Original Aricle Inhibition of osteoclast formation and function by CQ
Inflammation and Regeneration

Vol.32 No.5

NOVEMBER 2012

Original Article
Inhibitory effect of chloroquine on bone resorption reveals the key role of lysosomes in osteoclast differentiation and function
Md. Abdul Alim Al-Bari1, 2), Masahiro Shinohara1, 2),＊)
Yusuke Nagai1, 3) and Hiroshi Takayanagi1, 2, 3, 4,＊
1)
Department of Cell Signaling, Graduate School of Medical and Dental Sciences, 2)Global Center of Excellence
Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and
Dental University, Tokyo, Japan
3)
Japan Science and Technology Agency (JST), ERATO, Takayanagi Osteonetwork Project, Tokyo, Japan
4)
Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo,
Tokyo, JapanThe lysosome is an acidic compartment containing certain hydrolytic enzymes necessary forthe intracellular digestion of macromolecules. In terms of the activity of bone-resorbing osteoclasts, the secretion of lysosomal vesicles containing protons and matrix-degrading proteinases into the resorption lacunae is essential. Chloroquine (CQ), one of the lysosomotropic agents,
has an immunosuppressive effect and is used for the treatment for rheumatoid arthritis (RA) andsystemic lupus erythematosus (SLE). However, the direct effect of CQ on osteoclasts has notbeen reported. Here, we show that CQ suppresses the bone resorbing activity of osteoclasts by
inhibition of the acidification in the lysosomes, as well as osteoclast differentiation in vitro. CQ
treatment ameliorates the bone loss induced by RANKL injection in mice. These results suggestthat CQ has a bone-increasing effect by inhibiting osteoclast differentiation and function. In
addition, a lysosomal proton pump inhibitor bafilomycin A1 also inhibits osteoclast differentiation. Thus, this study revealed the importance of the lysosomes in osteoclast differentiation ando as well as in vitro, suggesting the therapeutic efficacy of immunosuppressivefunction in viv
vivo
CQ in osteoclast-mediated bone loss or destruction.
Rec.5/7/2012, Acc.5/27/2012, pp222-231＊

Correspondence should be addressed to:Hiroshi Takayanagi, Professor, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. Phone: +81-3-58035471, Fax: +81-3-5803-0192, E-mail: takayana@m.u-tokyo.ac.jp

Key w
ords
wo

chloroquine, lysosome, osteoclast, inflammatory bone disease222

Original Aricle Inhibition of osteoclast formation and function by CQ
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Introduction

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223

naling in B, T and other immune cells14-18). Thus, CQ andBone homeostasis is tightly regulated by the balanced

HCQ has become one of the most commonly prescribed

activities of bone formation and bone resorption. Osteo-

drugs in the treatment of many rheumatic diseases, includ-

clasts, which degrade bone matrix, are multinucleated cells,

ing RA19), systemic lupus erythematosus (SLE)20), palindro-and these cells are involved in the dynamic bone remodel-

mic arthritis21) and psoriatic arthritis22). Administration of CQ

ing which takes place in coordination with osteoblasts1, 2).

or HCQ reportedly results in a slowing or even arrest of

An imbalance in bone remodeling caused by increased

joint destruction in RA patients23) as well as the increasedbone resorption over bone formation leads to the loss ofbone mineral density (BMD) in SLE patients24, 25). Althoughbone mass in several bone disorders such as osteoporo-

the osteoclastic bone resorption in these diseases is enhanced, the direct effect of CQ on osteoclasts, in which3, 4)

sis and rheumatoid arthritis (RA).

Osteoclasts attach to the mineralized matrix and degrade
it by a secretion of protons and proteinases. The secretionthe lysosomal function is crucial for their activity, has yet tobe elucidated.

of protons is mediated by the proton pump localized at theIn this study, we investigated the effects of CQ on os-

lysosome and the ruffled border membrane, which is gen-teoclast function and differentiation. CQ suppresses both

erated by the fusion of lysosomal secretory vesicles withthe differentiation and bone resorbing activity of osteoclasts.

the plasma membrane that is in apposition to bone5). The

This study demonstrates the importance of the lysosome

lysosomal secretory vesicles contain proteinases, such as

in the function as well as the differentiation of osteoclasts.

the cysteine proteinase cathepsin K and the matrix metalloproteinase MMP9, both of which are essential for the deg6)

radation of bone matrix proteins . Thus, the lysosome plays

Materials and methods
1)Micea critically important role in osteoclastic bone resorption.

C57BL/6JJc1 mice (8-week-old) were purchased from

Osteoclast differentiation is regulated by macrophage

CLEA Japan. All of the animals were maintained in a spe-colony-stimulating factor (M-CSF) and receptor activator

cific-pathogen free environment and all of the animal ex-of nuclear factor-κB (NFκB) ligand (RANKL), a tumor ne-

periments were performed with the approval of the Institu-

2, 7). While M-CSF is crucial fortional Animal Care and Use Committee of Tokyo Medical

8)

the proliferation and survival of osteoclast precursor cells ,and Dental University and conformed to relevant guide-RANKL stimulation is essential for differentiation. The sig-lines and laws.

naling pathway mediated by the immunoglobulin (Ig)-like(1)In vitro osteoclast culture

crosis factor family cytokine

9)

receptors, such as OSCAR, TREM-2, SIRPβ1 and PIR-A ,In vitro osteoclast differentiation has been describedis also essential for osteoclast differentiation in order topreviously10). The bone marrow-derived macrophages

activate osteoclastogenic complex10). Finally, RANKL and(BMMs) were cultured in the osteoclastogenic medium

Ig-like receptor signals are integrated by the master tran-

containing 50 ng/ml RANKL (Peprotech) and 10 ng/ml M-

scription factor of osteoclast differentiation, NFATc1 . This

CSF in the presence of CQ (Sigma-Aldrich) or Bafilomycintranscription factor induces the expression of molecules

A1 (BafA1) (Tocris Bioscience) for 3 days. TRAP-positive

essential for the bone resorbing activity of osteoclasts, suchcells with more than three nuclei (TRAP+ MNCs) wereas cathepsin K, MMP9, the H+-ATPase subunits and car-counted.

11)

12)bonic anhydrase II .

(2)In vitro pit formation assay

Lysosomotropic agents chloroquine (CQ) and hydro-

Bone resorption activity of osteoclasts was assessed

xychloroquine (HCQ), both of which are derivatives of anti-

using a pit formation assay performed as previously

malarial drug quinacrine, penetrate acidic compartments

described26). For the pit formation assay, osteoclasts weresuch as the lysosome and raises the compartment pH tocultured on dentin slices with or without CQ in a 96-wellneutrality . Although CQ and HCQ was originally devel-

plate. The resorbed pit area was measured using an im-

oped as an anti-malarial drug, it has been shown that CQ

age analyzing software, Image J.

13)

and HCQ exert an anti-inflammatory effect and also inhibit
antigen presentation in dendritic cells, cytokine production
in macrophages, and calcium and Toll-like receptor sig-

2)TUNEL staining of mature osteoclasts
Manifestations of cell death were sought using fluores-

Original Aricle Inhibition of osteoclast formation and function by CQ
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224

cent in situ terminal deoxynucleotidyl transferase-mediatedcells as described previously29). After purification using

uridine 5'-triphosphate-biotin nick end labeling (TUNEL

glutathione-Sepharose 4B and Mono-Q (GE Healthcare),staining) using In Situ Cell Death Detection Kit, Fluores-

the protein was further purified with ToxinEraserTM En-

cein (Roche Diagnostics). The cells were analyzed using

dotoxin Removal Kit (GenScript). We confirmed that en-

a fluorescence microscope (Leica DMI3000B, Leica Mi-

dotoxin level is less than 50 EU/mg protein. Eight-week-

crosystems Ltd) with an appropriate filter set. The experi-

old mice were intraperitoneally injected with 1 mg/kg ofment was performed in duplicate on three independent

GST-RANKL three times at intervals of 24 h as previously

occasions in a 96-well plate.

reported29). CQ was injected 1 h before the every GSTRANKL injection. After the last injection, all of the mice3 ))Cell
Cell proliferation and apoptotic assays
Cell proliferation and apoptotic assays are performed de-were sacrificed and subjected to microcomputed tomography (microCT) analysis30).

10)

scribed previously . BMMs were cultured in the osteoclastogenic medium with or without CQ for 24 h. Before 1 h7)Statistical analysisof 24 h the BrdU (Roche Diagnostics) is added to the me-

Statistical analysis was performed using Student’s t -test

dium. Cells were labeled with FITC-conjugated anti-BrdU( p <0.05, ＊＊ p <0.01, ＊＊＊ p <0.005, n.s.: not significant,antibody (BD Pharmingen). Apoptosis of osteoclast pre-throughout the paper). All data are expressed as means

cursor cells was evaluated 24 h after RANKL stimulation± SEM.

＊

with or without CQ using an In Situ Cell Death Detection
Kit, Fluorescein (Roche Diagnostics) based on a TUNEL

Results

technique. In both experiments, cells were analyzed by flow

1)CQ inhibits osteoclastic bone resorption

cytometry, FACS Canto II and analyzed with Diva software

We evaluated the effect of CQ on the bone resorbing(BD Biosciences). In Fig. 3C, the number of nucleus was

activity of mature osteoclasts by pit formation assay. CQ

counted for the analysis of the cell viability after TRAP stain-

significantly inhibited the formation of pits in a dose-de-ing.

pendent manner (Fig.1A, B). Since it has been reported
that CQ induces cell apoptosis in various types of cells, we4)Acridine orange staining

analyzed the survival of CQ-treated osteoclasts by TUNEL

Intracellular acidification was determined by acridine or-assay. Although the cell survival was not affected at lower

ange (AO) quenching assay. Osteoclasts were exposed toconcentrations, the number of TUNEL-positive cells was

the lysosomotropic weak base, AO (Wako Pure Chemicalslightly but significantly increased at 7.5 μM and 10 μM

Industries, Ltd.) for staining as described previously27). The(Fig.1C). However, the pit area per osteoclast, which shows

accumulation of AO into lysosomes was visualized using a

the bone resorbing activity in a single cell, was dramati-

fluorescence microscope (Leica DMI3000B, Leica Micro-

cally decreased (Fig.1D). These results indicate that CQ

systems Ltd).

mainly affects the bone resorbing activity of osteoclasts,
and slightly affects the survival of osteoclasts. AO staining

5)Immunoblot analysis
Immunoblot analysis was performed as previously de28)

revealed that the CQ treatment results in a raising of the
pH in osteoclasts (Fig.1E), suggesting that CQ inhibits bone

scribed . Cells were solubilized in the lysis buffer (1%resorption through the neutralization of the protons in the

Nonidet P-40 in 150 mM NaCl, 20 mM Tris-HCl pH7.5,

lysosome. These observations indicate a significant role

1 mM EDTA, 10 mM NaF, 2 mM PMSF), supplemented withfor lysosomes in the bone resorbing activity of osteoclasts.

the complete protease inhibitor cocktail (Sigma-Aldrich).
Cell lysates were subjected to immunoblot analysis using
specific antibodies for NFATc1 (Santa Cruz Biotechnology)
and β-actin (Sigma-Aldrich).

2 ))Lysosomal
Lysosomal function and osteoclast differentiation
Next we examined the effect of CQ on osteoclast differentiation in vitro. Interestingly, the formation of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was6)RANKL-induced bone loss model
GST-RANKL recombinant protein was purified from BL21

suppressed by CQ in a dose-dependent manner (Fig.2A,
B). Consistent with this, the expression of NFATc1, theOriginal Aricle Inhibition of osteoclast formation and function by CQ
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Fig.1 Chloroquine suppresses osteoclastic bone
resorption(A)Effects of CQ on the bone resorbing activity of mature osteoclasts.
(B)Total pit area resorbed by osteoclasts.
(C)Effect of CQ on the survival of mature osteoclasts.
(D)Total pit area per osteoclast.
(E)Effects of CQ on the acidification of lysosomes inosteoclasts.

Fig.2 Inhibition of osteoclast differentiation by CQ(A)The formation of TRAP+ osteoclasts induced by RANKL and M-CSF in the presence of CQ.
(B)The number of TRAP+ osteoclasts in (A).
(C)Expression level of NFATc1 in (A).
(D)Proliferation of the CQ-treated osteoclast
precursor cells.
(E)Survival of the CQ-treated osteoclast precursor cells.

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Fig.3 Inhibition of osteoclast differentiation by BafA1(A)The formation of TRAP+ osteoclasts
induced by RANKL and M-CSF in thepresence of BafA1.
(B)The number of TRAP+ osteoclasts in(A).
(C)Cell viability of the BafA1-treated cells.

master transcription factor of osteoclast differentiation, wasclasts to resorb bone. Three-dimensional images of theinhibited by CQ treatment (Fig.2C). We also analyzed the

distal region of the femur showed robust trabecular bonecell proliferation and survival of osteoclast precursor cells

loss (Fig.4A), and microCT analysis revealed a marked

by BrdU incorporation and TUNEL assays. Although CQ

reduction in bone volume, thickness and the trabecular

tends to suppress the proliferation in a dose-dependent

number, and an increase in trabecular separation in themanner, we found no statistically significant difference inRANKL-injected mice (Fig.4B). The treatment with CQ pro-the proliferation (Fig.2D) and the survival (Fig.2E). These

tected against trabecular bone loss in these RANKL-in-

results indicate that CQ does not affect the survival signifi-

jected mice (Fig.4A). Consistent with this, the bone vol-cantly but slightly affects the proliferation in osteoclast pre-ume, the trabecular thickness and the trabecular number

cursor cells. To confirm the importance of lysosomal acidi-

were increased, and the trabecular separation was de-fication in osteoclast differentiation, we tested the effect of

creased (Fig.4B). Since osteoblastic bone formation is notanother lysosome inhibitor, BafA1, which selectively inhib-

affected in this model29), the protective effect of CQ on bone

its a vacuolar-type H+-ATPase. Similar to CQ, BafA1 also

loss is due to the suppression of osteoclast differentiation

inhibits osteoclast differentiation in a dose-dependent man-and function. In addition, osteoblast differentiation and func-ner (Fig.3A, B) without any defects in the cell survival

tion in vitro was not affected by the CQ at the concentra-(Fig.3C). These results suggest that lysosomal function hastion used in this study (M.S., unpublished data). Taken to-a critical role in osteoclast differentiation.

gether, our results suggest that CQ can protect against
the bone loss induced by the enhanced bone resorption of3 ))CQCQ protects against the bone loss induced byosteoclasts.

RANKL injection in mice
We investigated whether CQ has a preventive effect on

Discussion

the bone loss associated with enhanced osteoclastic bone

The lysosomal secretory vesicles in osteoclasts contain

resorption. We treated 8-week-old female mice that had

protons for decalcifying the mineralized matrix and enzymes

been intraperitoneally injected with GST-RANKL at 24 h

for digesting organic matrix. Since the lysosomal enzymes

intervals for 3 days. In this model, exogenous RANKL

such as cathepsins and MMPs are activated at a low pH,

mediates osteoclast differentiation and stimulates osteo-

acidification of the lysosome is required for the degrada-

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Fig.4 Protection against RANKLinduced bone loss by CQ
treatment(A)Microcomputed tomography(microCT) analysis of the femurs in micetreated with saline, GST-RANKL or GSTRANKL with 50, 100, 200 mg/kg CQ (upper photograph: longitudinal view; lower
photograph: axial view of the metaphyseal region).
(B)The parameters of the metaphyseal
region on microCT analysis. Data arerepresented as the mean ± SEM of 9
mice.

tion of bone matrix proteins. Previous reports have suggested+

can increase lysosomal volume and enlarge the plasmathat a selective inhibitor of the vacuolar-type H -ATPases

membrane surface35), which is a common event in apop-

BafA1, which suppresses acidification in lysosomes, inhibits

tosis36). It was previously reported that CQ induces cell

31, 32). Furthermore,

apoptosis in A549 lung cancer cells37), neurons38, 39), HeLa

mutations in the TCIRG1 gene, which encodes the a3 sub-cells40) and glioma cells41) at high concentrations (more thanunit of H+-ATPase, results in severe osteopetrosis in hu-

20 〜 30 μM), but not at low concentrations. However, ourmans and mice due to the impaired bone resorbing activity

data indicate that CQ induces apoptosis in mature osteo-

the bone resorbing activity of osteoclastsof osteoclasts

27, 33). These previous reports demonstrated

clasts at the concentration of 7.5 μM, suggesting that ma-

the importance of acidification in osteoclastic bone resorp-

ture osteoclasts are highly sensitive to CQ compared totion. The lysosomotropic agent CQ is a weak base, andother cell types. In contrast, CQ does not induce apoptosis

the unprotonated form of CQ passively diffuses into thein osteoclast precursor cells, even at 10 μM. This result

lysosomal compartment. In the lysosome it is protonated,indicates that the CQ sensitivity changes during the course

and the protonated form of CQ is unable to diffuse out ofof osteoclast differentiation. Although the detailed mecha-

the lysosome. As a result, intralysosomal proton consump-

nism remains to be elucidated, it may be caused by ation is carried out by the uncharged CQ and the intralyso-change in the expression level of genes related to lyso-

somal pH accordingly rises from 4.5 to 7.0, which in turnsome-dependent apoptosis during osteoclast differentia-

leads to alterations in lysosomal function34). AO stainingtion.

revealed that the intralysosomal pH is increased in CQ-

We also found that CQ and BafA1 have inhibitory ef-

treated osteoclasts. Thus, it is conceivable that CQ inhib-

fects on osteoclast differentiation. While M-CSF is neces-

its the bone resorbing activity by a suppression of protons

sary for the proliferation and survival of osteoclast precur-and hence the enzymatic activities of the bone-degrading

sor cells, RANKL and Ig-receptor signals are essential for

proteinases in osteoclasts.

differentiation. Since CQ inhibits osteoclast differentiation

CQ also induces apoptosis in mature osteoclasts. CQ

with minimum effects on the proliferation or survival of pre-

Original Aricle Inhibition of osteoclast formation and function by CQ
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228

cursor cells, CQ probably affects RANKL and/or Ig-recep-

efficacy in the treatment of SLE and lupus-related skin dis-

tor signaling. To explore the molecular mechanism by which

ease49, 50), and a phase III clinical trial of HCQ for these dis-

CQ inhibits osteoclast differentiation, we examined theeases has just started.

activation of the downstream pathways induced by RANKL,Previous studies have demonstrated that CQ and HCQsuch as the Erk, p38, JNK and IKK pathways, and foundcause retinal cell damage. The retinal cellular component,that CQ did not affect the activation of these pathways

retinal pigment epithelium (RPE) is the primary target of(unpublished observations). Although the molecular mecha-

CQ toxicity. The RPE is a single layer of cells adjacent tonism is unclear at present, the data suggest that lysoso-

the photoreceptor outer segment (POS) of the retina, andmal function is also important for osteoclast differentiation.

the RPE plays a critical role in the maintenance of the POS

A recent study showed that lysosomal function modulates

by removing shed rod and cone debris51). This debris is

glucocorticoid receptor signaling in macrophages42). This

endocytosed and then degraded in the lysosomes in thefinding, together with our results, indicates the importance

PRE52). Thus, the failure in the removal of the debris re-of the lysosome in signal transduction.

sults in dysfunction of the POS cells, leading to retinopa-

Inflammation perturbs normal bone homeostasis and isthy. CQ is also known to be an inhibitor of autophagy be-

known to induce bone loss, as it promotes local cartilage

cause it inhibits the function of autolysosomes, which isdegradation as well as both local and systemic bone de-generated by the fusion of autophagosomes and lysos-

struction by osteoclasts, along with inhibiting bone forma-omes. Therefore, it is likely that CQ induces retinopathy bytion by osteoblasts . Antirheumatic drugs such as meth-inhibiting autophagy. However, there is no direct evidence

otrexate (MTX), leflunomide (Lef), bucillamine (Buc) andthat autophagy is necessary for the function of the PRE insalazosulfapyridine (SASP), all of which are known to be

the maintenance of the POS, and the pathogenesis of ret-

immunomodulators (SASP/Buc) or immunosuppressantsinopathy can be explained by the inhibition of the lysos-

2)

19)

(MTX/Lef) , inhibit osteoclast differentiation and func-

omes.

under certain conditions. Previous reports sug-

Since retinopathy induced by CQ or HCQ is related togested that the production of inflammatory cytokines suchhigh daily dose (more than 750 mg per day) rather thanas TNFα and IL-6 are suppressed by CQ treatment in RA15)

long-term treatment47, 48), an effort to reduce the dose of

and SLE patients45) through the effects on macrophages

CQ or HCQ in the treatment is essentially required to re-and T-cells. Thus, CQ has been thought to ameliorate theduce the side effect. It has been shown that the treatment

course of arthritis by its inhibitory effect on these immuneof RA and SLE with a lower dose of HCQ in combination

cells. The improved BMD in SLE patients treated with

with MTX and/or SASP has a better therapeutic efficacy

tion

43, 44)has been attributed to the suppression of inflam-than a single drug treatment53, 54). We need to evaluate themation. However, in this study we found that CQ has aeffect of combined treatment with these drugs on the in-direct effect on osteoclast differentiation and function. Us-

flammation as well as osteoclastic bone resorption in theing a bone loss model in which exogenous RANKL stimu-

future. Considering that the treatment in a combination oflates osteoclast differentiation and function, we demon-

antirheumatic drugs exerts a marked inhibitory effect on

strated that CQ treatment protects against bone loss inosteoclasts even at a low dose55), the treatment by CQ orvivo. Our results clearly indicate that CQ plays a dual role

HCQ together with MTX and/or SASP would be more ef-

in that it is involved in both the suppression of inflamma-

fective than a single treatment in the inhibition of osteo-tion and bone destruction in RA.

clasts. Moreover, since osteoclasts also mediate the bone

CQ25, 26)

CQ and HCQ have the same effects on the function oflysosome. However, it has been suggested that there areloss in osteoporosis, CQ in combination with bisphosphonate
may be useful for the treatment of osteoporosis.

47)

differences in efficacy and toxicity between CQ and HCQ .
In RA therapy, it was reported that HCQ was one half to two

Acknowledgements

thirds as effective as CQ but one half in the toxicity48, 49).

We thank T. Ando, Y. Ogiwara, A. Yamamura, T. Koga, T.
Nakashima, M. Oh-hora, K Okamoto, M. Guerrini, A. Yamaguchi
and S. Kasugai for discussion and technical assistance. This work
was supported in part by Ministry of Education, Culture, Sports, Science and Technology (MEXT) Scholarship (B.A.); a grant for ERATO,

Thus, HCQ is thought to be less toxic and commonly prescribed than CQ in Canada, Australia and the USA. In Japan, it has been suggested that HCQ has a therapeuticOriginal Aricle Inhibition of osteoclast formation and function by CQ
Inflammation and Regeneration

Vol.32 No.5

Takayanagi Osteonetwork Project from the Japan Science and Technology Agency (H.T.); Grant-in-Aid for Young Scientist A and Challenging Exploratory Research from the Japan Society for the Promotion of Science (M.S.); grants for Global Center of Excellence
Program from MEXT (H.T.); and a grant from Kowa Life Science
Foundation (M.S.).

NOVEMBER 2012229

tion and activation of the transcription factor NFATc1(NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell. 2002; 3: 889-901.
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13)Poole B, Ohkuma S: Effect of weak bases on the

Conflict of interests
None

intralysosomal pH in mouse peritoneal macrophages.
J Cell Biol. 1981; 90: 665-669.

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