Cysteine-rich protein 61 (CYR61/CCN1) belongs to the family of CCN matricellular proteins. Most of the known effects of CCN proteins appear to be due to binding to extracellular growth factors or integrins, including alpha(v)beta(3) and alpha(v)beta(5). Although CYR61 can stimulate osteoblast differentiation, until now the effect of CYR61 on osteoclasts was unknown. We demonstrate that recombinant human CYR61 inhibits the formation of multinucleated, alpha(v)beta(3)-positive, or tartrate-resistant acid phosphatase-positive human, mouse, and rabbit osteoclasts in vitro. CYR61 markedly reduced the expression of the osteoclast phenotypic markers tartrate-resistant acid phosphatase, matrix metalloproteinase-9, calcitonin receptor, and cathepsin K. However, CYR61 did not affect the formation of multinucleated osteoclasts when added to osteoclast precursors prior to fusion or affect the number or resorptive activity of osteoclasts cultured on dentine discs, indicating that CYR61 affects early osteoclast precursors but not mature osteoclasts. CYR61 did not affect receptor activator of nuclear factor-kappaB (RANK) ligand-induced phosphorylation of p38 or ERK1/2 in human macrophages and did not affect RANK ligand-induced activation of nuclear factor-kappaB, indicating that CYR61 does not appear to inhibit osteoclastogenesis by affecting RANK signaling. Furthermore, a mutant form of CYR61 defective in binding to alpha(v)beta(3) also inhibited osteoclastogenesis, and CYR61 inhibited osteoclastogenesis similarly in cultures of mouse wild-type or beta(5)(-/-) macrophages. Thus, CYR61 does not appear to inhibit osteoclast formation by interacting with alpha(v)beta(3) or alpha(v)beta(5). These observations demonstrate that CYR61 is a hitherto unrecognized inhibitor of osteoclast formation, although the exact mechanism of inhibition remains to be determined. Given that CYR61 also stimulates osteoblasts, CYR61 could represent an important bifunctional local regulator of bone remodeling.
The interaction of receptor activator of NFκB (RANK), a member of the tumour necrosis factor receptor superfamily, with RANK ligand is crucial for the formation, function and survival of osteoclasts. The role of the cytoplasmic oligomerisation domain (pre-ligand assembly domain; PLAD or ‘IVVY’ motif) in the ligand-dependent activation of downstream NFκB signalling has not been studied previously. The discovery of truncating mutations of TNFRSF11A (W434X and G280X that lack the PLAD) as the cause of rare cases of osteoclast-poor osteopetrosis offered the opportunity for functional study of this region. Recapitulating the W434X mutation by transcription activator-like effector nuclease (TALEN)-mediated targeted disruption of Tnfrsf11a within the region homologous to W434X in the mouse macrophage-like cell line RAW264.7 impaired formation of osteoclast-like cells. Using overexpression studies, we demonstrated that, in contrast to WT-RANK, the absence of the PLAD in G280X-RANK and W434X-RANK prevented ligand-independent but not ligand-dependent oligomerisation. Cells expressing W434X-RANK, in which only two of the three TRAF6-binding motifs are present, continued to exhibit ligand-dependent NFκB signalling. Hence, the absence of the PLAD did not prevent ligand-induced trimerisation and subsequent NFκB activation of RANK, demonstrating that therapeutic targeting of the PLAD in the prevention of osteoporosis may not be as effective as proposed previously.
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