CHIP Regulates Osteoclast Formation through Promoting TRAF6 Protein Degradation

Bone tissue is constantly remodeled through bone resorption by osteoclasts and bone formation by osteoblasts (1–3). Abnormal osteoclastogenesis may result in bone disorders, such as osteoporosis, Paget’s disease and rheumatoid arthritis. Thus, the understanding of the mechanism of osteoclastogenesis is important for rational design of therapeutic approaches for the treatment of bone disorders. However, the regulatory mechanism of osteoclastogenesis is not fully understood. Nuclear factor of κB (NF-κB) plays a key role in osteoclast formation and bone resorption. During osteoclast differentiation, NF-κB is activated by RANKL, after RANKL association with its receptor, it initiates different cascades during activation of NF-κB pathways. A key event in NF-κB pathways is the activation of TRAF6, which functions as an E3 ligase. Once activated, TRAF6 synthesizes lysine 63-sepecific polyubiquitin chains leading to the activation of IKKβ (I-κB kinase), which phosphorylates I-κBα (the inhibitor of NF-κB), leading to its degradation. The degradation of I-κBα releases NF-κB from the I-κBα/NF-κB complex and allows NF-κB to enter the nucleus where it activates gene expression required for osteoclast formation (4–5). The importance of NF-κB in osteoclastogenesis has been highlighted by several mouse models. Mice lacking RANK, TRAF6, IKKβ and RelA/p65 (a subunit of NF-κB), lead to increased bone mass with absence of osteoclasts (6–9). Therefore, the TRAF6-NF-κB axis appears to be the critical molecular basis for the pathogenesis of abnormal osteoclastogenesis and bone diseases. Recent studies suggest that TRAF6 is critical in osteoclast formation and bone remodeling. For instance, TRAF6 interacts with RANK, while RANKL binds to RANK, to activate NF-κB signaling which in turn regulates osteoclastogenesis (2, 10–12). Moreover, TRAF6 knockout (KO) mice develop severe osteopetrosis and failure of bone resorption due to impaired RANK signaling pathway (7, 13). The suppressor of RANK signaling pathway, FHL2, inhibits the activated osteoclasts through blocking RANK/TRAF6 interaction. FHL2−/− osteoclasts are hyper-resorptive and FHL2−/− mice undergo RANKL-stimulated bone loss (14). Collectively, TRAF6 functions as a major adaptor molecule for RANK signaling pathway associated with osteoclastogenesis. To date, several factors have been identified to regulate TRAF6, including deubiquitination enzymes, such as A20, CYLD and USP4 (15–17). In contrast, three E3 ligases, including Smurf1, NUMBL and TRIM38, have been reported to regulate TRAF6 protein ubiquitination and degradation in vitro (18–20). However, the importance of TRAF6 regulation in osteoclast formation in vivo and detail molecular mechanism of TRAF6 degradation remain undefined. Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is a U-box containing protein that interacts with Hsp70 and functions as an E3 ligase for several protein substrates. CHIP induces the ubiquitination of proteins, such as p53, Smads, ER-α, Runx2, Src-3, p65 and TLR4 for proteasome-dependent degradation (20–27). Although it is known that CHIP plays a critical role in immunology and in tumor growth and metastasis, the role of CHIP in skeletal growth and bone remodeling in vivo has not been reported. In this study, we report the role of CHIP in TRAF6 degradation and in regulation of bone mass.