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. 2014 Jan;124(1):297-310.
doi: 10.1172/JCI66947. Epub 2013 Dec 9.

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

Chloroquine reduces osteoclastogenesis in murine osteoporosis by preventing TRAF3 degradation

Yan Xiu et al. J Clin Invest. 2014 Jan.

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.

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Figures

Figure 1
Figure 1. Mice with OC-specific deletion of TRAF3 have increased osteoclastogenesis and mild osteoporosis mediated by increased canonical and noncanonical NF-κB signaling.
(A) WBs of TRAF3 and NFATc1 expression in WT BM cells treated with M-CSF plus RANKL. Lanes were run on the same gel, but were noncontiguous. (B) OCs formed from FACSAria-sorted WT OCPs infected with GFP or TRAF3-IRES-GFP (TRAF3-GFP) retroviruses treated with RANKL. Original magnification, ×10. *OC centers. TRAP+ OCs were counted. (C) BM-derived C-cKO or WT cells cultured with RANKL. *P < 0.05. (D) Representative tibial μCT scans from 2-month-old CatK-Cre or C-cKO mice. *P < 0.05. (E) Representative TRAP-stained tibial sections and bone histomorphometry from 2-month-old WT or C-cKO mice; boxed areas in lower images. Scale bars: 500 μm (upper panels); 50 μm (lower panels). OcS/BS, OC surface/bone surface (%). *P < 0.05. (F) WBs of whole cell lysates of WT OCPs infected with GFP or TRAF3-IRES-GFP retroviruses and cultured with RANKL for 5 days. (G and H) WBs of WT and L-cKO OCPs treated with RANKL for 48 hours. Cyto, cytoplasmic.
Figure 2
Figure 2. RANKL-induced TRAF3 degradation is lysosome mediated.
(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.
Figure 3
Figure 3. CQ inhibits OC formation in vitro.
(A) Typical in vitro OC formation time-course. BM-derived WT OCPs were treated with M-CSF (M) for 2 days and with M-CSF plus RANKL (R) for 4 days. CQ was added on day 2 (B), or day 4 (C). Original magnification, ×4. *P < 0.05. (D) WBs of WT OCPs treated with RANKL with or without CQ (10 μM) for 3 days. (E) WBs of Traf3f/f OCPs infected with MSCV-GFP (GFP) or MSCV-Cre-GFP (Cre-GFP) retroviruses and GFP-FACS sorted or (F) cultured with RANKL with or without CQ (2 μM) for 5 days. (G) WBs of whole cell lysates from WT or TRAF3 L-cKO mouse tissues. (H) WBs of BM cells cultured with RANKL with or without CQ (2 μM) for 5 days. **P < 0.01 vs. PBS.
Figure 4
Figure 4. CQ affects OC function in vitro and in vivo.
(A) WT OCPs cultured with RANKL with or without CQ (5 μM) for 9 days. TRAP staining (left panels) and toluidine blue (right panels, to highlight resorption pits). Values are means + SEM of 4 wells. **P < 0.01 vs PBS. Original magnification, ×20. (B–D) Representative TRAP-stained sections and OC numbers in calvarial (B) and tibial (C) sections from 10- to 12-week-old male WT or L-cKO mice treated with CQ (50 mg/kg/d i.p. for 10 days) and given supracalvarial injections of hPTH(1-34 aa) (10 μg/mouse) 4×/d for 3 days beginning on day 7. (D) Representative H&E-stained tibial sections from the mice in C illustrating marrow fibrosis (arrows) and values for percentage of marrow space occupied by marrow fibrosis. Values are means + SEM of 4 mice/group. *P < 0.05. Boxed areas in B–D are illustrated at higher magnification in Supplemental Figure 5. Scale bars: 50 μm (B); 500 μm (C and D).
Figure 5
Figure 5. CQ prevents OVX-induced bone loss in WT, but not in C-cKO mice.
OVX or sham-operated 8- to 9-week-old WT and C-cKO mice treated with PBS or CQ (50 mg/kg/d i.p. for 28 days). (A) Representative μCT images of tibiae and trabecular BVs. (B) Representative TRAP-stained tibial sections and histomorphometric data for OC surface and numbers. Values are the mean + SEM of 5∼9 mice/group. *P < 0.05. Boxed areas are shown at higher magnification in Supplemental Figure 6B. Scale bar: 500 μm.
Figure 6
Figure 6. RelB positively regulates RANKL-induced TRAF3 degradation.
(A) WBs of WT and RelB–/– BM whole cell lysates and TRAF3 levels quantified densitometrically (mean + SEM of 3 blots). (B) WBs of RANKL-treated RelB–/– OCPs. (C) WBs of WT or RelB–/– OCPs infected with pMY-GFP or pMY-RelB-GFP retrovirus for 2 days and treated with RANKL for 8 hours. Arrowhead, specific RelB band. Lanes were run on the same gel, but were noncontiguous. (D) WT or RelB–/– OCPs infected with control GFP shRNA (Ctl-shRNA) or TRAF3 shRNA lentiviruses for 2 days were cultured with MCSF plus RANKL for 4 days. TRAF3 knockdown was confirmed by RT-PCR. Total numbers of OCs and large OCs were counted. (E) EM images of WT or RelB–/– OCPs treated with RANKL for 4 days in 2-well culture chambers showing autophagosomes (arrows).
Figure 7
Figure 7. RANKL-induced TRAF3 degradation is absent in BECN1, but not in ATG5/7 knockdown OCPs.
(A) WBs of WT OCPs treated with RANKL. (B–E) WT OCPs infected with Ctl-GFP, BECN1, or ATG5 and ATG7 shRNA lentiviruses for 2 days. (B) mRNA levels of BECN1, ATG5, and ATG7. (C) WB of infected cells treated with RANKL for 8 hours. (D) OCs formed from infected cells cultured with RANKL for 5 days. *P < 0.05 vs. Ctl-GFP shRNA. (E) EM images and numbers of autophagosomes (arrows, expressed/cell in at least 50 cells/preparation from 2 independent experiments) in infected cells.
Figure 8
Figure 8. RelB binds to the BECN1 promoter and regulates its expression.
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.
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