Chloroquine reduces osteoclastogenesis in murine osteoporosis by preventing TRAF3 degradation
Yan Xiu, … , Lianping Xing, Brendan F. Boyce
Yan Xiu, … , Lianping Xing, Brendan F. Boyce
Published December 9, 2013
Citation Information: J Clin Invest. 2014;124(1):297-310. https://doi.org/10.1172/JCI66947.
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Research Article Bone biology Article has an altmetric score of 45

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 8

RelB binds to the BECN1 promoter and regulates its expression.

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RelB binds to the BECN1 promoter and regulates its expression. Whole cel...
Whole cell lysate WBs (A) and RT-PCR–detected BECN1 mRNA levels (B) in WT or RelB–/– OCPs treated with RANKL or vehicle for 8 hours. (C) 293T cells transfected with RelA- or RelB-expressing plasmids or cotransfected with a human BECN1 promoter pGL3-Luc reporter and a Renilla luciferase plasmid and RelA- and/or RelB-expressing plasmids. RANKL-treated samples were cotransfected with a hRANK plasmid. (D) RelA- or RelB siRNAs were transfected into 293T cells 48 hours before cotransfection with pGL3-Luc and Renilla plasmids. Protein levels were assessed by WB. Dual-luciferase assays were performed 24 hours after transfection. Values in C and D are means + SEM from 3 independent experiments. *P < 0.05; **P < 0.01. (E) WT OCPs treated with RANKL for 8 hours and sheared chromatin precipitated with RelA- or RelB-specific Abs, or IgG (negative control [CTL]). Recovered DNA was used as a template for PCR. Primers for the A κB site inside the IκBα promoter and a non-κB site in the proximal BECN1 promoter region were positive and negative controls, respectively. RelA or RelB binding to the indicated promoters was quantified by real-time PCR. Data are representative of 2 independent experiments.