Chloroquine reduces osteoclastogenesis in murine osteoporosis by preventing TRAF3 degradation
Yan Xiu, Hao Xu, Chen Zhao, Jinbo Li, Yoshikazu Morita, Zhenqiang Yao, Lianping Xing, Brendan F. Boyce
Yan Xiu, Hao Xu, Chen Zhao, Jinbo Li, Yoshikazu Morita, Zhenqiang Yao, Lianping Xing, Brendan F. Boyce
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Research Article Bone biology

Chloroquine reduces osteoclastogenesis in murine osteoporosis by preventing TRAF3 degradation

Abstract

The cytokines RANKL and TNF activate NF-κB signaling in osteoclast precursors (OCPs) to induce osteoclast (OC) formation. Conversely, TNF can limit OC formation through NF-κB p100, which acts as an inhibitor, and TNF receptor–associated receptor 3 (TRAF3); however, a role for TRAF3 in RANKL-mediated OC formation is unknown. We found that TRAF3 limits RANKL-induced osteoclastogenesis by suppressing canonical and noncanonical NF-κB signaling. Conditional OC-specific Traf3-KO (cKO) mice had mild osteoporosis and increased OC formation. RANKL induced TRAF3 degradation via the lysosome/autophagy system. The autophagy/lysosome inhibitor chloroquine reduced RANKL-induced OC formation and function by increasing TRAF3 expression in OCPs in vitro and in vivo. Although chloroquine had no effect on basal bone resorption, it inhibited parathyroid hormone– and ovariectomy-induced OC activation in WT, but not cKO, mice. Deletion of the transcription factor gene Relb resulted in increased TRAF3 expression in OCPs, which was associated with decreased RANKL-induced TRAF3 degradation. RelB directly increased expression of BECN1, a key autophagy regulator, by binding to its promoter. These data indicate that autophagic/lysosomal degradation of TRAF3 is an important step in RANKL-induced NF-κB activation in OCPs. Furthermore, treatments that increase TRAF3 levels in OCPs, including pharmacological inhibition of its degradation with compounds such as chloroquine, may limit bone destruction in common bone diseases.

Authors

Yan Xiu, Hao Xu, Chen Zhao, Jinbo Li, Yoshikazu Morita, Zhenqiang Yao, Lianping Xing, Brendan F. Boyce

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Figure 2

RANKL-induced TRAF3 degradation is lysosome mediated.

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RANKL-induced TRAF3 degradation is lysosome mediated. (A) 293T cells tra...
(A) 293T cells transfected with HA-tagged WT or mutant TRAF3 constructs. L-cKO BM-derived OCPs infected with WT or mutant pMX-TRAF3 viruses were serum deprived or treated with RANKL for 8 hours. (B) Ubiquitinated (Ub) proteins from whole cell lysates of RANKL-treated (2 hours) WT OCPs using UbiQapture-Q Matrix blotted with anti-TRAF3 Ab. (C) WT OCPs pretreated with NH4Cl (50 mM) for 1 hour, CQ (50 mM) for 6 hours, or MG132 (20 mM) for 4 hours were treated with RANKL for 8 hours. For IκBα, WT OCPs were treated with DMSO or MG132 with or without RANKL for indicated times. Lanes were run on the same gel, but were noncontiguous in B and C. (D) WT OCPs pretreated with bafilomycin (Bafilo; 50 ng/ml) for 16 hours or 3-MA (3-MA (5 mM) for 2 hours were treated with or without RANKL for 8 hours in serum-enriched or -deprived conditions. (E) TRAF3-GFP retrovirus–infected WT OCPs treated with RANKL for 8 hours were fixed and double-stained with TRAF3 and LAMP2 Abs. Left plot shows background staining with isotype control Abs. (F) TRAF3-GFP retrovirus–infected WT OCPs stained with anti-LAMP2 Ab. Colocalization assessed using confocal microscopy. Original magnification, ×60. (G) TRAF3-GFP retrovirus–infected WT OCPs treated with or without RANKL or CQ (2 mM) were stained with anti-LAMP2 Ab. TRAF3/LAMP2 double-positive cells were counted. Original magnification, ×20. *P < 0.05.