A Novel Receptor Activator of NF-κB (RANK) Cytoplasmic Motif Plays an Essential Role in Osteoclastogenesis by Committing Macrophages to the Osteoclast Lineage

Abstract

Receptor activator of NF-B (RANK) ligand (RANKL) and its receptor RANK play an essential role in osteoclastogenesis and osteoclast function by activating several signaling pathways. However, several lines of evidence suggest that RANK also transmits an unidentified signaling pathway(s) essential for osteoclastogenesis. To identify the novel pathway(s), we carried out a detailed structure/function study of the RANK cytoplasmic domain. A series of studies using numerous deletion/point mutants elucidated a specific 4-amino acid motif ( 535 IVVY 538 ) essential for osteoclastogenesis. This novel motif plays a crucial role in committing macrophages to the osteoclast lineage but is not implicated in osteoclast function or survival. Moreover, this motif does not activate the known RANK signaling pathways, indicating that it initiates a novel pathway(s). The identification of the novel motif not only provides critical insight into RANK signaling in osteoclastogenesis, but more importantly, the RANK motif and its downstream signaling pathways may represent specific therapeutic targets for various bone diseases, including postmenopausal osteoporosis.Receptor activator of nuclear factor B ligand (RANKL), 3 also known as OPGL (osteoprotegerin (OPG) ligand)/ODF (osteoclast differentiation factor)/TRANCE (tumor necrosis factor (TNF)-related activationinduced cytokine), was identified as a member of the TNF superfamily independently by two bone groups (1, 2) and two immunology groups (3, 4) in the late 1990s. To date, RANKL has been shown to play pivotal roles in regulating various biological processes such as bone homeostasis (2, 5), immune function (3, 6) and mammary gland development (7).In bone, RANKL is not only an essential regulator of osteoclast formation (osteoclastogenesis) but also a potent modulator of osteoclast function and survival (5,8,9). RANKL exerts its effects on osteoclast formation and function by binding to its receptor RANK (receptor activator of nuclear factor B) expressed on osteoclast precursors and mature osteoclasts (1, 10, 11). RANKL also has a decoy receptor, OPG, which inhibits RANKL action by competing with RANK for binding RANKL (5,12). The essential role of both RANKL and RANK in the osteoclastogenic process has been unambiguously demonstrated by the findings that mice lacking the gene for either protein develop osteopetrosis due to failure to form osteoclasts (13)(14)(15).Because the unraveling of the RANKL/RANK system, enormous efforts have been focused on elucidating RANK-initiated intracellular signaling. RANK was identified as a member of the TNF receptor (TNFR) family (3). Members of the TNFR family lack intrinsic enzymatic activity, and hence, they transduce intracellular signals by recruiting various adaptor proteins including TNF receptor-associated factors (TRAFs) through specific motifs in the cytoplasmic domain (16,17). Six TRAF proteins (TRAF1, -2, -3, -4, -5, and -6) have been identified so far, and they play important but distinct roles in TNFR member-induced intracellular sign...

Osteoclasts: New Insights

“…Thirdly, RANK cytoplasmic domain shares no homology with any known members of the TNFR family (74), further suggesting that it may activate downstream signals different from those arising from other members of the TNFR family. A systematic structure/function study of the RANK cytoplasmic domain has identified a specific 4-a.a. RANK motif (IVVY535-538), which function to mediate commitment of macrophages to the osteoclast lineage in osteoclastogenesis (119). This finding is consistent with the previous study that RANKL plays an essential role in committing macrophages to the osteoclast lineage (120).…”

Interaction of Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) and Vav3 in the Receptor Activator of Nuclear Factor κB (RANK) Signaling Complex Enhances Osteoclastogenesis

“…9B, left panel). Similar to previous reports that both T6BSs and the IVVY motif are crucial for osteoclastogenesis (12,13,16,17), OC formation was not detected in hCD40/ mRK mutants that lacked either T6BSs (hCD40/mRK-AAA) or the IVVY motif (hCD40/mRK-LAAF) after 3-day culture (Fig. 9B, left panel).…”

“…Studies of deletion mutations of RANK cyto have revealed that RANK cyto harbors a unique domain named the highly conserved domain in RANK (HCR, amino acids 487-546) that functions as a crucial regulatory motif in RANK-mediated osteoclastogenesis (16,17). Upon RANKL stimulation, HCR recruits additional adaptor/kinase molecules, such as Vav3, Grb-2-associated binder 2 (Gab2), and phospholipase C ␥2 (PLC␥2), to induce the late phase of RANK-mediated signaling (16,18,19).…”

Selective inhibition of RANK blocks osteoclast maturation and function and prevents bone loss in mice

“…To overcome this issue, we have characterized a signaling pathway that is initiated by a specific motif of RANK (27). While point mutation analyses suggested that this motif might play an important role in OC differentiation in vitro, how it regulates OC formation or whether this motif is also important for the fate of OC in vivo has not been investigated.…”