Enoxacin Directly Inhibits Osteoclastogenesis withoutInducing Apoptosis*□S

Received for publication, July 7, 2011, and in revised form, March 9, 2012 Published, JBC Papers in Press, April 2, 2012, DOI 10.1074/jbc.M111.280511

Edgardo J. Toro‡1, Jian Zuo‡, David A. Ostrov§, Dana Catalfamo¶, Vivian Bradaschia-Correa�, Victor Arana-Chavez�,Aliana R. Caridad‡, John K. Neubert‡, Thomas J. Wronski**, Shannon M. Wallet¶, and L. Shannon Holliday‡ ‡‡2

From the ‡Department of Orthodontics, University of Florida College of Dentistry, Gainesville, Florida 32610, the §Department ofPathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida 32610, the¶Department of Oral Biology, University of Florida College of Dentistry, Gainesville, Florida 32610, the �Laboratory of Oral Biology,Department of Dental Materials, School of Dentistry, University of São Paulo, 05508-900 São Paulo SP, Brazil, the **Department ofPhysiological Sciences, University of Florida, Gainesville, Florida 32610, and the ‡‡Department of Anatomy and Cell Biology,University of Florida College of Medicine, Gainesville, Florida 32610

Background: Enoxacin inhibits vacuolar H�-ATPase binding to microfilaments and bone resorption.Results: Enoxacin inhibits osteoclastogenesis without triggering apoptosis, induces changes in the proteolytic regulation ofproteins, and alters the localization of key proteins involved in osteoclast function.Conclusion: Enoxacin inhibits osteoclastogenesis by a mechanism that involves changes in post-translational processing andtargeting of proteins.Significance: A new type of direct inhibitor of osteoclasts has been identified.

Enoxacin has been identified as a small molecule inhibitor ofbinding between the B2-subunit of vacuolar H�-ATPase(V-ATPase) and microfilaments. It inhibits bone resorption bycalcitriol-stimulated mouse marrow cultures. We hypothesizedthat enoxacin acts directly and specifically on osteoclasts by dis-rupting the interaction between plasma membrane-directedV-ATPases, which contain the osteoclast-selective a3-subunitof V-ATPase, and microfilaments. Consistent with this hypoth-esis, enoxacin dose-dependently reduced the number of multi-nuclear cells expressing tartrate-resistant acid phosphatase(TRAP) activity produced by RANK-L-stimulated osteoclastprecursors. Enoxacin (50 �M) did not induce apoptosis as meas-uredbyTUNELandcaspase-3 assays.V-ATPases containing thea3-subunit, but not the “housekeeping” a1-subunit, were iso-lated bound to actin. Treatment with enoxacin reduced theassociation of V-ATPase subunits with the detergent-insolublecytoskeleton. Quantitative PCR revealed that enoxacin trig-gered significant reductions in several osteoclast-selectivemRNAs, but levels of various osteoclast proteins were notreduced, as determined by quantitative immunoblots, evenwhen their mRNA levels were reduced. Immunoblots demon-strated that proteolytic processing of TRAP5b and the cytoskel-etal protein L-plastin was altered in cells treated with 50 �M

enoxacin. Flow cytometry revealed that enoxacin treatmentfavored the expression of high levels of DC-STAMP on the sur-face of osteoclasts.Our data show that enoxacin directly inhibits

osteoclast formation without affecting cell viability by a novelmechanism that involves changes in posttranslational process-ing and trafficking of several proteins with known roles in oste-oclast function. We propose that these effects are downstreamto blocking the binding interaction between a3-containingV-ATPases and microfilaments.

Enoxacin is a fluoroquinolone antibiotic that has been usedextensively in humans for the treatment of urinary tract infec-tions and gonorrhea with minimal side effects (2). Recently,unexpected properties of enoxacin have come to light. Ourgroup, making use of a rational reverse chemical genetic strat-egy, identified enoxacin as an inhibitor of vacuolar H�-ATPase(V-ATPase)3-microfilament binding and of osteoclast forma-tion and bone resorption in cell culture (1). Concurrently, oth-ers have identified enoxacin in screens for small molecule stim-ulators of RNA interference andmicroRNA activity (3, 4). Veryrecently enoxacin was reported to be a “cancer-specific” inhib-itor that blocks the growth andmetastases of human colorectalcancers in amousemodel (5). Because of the therapeutic poten-tial of enoxacin, it is vital to understand the mechanisms bywhich it selectively affects osteoclasts.V-ATPases are essential “housekeeping” enzymes in all

eukaryotic cells that are necessary for the acidification of com-partments of the endocytic and phagocytic pathways (6, 7).Most cell types express only the low levels of V-ATPaserequired to carry out housekeeping functions, but some celltypes also contain an additional subset of V-ATPases that plays* This work was supported, in whole or in part, by National Institutes of Health

Grant R21-DE19862 01A1 from NIDCR (to L. S. H.). This work was also sup-ported by a grant from the University of Florida Opportunity Fund (toL. S. H., D. A. O., and T. J. W.).

□S This article contains supplemental Figs. S1–S4.1 Supported by Grant T32 DE07200-15 from NIDCR.2 To whom correspondence should be addressed: Dept. of Orthodontics,

D7-18, CB100444, 1395 Center Dr., University of Florida College of Den-tistry, Gainesville, FL 32611. Tel.: 352-273-5689; Fax: 352-846-0459; E-mail:sholliday@dental.ufl.edu.

3 The abbreviations used are: V-ATPase, vacuolar H�-ATPase; TRAP, tartrate-resistant acid phosphatase; DC-STAMP, dendritic cell-specific transmem-brane protein; L-plastin, leukocyte-plastin; RANK, receptor activator ofnuclear factor �B; RANK-L, RANK ligand; CSF, macrophage-colony stimu-lating factor; M-CSF, murine CSF; WASH, Wiscott-Aldrich and Scar homo-log; DMSO, dimethyl sulfoxide; qPCR, real-time quantitative PCR; ANOVA,analysis of variance.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 21, pp. 17894 –17904, May 18, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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a role in the unique functions of the cell. V-ATPases are com-posed ofmore than 10 subunits, and a number of these subunitshave multiple isoforms. Housekeeping V-ATPases are com-posed of a specific subset of subunit isoforms, whereas non-housekeeping V-ATPases are marked by the inclusion of celltype-restricted isoforms of one or more of the subunits. Thesesubpopulations are targeted and utilized differently than thehousekeeping enzymes. Although it is well documented thatparticular isoforms of certain V-ATPase subunits are found inV-ATPases that are targeted to atypical cellular locations, theunderlyingmechanisms bywhich isoforms contribute to differ-ential targeting and use of V-ATPases are not understood (7).Osteoclasts are highly specialized cells that have the capacity

to resorb bone or, perhaps more accurately, to invade mineral-ized tissue in a regulated manner (8). They express very highlevels of a subpopulation of V-ATPases (a3- and d2-containingV-ATPases) that are targeted to the ruffled plasma membrane,a subdomain of the plasma membrane, when osteoclastsencounter bone (9–11). The targeting of V-ATPases to the ruf-fled plasma membrane is absolutely required for bone resorp-tion (11).Mutations in the a3 and d2 isoforms ofV-ATPase leadto osteopetrosis in mice (a3 and d2) and humans (a3) (11–13).Recently, interest has increased in osteoclast V-ATPase-di-rected therapeutic development because it has been demon-strated that the osteopetrotic pathology associated with the a3and d2 mutations involves both reductions in bone resorptionand increases in bone formation (13, 14). Because neither a3nor d2 is expressed in osteoblasts (11, 13), it is thought thatthese mutations set up a situation where osteoclasts are ren-dered unable to resorb bone efficiently but continue to producesignals that recruit and activate osteoblasts to form bone. It hasbeen proposed that osteoclast inhibitors that mimic the effectsof these mutations might prove to be bone anabolic and there-fore particularly useful for the treatment of osteoporosis (14). Arecent mouse study directly supports this idea (15). Unfortu-nately, the nature of the unique activities conferred on V-ATPases by the presence of a3 or d2 has not been identified.Cancer cells also express relatively high levels of V-ATPase,

and some have high levels of the a3-subunit (16). V-ATPaseshave been shown to enter the plasmamembrane of cancer cells,which is proposed to be vital for the survival and metastasis ofcancer cells (17). A number of studies have demonstrated thatcancer cells are sensitive to V-ATPase inhibitors (18–23), andvery recently it was shown that cancer growth and metastasiscould be blocked inmice by knocking downof the a3-subunit inB16 melanoma cells (24).We have found that in osteoclasts, but not most other cell

types, a high proportion of V-ATPases is bound to microfila-ments (25). This interaction correlates with the activation stateof osteoclasts and ismediated by a specific region of the B2-sub-unit that includes the “profilin-like domain,” so named becauseof its sequence and structural similarity to the actin-bindingdomain of mammalian profilin 1 (26–29). The total actin bind-ing domain region is composed of 44 amino acids (amino acids29–73 in mammalian B2-subunits) (26). The profilin-likedomain is smaller and is composed of amino acids 55–68 in B2.Small changes in the profilin-like domain of the B2-subunitdisrupt actin binding activity without interfering with the

capacity of the altered B2-subunit to contribute to the enzy-matic activity of themultisubunit V-ATPase (30). B-subunits soaltered are not targeted to the ruffled plasma membrane ofosteoclasts (28).Although the actin-binding site in the B-subunits (both the

B1 and B2 isoforms bind actin (27)) is present in all mammalianV-ATPases, they are not recovered from most cells bound tomicrofilaments. In yeast, molecular genetic studies have dem-onstrated that actin binding activity is only important for thegrowth and survival of yeast in the presence of specific environ-mental stressors (30). These data suggest that actin bindingactivity may not be required for the function of housekeepingV-ATPases.Together, these data led us to hypothesize that small mole-

cules that bind to the profilin-like domain of subunit B2 andcompetitively interfere with its binding to microfilamentsmight represent a new class of osteoclast-selective anti-resorp-tive therapeutic agents. Computational chemistry techniqueswere used to conduct a virtual screen to identify small mole-cules predicted to bind the actin binding surface of the B2-sub-unit and thus block its interaction with microfilaments (1).From the candidates identified, we reported that the fluoro-quinolone antibiotic enoxacin blocks the interaction betweenthe recombinant B2-subunit and microfilaments in the testtube and also blocks osteoclast differentiation and bone resorp-tion in mixed cell cultures (1). V-ATPase transport to ruffledplasma membranes of osteoclasts on bone slices in mixed cul-tures is also disrupted. Enoxacin does not prevent growth ormineralization by osteoblasts at concentrations where oste-oclast activity is inhibited. In the current study, we tested thehypothesis that enoxacin acts directly and specifically on oste-oclasts by disrupting the interaction between plasma mem-brane-directed V-ATPases, which contain the osteoclast-selec-tive a3-subunit of V-ATPase, andmicrofilaments. Based on thephenotype of osteoclasts in which mutations of the a3- and d2-subunits of V-ATPase occur, we anticipated that enoxacinwould not provoke apoptosis (11, 13). Because enoxacin inhib-ited cell fusion and expression of tartrate-resistant acid phos-phatase (TRAP) activity, we suspected that the expression lev-els of certain proteins, including ADAM (a disintegerin andmetalloproteinase) 8, ADAM12, and dendritic cell-specifictransmembrane protein (DC-STAMP), which are linked to cellfusion, and TRAP5b, might be reduced.

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—The polyclonal anti-E-, anti-a3-,and anti-B2-subunit antibodies were described previously (26,31). The other antibodies used were obtained from the follow-ing suppliers as indicated: anti-TRAP antibody (BioLegend, SanDiego, catalog No. 648401); anti-leukocyte (L)-plastin (alsoknown as plastin-2; Abcam, Cambridge, MA, catalog No.ab83496); anti-cortactin (sc-25577), anti-DC-STAMP (sc-25579), anti-ADAM12 (sc25579), anti-nuclear factor activatedin T-cells c1 (NFAT-c1; sc7294), and anti-lamin A/C (sc-6215)(all from Santa Cruz Biotechnology, Santa Cruz, CA); anti-cal-citonin receptor (Abbiotec, San Diego); anti-Src homologyregion 2 domain-containing phosphatase-1 (Shp1; Abcam,ab18708); and anti-cathepsin K (Santa Cruz Biotechnology,

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sc-6506). Unless otherwise noted, other antibodies andreagents were obtained from Sigma-Aldrich. Recombinantreceptor activator of nuclear factor �B ligand (RANK-L) wasproduced in Escherichia coli as described previously (33) orobtained fromPeprotech (RockyHill, NJ).Macrophage colony-stimulating factor (CSF-1) was obtained from Peprotech.Enoxacin was obtained from Sigma-Aldrich and dissolved inDMSO (1) or 0.1 M sodium hydroxide (32).Osteoclast Differentiation—To grow primary osteoclasts, the

femora and tibiaewere removed andmarrowwas expelled frombones using a syringe with �-MEM complete medium (Sigma-Aldrich) (10% fetal bovine serum (Mediatech), 1% L-glutamine(Thermo Scientific), and 1% penicillin/streptomycin/ampho-tericin B (Fisher)). Cells were seeded in T75 flasks at a concen-tration of 1.5� 106 cells/ml supplementedwith 5 ng/ml recom-binant murine CSF-1 (M-CSF; Peprotech) and allowed toculture for 24 h at 37 °C in 5% CO2. Non-adherent cells wereremoved, and adherent cells (5.9� 105 cells/ml) were seeded instandard 24-well plates or on UpCell tissue culture plastic(Nunc). All cultures were supplemented with 10 ng/ml CSF-1and -5 (33) or 50 ng/ml recombinant murine RANK-L (Pepro-tech). Cells were treated with vehicle or enoxacin, as indicated,and cultured for 5 or 6 days with complete medium refreshedevery 3 days.Raw 264.7 cells were stimulated to differentiate into oste-

oclast-like cells with recombinant RANK-L as described previ-ously (33). Raw 264.7 were seeded on 24-well plate at a densityof 1.25 � 104 cells/well or on 6-well plates at 1.8 � 105 cells/well. These cells were cultured for 5 days with 5 ng/ml RANK-Land fed on day 3 in culture.TRAP Activity Assay—TRAP activity was detected using the

leukocyte acid phosphatase kit (Sigma-Aldrich, catalog No.387A-KT) following the instructions from the manufacturer.Osteoclasts were detected as staining positive for TRAP activ-ity. TRAP� cells were counted and classified according to thenumber of nuclei present as mono- or multinuclear (2–10nuclei) or as giant cells (more than 10 nuclei).Terminal Deoxynucleotidyl Transferase dUTP Nick End

Labeling (TUNEL) and Caspase-3 Assays—After 5 days of cellculture with 50 �M enoxacin, the cells were rinsed twice withPBS and fixed for 2 min in 4% paraformaldehyde at room tem-perature. Apoptotic cells were detected by using an in situ apo-ptosis detection kit (Promega, G7132) according to the manu-facturer’s instructions.To assay for caspase-3 activity cells were plated in 24-well

plates at a density of 0.5 � 104 cells/cm2 and treated with 50ng/RANK-L plus orminus 50�M enoxacin for 24, 48, and, 72 h.Caspase-3 assays were performed following themanufacturer’sinstructions (catalog No. APT131, Millipore, Temecula, CA).At the end of each time point, the plate was centrifuged at 1200rpm for 10 min. Cells were treated with 100 �l of chilled celllysis buffer, and cell lysates were centrifuged at 1200 rpm for 10min. Cell lysates were treated according to the manufacturer’srecommendations and the colorimetric reactionwas quantifiedusing a BioTek KC4 spectrophotometer (Winooski, VT) at405 nm.Immunoprecipitations—Immunoprecipitations were per-

formed as described previously (26, 34). Osteoclasts were gen-

erated from Raw 264.7 cells or primary osteoclasts. Cells werewashed in PBS and solubilized in Triton X-100 buffer (1% Tri-ton X-100, 20 mM Tris, pH 7.4, 1 mM EDTA, 1 mM dithiothre-itol, 0.1% SDS, 10% glycerol, 5 mM sodium azide, and proteaseinhibitors). After centrifugation at 20,000 � g for 10 min toremove insoluble material, the extracts were incubated for 1 hat 4 °C with 1:200�g of an anti-a3 or anti-a1 subunit polyclonalantibodies (31). Fiftymicroliters of proteinA beads (Sigma)wasadded, and the mixture was incubated for 1 h at 4 °C with rock-ing. The protein A beads were collected by centrifugation at10,000 � g for 15 s at 4 °C and washed three times with theTriton X-100 buffer. The wash buffers were removed by aspi-ration with a bent 23-gauge needle, and Laemmli sample bufferwas added. The sampleswere heated to 85 °C for 10min, cooledto room temperature, and centrifuged at 10,000 � g for 1 min,and the supernatants were subjected to SDS-PAGE, transferredto nitrocellulose, andprobedwith antibodies as described in thelegends to Fig. 1–5.Quantitative Immunoblotting—Immunoblots were per-

formed by standard procedures using the SuperSignal West-Dura chemiluminescence detection system (Thermo Scientif-ic/Pierce). To determine the association of V-ATPase with thedetergent-insoluble cytoskeletal fraction, blots of supernatantsand pellets were obtained by extraction of osteoclasts with 20mMTris-HCl, pH 7.4, 100mMNaCl, 5 mMMgCl2, 0.5 mMATP,0.2 mM CaCl2, and 0.2 mM dithiothreitol (Buffer F) plus 1%Triton X-100 and protease inhibitors with ultracentrifugation(100,000 � g for 45 min). The samples were heated at 85 °C for10 min, cooled to room temperature, and centrifuged at10,000 � g for 1 min, and the supernatants were applied toSDS-polyacrylamide gels, transferred to nitrocellulose, andprobed with antibodies as described in the legends to Fig. 3 and4.Cell sample dilutionswere applied to SDS-polyacrylamide geland stained with Coomassie Blue stain (Bio-Rad, catalog No.161-0436) using staining levels of multiple proteins to establishequal loading between treatment groups. Blots were then per-formed to identify and put target proteins into the linear rangeof detection by immunoblot. Samples from enoxacin treatmentwere compared with vehicle controls to correlate changes inprotein levelswith dosages of enoxacin. Bandswere quantitatedby densitometry using an Alpha Innotech FluorChem 8000(Alpha Innotech, San Leandro, CA).Real-time PCR—Total RNA from Raw 264.7 cells was iso-

lated with an RNeasy mini kit (Qiagen, Tokyo) and real-timequantitative PCR (qPCR) was performed using the TaqManOne-Step RT-PCR master mix reagents kit and an ABI Prism7700 HT detection system (Applied Biosystems, Foster City,CA) (33). Probes and primers for the mouse genes that encodeTRAP5b (Mm00475698_m1), a3-subunit (Mm00469406_m1),L-plastin (Mm01310735_m1), ADAM8 (Mm00545762_m1),B2-subunit (Mm00431987_m1), Wiscott-Aldrich and Scarhomolog (WASH; Mm01239734_m1), calcitonin receptor(Mm00432271_m1), cathepsin K (Mm00484039_m1), FBJosteosarcoma oncogene (c-fos; Mm00487425_m1), integrin�3 (Mm00443980_m1), nuclear factor activated in T-cells(NFATc1; Mm00479445_m1), SFFV proviral integration 1(Pu.1; Mm00488142_m1), and hypoxanthine phosphoribosyl-transferase 1(HPRT1; Mm00446968_m1) were obtained from

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Applied Biosciences. Raw 264.7 cells were treated as indicatedin the legend to Fig. 4; RNA was isolated and assayed on day 5,and the level of each of the genes relative to HPRT1 was deter-mined. The experimentwas analyzed statistically using t tests ofthe �Ct values. p � 0.05 was considered significant.Flow Cytometry—Cells were allowed to dissociate from the

bottom of UpCell coated plates (Thermo Scientific) at roomtemperature for 30–45min. Suspended cells were washedwithfluorescence-activated cell sorter (FACS) buffer (1� PBS, 5%FBS, and 0.372 g of EDTA) and treated with 1:200 dilutions ofantibodies for 1 h at 4 °C in the dark. Biotin-conjugated ratanti-mouse RANK (eBioscience) with Alexa Fluor 647-conju-gated streptavidin (Invitrogen) and rabbit anti-mouseDC-STAMP (Invitrogen)withAlexa Fluor 488-conjugated goatanti-rabbit IgG (Invitrogen) were used in this study. Cells wereacquired using a FACSCalibur flow cytometer (BDBiosciences)and analyzed using FCS Express (De Novo Software).Statistics—Counters were precalibrated for their ability to

identify TRAP� multinuclear cells and then blinded to treat-ment groups. Results are expressed as mean � S.E. Sampleswere compared by one-way ANOVA and Student’s t test using

the program GraphPad Prism 5 (GraphPad Software, La Jolla,CA). p values � 0.05 were considered significant.

RESULTS

Enoxacin Inhibits Differentiation of Primary Marrow Cellsand Raw 264.7 Cells into Osteoclasts without Inducing ExcessApoptosis—Primary marrow-derived osteoclast precursors orRaw 264.7 cells were stimulated with recombinant RANK-LandM-CSF in 24-well plates and treated with vehicle or differ-ent concentrations of enoxacin. After 5 days, the cells werefixed and stained for TRAP activity and then examined forTRAP activity and fusion intomultinuclear cells (both are char-acteristics of osteoclasts). A significant dose-dependent reduc-tion in the number of TRAP-positive, multinucleated cells wasdetected in response to enoxacin. The IC50 value was the samefor both primary osteoclasts (Fig. 1, A and C–F) and Raw 264.7cells (Fig. 1, B and G–J).

To determinewhether enoxacin had disrupted cell growth orsurvival, we counted the number of nuclei after 5 days in cultureof the Raw 264.7 cells treated with RANK-L plus various con-centrations of enoxacin. Although the phenotype of the cells

FIGURE 1. Enoxacin dose-dependently reduces the number of TRAP� multinuclear osteoclast-like cells that form as the result of RANK-L stimulationof primary marrow and Raw 264.7 cells. A, primary mouse marrow cells were stimulated with 5 ng/ml recombinant RANK-L and 5 ng/ml CSF-1 for 5 days inthe presence of vehicle (DMSO) or enoxacin at the micromolar concentrations indicated. At the end of 5 days, cells were fixed and stained for TRAP activity, andthe number of mononuclear, multinuclear (2–10 nuclei), and giant cells (�10 nuclei) was counted. The average number of TRAP� cells in each category in thevehicle-treated cultures was defined as 100%. The numbers in enoxacin-treated cultures are depicted as a percentage of the vehicle controls. The averagenumbers for the vehicle control cultures were 1299 for mononuclear cells, 845 for multinuclear cells, and 103 for giant cells. B, raw 264.7 cells were stimulatedwith 5 ng/ml recombinant RANK-L for 5 days in the presence of vehicle (DMSO) or enoxacin at the micromolar concentrations indicated. At the end of 5 days,cells were fixed and stained for TRAP activity, and the number of mononuclear, multinuclear (2–10 nuclei), and giant cells (�10 nuclei) was counted. Theaverage number of TRAP� cells in each category in the vehicle-treated cultures was defined as 100%. The numbers in enoxacin-treated cultures are depictedas a percentage of the vehicle controls. The average numbers for the vehicle control cultures were 3810 for mononuclear cells, 1141 for multinuclear cells, and244 for giant cells. One-way ANOVA was utilized to determine whether there were differences within groups, and Student’s t test was used to test fordifferences between specific treatment groups and the vehicle control. p � 0.05 was considered significant. C–F, representative photographs from theexperiment documented in A: C, vehicle control; D, 10 �M enoxacin; E, 50 �M enoxacin; F, 100 �M enoxacin. G–J, representative photos from the experimentdocumented in B: G, vehicle control; H, 10 �M enoxacin; I, 50 �M enoxacin; J, 100 �M enoxacin. Scale bar � 50 �m.

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was very different when they were treated with enoxacin, therewere no significant differences in the number of nuclei at con-centrations as high as 50 �M.To test whether enoxacin induced apoptosis during themat-

uration process, Raw264.7 cells were treatedwith either vehicleor 50 �M enoxacin and stimulated with RANK-L. TUNELassays were performed. No significant difference was observedin the number of apoptotic cells that was attributable to enoxa-cin (Fig. 2, B–D). To confirm this result, we assayed RANK-L-stimulated Raw 264.7 cells treated with either vehicle or 50 �M

enoxacin for caspase-3 activity (a marker of apoptosis (35)) atvarious time points after stimulation with RANK-L (Fig. 2E).Enoxacin significantly reduced the level of caspase-3 activity at72 h in this assay compared with RANK-L-stimulated cellstreated with vehicle. Cells that were not stimulated withRANK-L displayed less caspase-3 activity, and this was not sig-nificantly affected by enoxacin.V-ATPases That BindMicrofilaments in Osteoclasts Contain

the a3-subunit but Not the a1-subunit—B2 is expressed in allcell types, but usually V-ATPases are not detected bound tomicrofilaments (25, 34). We hypothesized that the a-subunitisoform, a3, which is selectively expressed in osteoclasts and isplasma membrane-directed, might be preferentially associatedwith actin binding activity. To test this idea, primary mousemarrow osteoclasts were extracted with buffer F (20 mM Tris,pH 7.5, 100 mM NaCl, 0.5 mM MgCl2, 0.2 mM CaCl2 0.2 mM

ATP, and 0.2 mM DTT) plus 1% Triton X-100 and subjected toultracentrifugation to pellet the detergent-insoluble cytoskel-etal fraction. The proteins from the supernatant and pellet frac-tions were collected in equal volumes of SDS-PAGE samplebuffer, loaded equivalently on gels, separated by SDS-PAGE,blotted to nitrocellulose, and probed with antibodies to a3 anda1 (Fig. 3A). 92� 4% (n� 3; by densitometry of blots) of a3 wasfound in the pellet, whereas only 7� 5% of a1was present in thepellet. To confirm that a3 was present in the pellets because itwas bound to F-actin, primary osteoclasts were homogenizedwith buffer F plus 1% Triton X-100 and spun at 5,000 � g toremove nuclei and large insoluble material. In preliminaryexperiments, we found that the amount of actin orV-ATPase inthis pellet was less that 5% of the total. We then performedimmunoprecipitations from the soluble fraction using eitheranti-a1 or anti-a3 antiserum. Anti-a3 pulled down subunit E, aperipheral subunit of V-ATPase, demonstrating that a3 wasassembledwith peripheral subunits andwith actin. Anti-a1 alsopulled down subunit E, but actin was not detected in the a1immunoprecipitations (Fig. 3B). As a further control, F-actinwas depolymerized in detergent extracts by dialyzing into 20mM Tris, pH 7.5, 0.2 mM CaCl2 0.2 mM ATP, and 0.2 mM DTTplus 50�M latrunculin A (36) for 8 h at 4 °C and then subjectingthe samples to ultracentrifugation. The amount of subunit a3that pelleted from latrunculin-treated extracts (Lat.) wasreduced to 24% of the untreated extracts (Ct) (Fig. 3C) as meas-ured by densitometry. Subunit a3, but not a1, was also associ-ated with the detergent-insoluble cytoskeleton of Raw 264.7cells (supplemental Fig. S1).To determine whether enoxacin reduced the amount of

V-ATPase associated with the cytoskeleton, we examined thelevel of B2-, E-, and a3-subunits associated with the detergent-

insoluble cytoskeleton plus or minus 50 �M enoxacin for 5 daysin both RANK-L-stimulated Raw 264.7 osteoclast-like cells andprimary osteoclasts. There was no difference detected in thetotal level of any of the V-ATPase-subunits, but a shift wasdetected in the B2-, E-, and a3-subunits from the detergent-insoluble cytoskeleton to the soluble fraction (Fig. 3D and sup-plemental Fig. S2). This result is consistent with our findingthat enoxacin blocks binding between recombinant B2-subunitand microfilaments (1). Although all three subunits assayedwere shifted by enoxacin from the cytoskeletal fraction towardthe supernatant, the shift was most profound with respect tothe B2-subunit.Enoxacin Reduced Levels of Several Osteoclast andV-ATPase-

selective mRNAs, but Protein Levels Were Not Reduced—To examine the molecular mechanisms underlying the effects ofenoxacin, we analyzed a number of V-ATPase and osteoclast-se-lective genesbyqPCR(Fig. 4A). Raw264.7 cellswere cultured for 5days with RANK-L and treated with vehicle or 50 �M enoxacin.We detected small, but significant reductions in the a3-, B2-, andE-subunits of V-ATPase, TRAP5b, L-plastin, c-Fos, and WASH.Larger reductions in the levels of�3 integrin, cathepsin K, and thecalcitoninreceptorweredetected.Asareference,weobserved thatthe expression level of TRAP5b in enoxacin-treated cells wasreduced by 1.96-fold compared with control, whereas in RANK-L-stimulation increased its level was 350-fold comparedwith cellsnot stimulated with RANK-L. The levels of ADAM8 and PU.1mRNAwerenot alteredby enoxacin.The level ofNFATc1mRNAwas significantly increased in enoxacin-treated cells. Despite thedifferences in mRNA levels, we were unable to detect any reduc-tions in protein levels in total protein extracts separated by SDS-PAGEandanalyzedbyquantitative immunoblottingusingactinasan internal standard (Fig. 4B and supplemental Fig. S3). Theseincluded the a3-, E-, andB2-subunits ofV-ATPase, cortactin,DC-STAMP, calcitonin receptor, Shp1, and �3 integrin. Cathepsin KandNFAT-c1displayedslight increases. Inaddition,we foundthatTRAP5b, and L-plastin showed staining patterns most consistentwith altered proteolytic processing (Fig. 4,C andD). Dose-depen-dent reduction in thenumberofRANK-L-stimulatedcellsdisplay-ingTRAPactivity is a characteristic effect of enoxacin, but surpris-ingly, enoxacin only slightly reduced the level of TRAP5bmRNA.TRAP5b is expressed as a 38-kDa latent pro-enzyme and is pro-teolytically activated to the active form, which is characterized inblots by a 16-kDa band (37). Anti-TRAP immunoblots displayedthe expected 16-kDaband in vehicle controls, but a series of bandsstarting at 38 kDa, and very little at 16 kDa, was detected in thecultures treated with 50 �M enoxacin (Fig. 4C and supplementalFig. S4).Enoxacin was shown previously to inhibit actin ring forma-

tion (1). L-plastin, a protein tied to actin ring formation (38–41), was assayed for changes associated with enoxacin treat-ment. The anti-L-plastin antibody detected a dose-dependentshift from the expected 67-kDaband to a 57-kDaband (Fig. 4D).The amount of the 67-kDa band was reduced by 88% (deter-mined by densitometry). Mass spectroscopy confirmed thepresence of L-plastin at 57 kDa (data not shown). Because theanti-L-plastin antibody detects the C terminus, this shift mayinvolve proteolytic cleavage of the N-terminal domain.

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FIGURE 2. Enoxacin does not alter the number of viable nuclei or increase the levels of apoptosis as measured by TUNEL and caspase-3 assays. A, fiverandomly selected fields/well of a 24-well plate were counted and averaged. Four wells were counted per treatment group. One-way ANOVA was utilized todetermine whether there were differences within groups, and this indicated that there were no differences between any of the groups. Enoxacin at aconcentration of 100 �M appeared to reduce the nuclei number, although it did not reach significance. Because of this we focused our studies on the effectsof 50 �M enoxacin, a concentration at which osteoclastogenesis and bone resorption (1) were strongly inhibited but numbers of nuclei were not affected.B, quantitative analysis of counts of the number of TUNEL-positive cells after 5 days in cultures treated with vehicle or 50 �M enoxacin. C and D, Raw 264.7 cellsstimulated with RANK-L for 5 days and treated with vehicle (C) or 50 �M enoxacin (D). Apoptotic nuclei are dark. Arrows point the edges of giant cells in C andthe edge of a multinuclear cell in D. Bar � 110 �m. E, quantitative analysis of caspase-3 activity of Raw 264.7 cells treated with vehicle, RANK-L (5 ng/ml), andenoxacin 50 �M as noted for the culture times indicated. Units are defined as the amount of enzyme that cleaves 1 nM colorimetric substrate/h. DMSO was usedas the vehicle control. No statistically significant differences exist between vehicle and enoxacin treatment in unstimulated (�RANKL) cells. At 48 and 72 h, asignificant increase in caspase-3 activity was detected in both treatment groups after stimulation with RANK-L. Enoxacin (ENX) significantly decreased thelevels of caspase-3 activity at 72 h compared with control. *, p � 0.05 by one-way ANOVA. The data represent mean � S.E. (n � 3).

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Enoxacin Stimulates an Increase in the Surface Expression ofDC-STAMP and RANK—The seven-transmembrane regionreceptor DC-STAMP is preferentially expressed in osteoclastsafter stimulation by RANK-L. Osteoclasts from DC-STAMP-deficient mice display reduced fusion but comparable TRAPactivity, formation of ruffled plasma membranes, and actinrings, indicating that DC-STAMP is not involved in osteoclastactivation except for cellular fusion (42). We examined DC-STAMP surface protein expression by immunoblot and FACSanalysis. Osteoclasts were stimulated to differentiate for 6 daysand then analyzed after 3 additional days of culture with 50 �M

enoxacin or vehicle and continued stimulation with RANK-L.Immunoblots had indicated that enoxacin did not affect theoverall expression of DC-STAMP in RANK-L-stimulated cells(Fig. 4B). Flow cytometry revealed that after treatment withenoxacin, a significantly higher portion of the cells expressedhigh levels of DC-STAMP on the cell surface (Fig. 5). Surpris-ingly, significantly more cells from enoxacin-treated culturesalso expressed high levels of RANK on their surfaces (Fig. 5D).This is consistent with enoxacin altering the cellular vesiculartrafficking pattern of DC-STAMP and RANK to promote theexpression of higher levels of these proteins on the cell surface.

DISCUSSION

In this study, we have shown for the first time that enoxacininhibits osteoclastogenesis directly with an IC50 of about 10 �M

without inducing apoptosis.We demonstrated that V-ATPasescontaining a3, but not a1, bind to the actin cytoskeleton, whichprovides a possible explanation of why blocking the interactionbetween the ubiquitous B2-subunit andmicrofilaments has cell

type-selective consequences. This also provides a first exampleof an activity (interaction with microfilaments) that is associ-ated with the presence of a specific isoform of the V-ATPase ina cell. Moreover, we directly demonstrated reductions in therelative amounts of V-ATPase subunits associated with thedetergent-insoluble “cytoskeletal” fraction that were inducedby enoxacin treatment. Although 50 �M enoxacin shifted mostof the B2-subunit from the cytoskeletal fraction to the solublefraction, a smaller proportion of the E-subunit and a3-subunitwas solubilized by enoxacin. This indicates that a significantportion of these subunits is not assembledwith B2 and that theymay be associatedwith the cytoskeleton by an alternate linkage.One possibility is that these subunits are associated with theC-subunit, which is thought to bind microfilaments whenV-ATPases are not fully assembled (43–45).The protein levels of several V-ATPase subunits and a selec-

tion of other osteoclast proteins were not affected by enoxacin.The mRNA levels of a number of osteoclast-selective geneswere significantly reduced in enoxacin-treated cells, but otherswere unchanged, and one, NFATc1, increased. In a number ofcases we showed that changes in mRNA levels were notreflected by altered levels of the protein product. For example,although cathepsin K mRNA levels were very significantlyreduced, the amount of cathepsin K protein recovered fromcells was not reduced. Enoxacin triggered changes in oste-oclasts including reduction in the proteolytic activation ofTRAP5b, a shift in L-plastin from the 67- to 57-kDa form, andalterations in the transport of DC-STAMP and RANK to theosteoclast plasma membrane.

FIGURE 3. Subunits a1 and a3 of V-ATPases partition to distinct fractions after extraction of osteoclasts with 1% Triton X-100 in PBS, and enoxacinreduces the amount of B2-, E-, and a3-subunits associated with the detergent-insoluble cytoskeletal fraction. A, mouse marrow osteoclasts were grownto maturity, extracted in ice-cold buffer F plus 1% Triton X-100 for 20 min, and spun at 4 °C at 100,000 � g for 60 min. Supernatants (S) and pellets (P) werecollected, subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antibodies against subunit a1 and a3. B, marrow osteoclasts were immuno-precipitated with preimmune serum (PI), anti-a3, or anti-a1 antibodies as indicated and then subjected to SDS-PAGE and immunoblotted with anti-actin oranti-E subunit antibodies as indicated. C, extracts from marrow osteoclasts were prepared in ice-cold buffer F plus 1% Triton X-100 and treated on ice with 50�M latrunculin A (Lat., which binds monomeric actin and prevents its polymerization (36)) or vehicle (Ct) for 8 h. At the end of the incubation, the extracts weresubjected to ultracentrifugation at 4 °C at 100,000 � g for 60 min, and pellets were collected, separated by SDS-PAGE, and immunoblotted with anti-a3 andanti-actin antibodies. Band densities were measured by densitometry. D, cells were treated with vehicle or 50 �M enoxacin as they differentiated in responseto RANK-L for 5 days and then extracted with buffer F containing 1% Triton X-100 and centrifuged at 100,000 � g for 30 min; the supernatants and pellets weresuspended in equivalent volumes and the proteins separated by SDS-PAGE, blotted, and probed with anti-B2, anti-E, and anti-a3 antibodies. The V-ATPasesubunits were found primarily in the detergent-insoluble fraction in the vehicle-treated osteoclasts but shifted toward the soluble fraction in the osteoclaststreated with enoxacin.

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Changes in the sorting of DC-STAMP to the plasma mem-brane represent direct evidence that a vesicle sorting pathwayin osteoclasts is perturbed by enoxacin. Because DC-STAMP isknown to be involved in the fusion of osteoclast precursors (42,46–48), the change could be linked functionally to the reduc-tion in multinuclear and giant cells detected in enoxacin-treated cultures. Levels of cell surface DC-STAMP have been

shown to gradually decline during osteoclastogenesis (49).These results indicate that enoxacin influences DC-STAMPtoward a cell surface expression pattern of DC-STAMP associ-ated with less mature osteoclasts.The failure of TRAP5b to be activated by proteolytic cleavage

could also be explained if the interaction between V-ATPaseand microfilaments plays a role in directing vesicular traffick-

FIGURE 4. Enoxacin significantly reduces mRNA levels of numerous osteoclast marker mRNAs by qPCR, but immunoblots fail to detect reductions inprotein levels but do detect changes in proteolytic processing. A, Raw 264.7 cells were treated with RANK-L and either vehicle or 50 �M enoxacin for 5 days,and mRNA was prepared and analyzed by qPCR. Relative expression of the various mRNAs is shown in the bar graph. B, Raw 264.7 cells were treated with RANK-Land either vehicle or 50 �M enoxacin for 5 days, and total protein was prepared. From 10 to 100 �g of total protein was separated by SDS-PAGE, blotted tonitrocellulose membranes, and probed with antibodies directed against the indicated proteins. Preliminary experiments were performed to ensure that thetarget proteins were at levels within the linear detection range of the antibodies. No major differences in the amount of any of the proteins present weredetected. C, two independent experiments (lanes 1 and 2 and lanes 3 and 4 are paired) in which Raw 264.7 cells were treated with RANK-L for 5 days inthe presence of vehicle or 50 �M enoxacin, total protein was prepared, separated by SDS-PAGE, blotted, and probed with an anti-TRAP antibody. In the vehicle(lanes 1 and 3), most of the protein that was reactive to the antibody was in a band at about 16 kDa. Enoxacin treatment (lanes 2 and 4) led to a reduction inthe 16-kDa band and a large increase in the higher molecular weight bands up to the 38-pkDa band, which is likely the full-length TRAP. D, Raw 264.7 cells werestimulated with RANK-L and treated with vehicle or enoxacin at the indicated concentrations (�M) for 5 days. Proteins were extracted, 100 �g of protein wasloaded onto SDS-polyacrylamide gels, and the proteins were separated, blotted, and probed with the indicated antibodies. In the vehicle, the expected 67-kDaband was detected but at higher concentrations of enoxacin; the level of 67-kDa was reduced, and a 57-kDa band appeared. Actin was utilized as a loadingcontrol.

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ing. Acid cysteine proteinase activity has been implicated in theactivation of TRAP, suggesting that failure of V-ATPases to besorted to the same pathway as TRAP5b, and the consequentfailure to acidify vesicles might disrupt the action of activatingacid proteinases.A change in L-plastin is consistent with a reduction in the

formation of actin rings detected previously in response toenoxacin (1). L-plastin is an actin-cross-linking protein that hasbeen shown to be present in the core of podosomes and in actinrings (50). Regulation of L-plastin levels has been tied to thecapacity of osteoclasts to form actin rings and resorb bone (41).Our data suggest that L-plastin is likely cleaved proteolyticallyso that the N-terminal EF-hand domain is removed in responseto enoxacin. The EF-hand binds calcium and mediates inhibi-tion of binding of L-plastin tomicrofilaments in the presence ofcalcium (51–53).The mRNA levels of several osteoclast- and V-ATPase-spe-

cific genes were significantly reduced in response to enoxacin.However, reductions in themRNA levels did not correspond tosignificant differences in the protein levels. There are a varietyof explanations for the observed disconnect including alteredtranslation from mRNAs (54), altered life span of proteins (55)in enoxacin-treated cells and changes in the amount of a spe-cific proteins being secreted. For example, cathepsin K andTRAP5b are both secreted. A reduction in the secretion of theseenzymes triggered by enoxacin could lead to maintaining sim-ilar cellular levels compared with controls even if mRNA levelsand/or protein translation are reduced.

Could the diverse effects we detected all be the result ofblocking the interaction between V-ATPase and microfila-ments? One explanation for the results relies in the suggestionmade by Brown et al. (56) that V-ATPasesmay serve, like clath-rin, caveolin, and coatamer protein complexes, to regulatevesicular trafficking. Brown and colleagues (57) propose thatthe recently identified interactions between V-ATPases andARF6 and ARNO could work in coordination with the actinbinding activity of B2 to modulate vesicular trafficking andcytoskeletal reorganization. This hypothesis implies that theV-ATPase/microfilament interaction could be a vital nodeintegrating multiple regulatory pathways. This could explainthe diverse, but selective, effects that enoxacin has onosteoclasts.Very strong support for the notion that the V-ATPase/mi-

crofilament interaction can have a vital role in vesicular sortinghas appeared recently (58). It was reported that the capacity ofV-ATPase to bind microfilaments works in coordination withlocalized actin polymerization nucleated by actin-related pro-tein 2/3 (ARP2/3)-WASH protein complex to sort V-ATPasesfrom exocytic vesicles in Dictyostelium. Although this sortingwas in the context of a constitutive exocytic pathway that allowsthe amoeba to rid itself of indigestible material, WASH and theactin-binding site in the B-subunit are both evolutionarilyhighly conserved (1, 58). It is plausible that this basic sortingmechanism has been adapted in mammalian cells, like oste-oclasts, for cell type- and situation-specific functions. Efforts

FIGURE 5. Enoxacin alters its distribution of the plasma membrane surface of DC-STAMP in the plasma membrane of RANK-L-stimulated cells. A, bonemarrow-derived osteoclasts seeded on UpCell plates were stimulated for 6 days with M-CSF and RANK-L to produce osteoclasts. Osteoclasts were then treatedwith 50 �M enoxacin or vehicle for 72 h. Cells were lifted, probed with anti-RANK and anti-DC-STAMP antibodies, and acquired using a FACSCalibur flowcytometer. Data were analyzed using FCS Express software. A, forward versus side scatter of osteoclasts showing gating on live cells (red) with or withoutenoxacin treatment. B, RANK versus DC-STAMP mean fluorescence intensity (MFI) gated on live cells with or without enoxacin treatment. The blue squareindicates the boundary for high level RANK-expressing cells used for graphs. C, the top graph shows mean fluorescence intensity for the osteoclasts from arepresentative mouse treated with vehicle (red lines) or 50 �M enoxacin (black lines), gated on RANK� live cells measuring the fluorescence intensity ofDC-STAMP. The bottom graph is identical, except it measures the fluorescence intensity of RANK. D, significantly more cells express high levels of DC-STAMP inenoxacin-treated cultures. Significantly higher levels of cells from vehicle-treated cultures express low levels of DC-STAMP and RANK.

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are currently under way in our laboratory to test that idea inosteoclasts.Enoxacin was recently identified as a stimulator of RNA

interference andmicroRNA activity (3). We have reported thatpefloxacin, which does not havemicroRNA stimulating activity(3) but does block the B2-microfilament binding interaction,has similar inhibitory effects on osteoclasts compared withenoxacin (1). We have also have identified a non-fluoroquin-olone inhibitor of the B2-microfilament interaction, which werefer to as Binhib16, with similar inhibitory effects on oste-oclasts.4 Given these results, we have tentatively concluded thatthe effects on microRNAs are not primarily responsible for theability of enoxacin to inhibit osteoclasts. However, the com-plexity of the response of osteoclasts to enoxacin, as well as thedearth of information regarding bothV-ATPase trafficking andmicroRNA function in osteoclasts, currently makes a definitiveconclusion impossible.In summary, enoxacin directly inhibited osteoclasts without

triggering apoptosis. It reduced the amount of the B2-subunitbound to the detergent-insoluble cytoskeletal fraction, alteredthe transport of DC-STAMP to the plasma membrane, andblocked proteolytic activation of TRAP5b from its latent pro-enzyme to the active form. Enoxacin triggered a reduction inthe size of L-plastin, likely because of proteolytic cleavage of theN-terminal calcium-binding EF-hand domain. Enoxacin maybe selectively active toward osteoclasts because only thoseV-ATPases that contain the a3 isoform of subunit a, which isselectively expressed in osteoclasts and required for osteoclastfunction, are detected bound to microfilaments in osteoclasts.We propose that enoxacin selectively blocks elements of theosteoclast differentiation and activation program that aredownstream of the binding interaction between a3-containingV-ATPases and microfilaments, which is mediated by theactin-binding site on the B2-subunit.This report shows clearly that enoxacin is a novel type of

selective inhibitor of osteoclasts. Enoxacin, or other moleculeswith similar activity, may prove useful for the treatment ofosteoporosis and other bone pathologies. Because enoxacin is apotent inhibitor of the growth and metastasis of cancers (5),and selectively inhibits osteoclasts (1), it is a particularly attrac-tive candidate to test as a therapeutic agent for the treatment ofbone cancer.

Acknowledgments—We thankDr. Beth S. Lee (Ohio State University),Drs. Stephen L. Gluck and Ming Lu (University of California, SanFrancisco), and Dr. Michael R. Bubb (North Florida/South FloridaVeterans Affairs Medical Center) for their vital roles in the develop-ment of this project.

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Enoxacin Directly Inhibits Osteoclast Formation

17904 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 21 • MAY 18, 2012

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J. Wronski, Shannon M. Wallet and L. Shannon HollidayBradaschia-Correa, Victor Arana-Chavez, Aliana R. Caridad, John K. Neubert, Thomas

Edgardo J. Toro, Jian Zuo, David A. Ostrov, Dana Catalfamo, VivianEnoxacin Directly Inhibits Osteoclastogenesis without Inducing Apoptosis

doi: 10.1074/jbc.M111.280511 originally published online April 2, 20122012, 287:17894-17904.J. Biol. Chem. 

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