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

Abstract

The signaling pathway downstream of stimulation of receptor activator of nuclear factor B (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANK cyto ) to trigger downstream osteoclastogenic signaling cascades. RANK cyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANK cyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANK cyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANK cyto enhances downstream NF-B, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex.The integrity of the skeletal system is maintained by the process of bone remodeling, in which old and damaged bone is continuously replaced with new bone through the balanced action of bone-resorbing osteoclasts (OCs) 3 and bone-forming osteoblasts (1-3). As the only cells with bone-resorbing activity, OCs are clinically important in bone-related diseases; accelerated bone destruction by OCs results in pathological bone loss associated with rheumatoid arthritis, multiple myeloma, metastatic cancer, and osteoporosis, whereas impaired function of OCs leads to osteopetrotic disorders (2, 4).OCs are multinucleated giant cells that are formed by the fusion of monocyte/macrophage lineage precursors through the process of OC differentiation or osteoclastogenesis (2). The signaling pathways of receptor activator of nuclear factor B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) play key roles in osteoclastogenesis (2, 5). Mice lacking either RANK or RANKL exhibit severe osteopetrotic phenotypes, highlighting the importance of RANK-RANKL signaling as a crucial regulator of OC differentiation, activation, and survival (6, 7). RANK-RANKL signaling is initiated primarily by the recruitment of cytoplasmic TNF receptor-associated factors (TRAFs) that trigger the activation of signaling cascades downstream of adaptors/kinases, such as nuclear fa...

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Regulation of Osteoclast Differentiation by Cytokine Networks

“…RANKL-RANK binding recruits TNFR-associated factors (TRAFs) to initiate the activation of downstream signaling cascades of adaptors/kinases such as NF-κB essential modulator, inhibitor of IκB kinases, c-Src, Vav3, and mitogen-activated protein kinases (MAPKs), including p38, JNK, and ERK ( Fig. 2 ) ( 1 3 10 ). The final consequence of RANKL-RANK signaling is the activation of osteoclastogenic transcription factors such as NF-κB, activator protein 1 (AP-1), cyclic adenosine monophosphate response element-binding protein (CREB), and nuclear factor of activated T cells 1 (NFATc1), all of which induce the expression of osteoclastogenic markers, such as tartrate-resistant acid phosphatase (TRAP), dendritic cell-specific transmembrane protein (DC-STAMP), v-ATPase subunit d2 (Atp6v0d2), OC-associated receptor (OSCAR), β3 integrin, osteopetrosis-associated transmembrane protein 1 (OSTM1), B-lymphocyte induced maturation protein 1 (BLIMP1), and cathepsin K ( 2 3 11 12 13 14 ).…”