osteoclastogenesis and bone loss induced by either ovariectomy or low dietary calcium. As measured by surface plasmon resonance, WP9QY bound to sRANKL but had little effect on the ability of sRANKL to bind to RANK. Molecular modeling studies suggest that the binding of WP9QY to RANKL blocks only one of the two receptor-ligand contact points, allowing the two proteins to bind while forcing a conformational change in the membrane-proximal portion of the extracellular domain of ligand-bound RANK. Conclusion: The conformational change is predicted to antagonize the clustering of RANK cytoplasmic domains that normally initiates signaling downstream of RANK, thereby WP9QY could block osteoclastogenesis and bone resorption induced by RANKL.