HDAC7 Inhibits Osteoclastogenesis by Reversing RANKL-Triggered β-Catenin Switch

HDAC7 Inhibits Osteoclastogenesis by ReversingRANKL-Triggered �-Catenin Switch

Zixue Jin, Wei Wei, Paul C. Dechow, and Yihong Wan

Department of Pharmacology (Z.J., W.W., Y.W.), University of Texas Southwestern Medical Center,Dallas, Texas 75390; and Department of Biomedical Sciences (P.C.D.), Baylor College of Dentistry, TexasA & M University Health Sciences Center, Dallas, Texas 75246

The bone-resorbing osteoclast is essential for skeletal remodeling, yet its deregulation contributesto diseases such as osteoporosis and cancer bone metastasis. Here we identify histone deacetylase7 (HDAC7) as a key negative regulator of osteoclastogenesis and bone resorption using both invitro cellular and molecular analyses and in vivo characterization of conditional HDAC7-knockoutmice. Bone marrow osteoclast differentiation assays reveal that HDAC7 overexpression sup-presses, whereas HDAC7 deletion enhances, osteoclastogenesis. Mechanistically, in the absence ofreceptor activator of nuclear factor �-B ligand (RANKL), HDAC7 attenuates �-catenin function andcyclin D1 expression, thereby reducing precursor proliferation; upon RANKL activation, HDAC7suppresses NFATc1 and prevents �-catenin down-regulation, thereby blocking osteoclast differ-entiation. Consequently, HDAC7 deletion in the osteoclast lineage results in a 26% reduction inbone mass (P � 0.003) owing to 102% elevated bone resorption (P � 0.01). These findings areclinically significant in light of the remarkable therapeutic potentials of HDAC inhibitors forseveral diseases such as cancer, diabetes, and neurodegeneration. (Molecular Endocrinology 27:325–335, 2013)

Skeletal homeostasis is controlled by the delicate bal-ance between osteoclast-mediated bone resorption

and osteoblast-mediated bone formation. Osteoclasts aremultinucleated cells differentiated from myeloid progen-itors by mainly two cytokines, macrophage colony-stim-ulating factor (MCSF) and receptor activator of nuclearfactor �-B ligand (RANKL) (1–3). RANKL induces theexpression of key transcription factors such as NFATc1and c-fos that are essential for osteoclast differentiationand maturation (4, 5). Osteoclasts are required for nor-mal functions such as bone remodeling and fracture re-pair. However, excessive osteoclast activity can lead toseveral disorders such as osteoporosis, arthritis, and can-cer bone metastasis. Therefore, discovery of novel mech-anisms underlying the regulation of osteoclastogenesis ispivotal to enhance our understanding of bone physiology,and the identification of key modulators of bone resorp-

tion may reveal new therapeutic targets for the preventionand treatment of bone diseases.

Wnt/�-catenin signaling is a key regulator of skeletalphysiology. This pathway is clinically important becauseneutralizing antibodies against Wnt antagonists arepromising new drugs for bone diseases (6–11). Previousstudies have shown that activation of Wnt/�-catenin sig-naling can increase osteoblast-mediated bone formationand modulate osteoblast functions (12–15). Our recentstudy has identified �-catenin as a novel and critical reg-ulator of osteoclast differentiation and bone resorption(16). �-Catenin protein and its target gene cyclin D1 areinduced during the MCSF-mediated precursor prolifera-tion but must be down-regulated during RANKL-medi-ated osteoclast differentiation; �-catenin constitutive ac-tivation blocks, whereas �-catenin dosage reductionenhances, osteoclast differentiation (16, 17). However,

ISSN Print 0888-8809 ISSN Online 1944-9917Printed in U.S.A.Copyright © 2013 by The Endocrine Societydoi: 10.1210/me.2012-1302 Received September 4, 2012. Accepted October 30, 2012.First Published Online November 30, 2012

Abbreviations: BrdU, Bromodeoxyuridine; BS, bone surface; BV, bone volume; �CT, mi-cro-computed tomography; HDAC, histone deacetylase; HEK, human embryonic kidney;MCSF, macrophage colony-stimulating factor; P1NP, serum amino-terminal propeptide oftype I collagen; PPAR�, peroxisome proliferator-activated receptor �; RANKL, receptoractivator of nuclear factor �-B ligand; TRAP, tartrate-resistant acid phosphatase; TV, tissuevolume.

O R I G I N A L R E S E A R C H

Mol Endocrinol, February 2013, 27(2):325–335 mend.endojournals.org 325

the upstream factors that govern �-catenin regulation inthis process are unknown.

Histone deacetylases (HDACs) are important regula-tors of a wide variety of biological processes, such asmuscle differentiation and neuronal survival, by deacety-lating both histone and nonhistone proteins (18). Basedon structural and functional similarities, the 18 HDACsin the human genome are classified into four groups.Class I HDACs (HDACs 1, 2, 3, and 8) are broadly ex-pressed and represent the major enzymes for histonedeacetylation. In contrast, class II HDACs (HDACs 4–7,9, and 10) are expressed in a more tissue-restricted man-ner and appear to have less contribution to histonedeacetylation. Class III HDACs (Sirtuins 1–7) require re-duced nicotinamide adenine dinucleotide for enzymaticactivity. Class IV HDAC (HDAC11) is still poorly under-stood (19).

HDACs are also emerging as key regulators of skeletalhomeostasis (19). Several in vitro studies report that os-teoclast differentiation can be suppressed by HDAC in-hibitors (20–24), which are broad-spectrum compoundsthat target several HDAC members (18). Nonetheless, thespecific physiological roles of individual HDAC in bone,particularly in osteoclasts, are largely unknown (19). Thisis an important question because HDAC inhibitors, manyof which are currently in clinical trials, hold tremendoustherapeutic potential for numerous diseases such as can-cer, diabetes, and neurodegeneration (25, 26).

HDAC7 is a member of the class IIa HDACs. In vitrostudies suggest that HDAC7 regulates the maturation ofboth osteoclast and osteoblast (27, 28). However,whether HDAC7 is a physiologically relevant regulator ofosteoclastogenesis and bone resorption in vivo is still anopen question, and the mechanisms underlying potentialHDAC7 regulation of osteoclast is unclear. In this study,by generating two new mouse genetic models that targetHDAC7 in the osteoclast lineage, we reveal a critical roleof HDAC7 in suppressing osteoclastogenesis and boneresorption via a novel molecular mechanism that involvesthe dual regulation of �-catenin and NFATc1.

Materials and Methods

MiceHDAC7 flox mice (29) and peroxisome proliferator-acti-

vated receptor � (PPAR�)-tTA;TRE-cre mice (16, 30, 31) werepreviously described. Lysozyme-cre mice (32) were purchasedfrom The Jackson Laboratory (Bar Harbor, ME). All experi-ments were performed with littermates. All protocols for mouseexperiments were approved by the Institutional Animal Careand Use Committee of University of Texas Southwestern Med-ical Center.

Bone analysesMicro-computed tomography (�CT) was performed on tib-

ias to evaluate bone volume and architecture using a Scanco�CT-35 instrument (SCANCO Medical, Wayne, PA) as previ-ously described (33, 34). Histomorphometry of femoral sectionswas conducted using the BIOQUANT Image Analysis software(Bioquant, Nashville, TN). Tartrate-resistant acid phosphatase(TRAP) staining of osteoclasts was performed using a Leuko-cyte Acid Phosphatase staining kit (Sigma, St. Louis, MO). As abone resorption marker, serum CTX-1 was measured with theRatLaps enzyme immunoassay kit (Immunodiagnostic Systems,Fountain Hills, AZ) (33). As a bone formation marker, serumamino-terminal propeptide of type I collagen (P1NP) was mea-sured with the Rat/Mouse P1NP enzyme immunoassay kit (Im-munodiagnostic Systems) (33).

Ex vivo bone marrow osteoclast differentiationOsteoclasts were differentiated from mouse bone marrow

cells as described previously (16, 35). Briefly, hematopoieticbone marrow cells were purified with a 40-�m cell strainer toremove mesenchymal cells, and differentiated with 40 ng/ml ofMCSF (R&D systems, Minneapolis, MN) in �-MEM contain-ing 10% FBS for 3 d and then with 40 ng/ml of MCSF and100ng/ml of RANKL (R&D systems) for 3 d (for mRNA anal-ysis) or 9–12 d (for TRAP staining), in the presence or absenceof 1 �M rosiglitazone (Cayman Chemical, Ann Arbor, MI). Ma-ture osteoclasts were identified as multinucleated (�3 nuclei)TRAP� cells. Osteoclast differentiation was quantified by theRNA expression of RANKL-induced transcription factors andosteoclast function genes using quantitative RT-PCR analysis.For HDAC7 transfection, bone marrow cells were cultured withMCSF for 2 d, and the attached cells were transfected withHDAC7 expression plasmid or vector control with FuGENEHD (Roche, Indianapolis, IN) for 24 h before changing to me-dium containing MCSF and RANKL for osteoclastdifferentiation.

Osteoclast precursor proliferation assayOsteoclast precursor proliferation was quantified using a

bromodeoxyuridine (BrdU) Cell Proliferation Assay Kit (GEHealthcare Life Sciences, Piscataway, NJ) (16, 36). Mouse bonemarrow cells were treated with MCSF (40 ng/ml) for 1 or 3 d.The cells were MCSF-starved for 6 h, and then restimulated withMCSF for 4 h to induce S phase, during which BrdU was pro-vided in the culture medium. Cell proliferation was quantified asBrdU incorporation using the BrdU ELISA assay in the kit.

Gene expression analysesRNA expression was analyzed by quantitative RT-PCR.

RNA was reverse transcribed into cDNA using an ABI HighCapacity cDNA RT Kit, and analyzed using real-time quantita-tive PCR (SYBR Green) in triplicate. All RNA expression wasnormalized by L19. Genes analyzed and PCR primer sequencesare listed below. Protein expression was analyzed by Westernblot using whole-cell extract or nuclear extract. The followingantibodies were used: anti-�-catenin and anticyclin D1 (BD Bio-sciences, Palo Alto, CA); anti-HDAC7 (Abcam, Inc., Cam-bridge, MA) and anti-�-actin (Sigma).

HDAC7a-forward (F) CTCGGCTGAGGACCTAGAGAHDAC7a-reverse (R) CAGAGAAATGGAGCCTCTGC

326 Jin et al. HDAC7 Inhibits Bone Resorption in Mice Mol Endocrinol, February 2013, 27(2):325–335

NFATc1-F AGTCTCTTTCCCCGACATCANFATc1-R GATCCGAAGCTCGTATGGACc-fos-F AAGTATGCCCACACCAACTGATCc-fos-R GAAAGCCCGTTCCCAAGAAATRAP-F AAGTATGCCCACACCAACTGATCTRAP-R GAAAGCCCGTTCCCAAGAAA�-catenin-F AGCTGATATTGACGGGCAGTATG�-catenin-R GCCAAGCGCTGGACATTAGTCyclinD1-F TCCCTAGCAAGCTGCCAAACCyclinD1-R TGGACCCACCACCAGTCTATGCTSK-F AGCAGGCTGGAGGACTAAGGTCTSK-R GATTTGTGCATCTCAGTGGAAGACCAR2-F TGGTCAACTTAGGGCATCTTTTCCAR2-R TCCTATGGCTGTGAAGAGAAGCAMMP-9-F CCAAGGGTACAGCCTGTTCCTMMP-9-R GCACGCTGGAATGATCTAAGCAtp6v0d2-F GACCAACACACCTTCTCAACCAAtp6v0d2-R GCCACAAAATACCTGAAACAACAGClcn7-F TCTGTCAGGGAACCCACATGTClcn7-R GGGCAGATGTTGGTGAATTCA

Transient transfection and reporter analysisTo quantify �-catenin activity, human embryonic kidney

(HEK)293 cells were transfected with a TOP-flash luciferasereporter, or FOP-flash negative control, together with a cyto-megalovirus-�-gal reporter (as internal control for transfectionefficiency), as well as expression plasmids for �-catenin-�ex3,HDAC7, NFATc1, or vector control. To quantify NFATc1 ac-tivity, HEK293 cells were transfected with an NFATc1-lu-

ciferase reporter (Stratagene, La Jolla, CA), together with a cy-tomegalovirus-�-gal reporter, as well as expression plasmids forNFATc1, HDAC7, or vector control. HDAC7 plasmid was gen-erously provided by Dr. Eric Olson (University of Texas South-western). �-Catenin-�ex3 plasmid was generously provided byDr. Chi Zhang (Texas Scottish Rite Hospital for Children).NFATc1 plasmid was purchased from Open Biosystems (Hunts-ville, AL). All transfection was performed using FuGENE HD(Roche) (n � 6) and repeated for at least three times. Reporterassays were conducted 48 h after transfection, and luciferaseactivity was normalized by �-gal activity.

Statistical analysesStatistical analyses were performed with Student’s t test and

represented as mean � SD; *, P � 0.05; **, P � 0.01; ***, P �0.005; ****, P � 0.001; n.s., nonsignificant, P � 0.05.

Results

HDAC7 expression during osteoclastdifferentiation

We first examined HDAC7 expression during a timecourse of osteoclast differentiation. Bone marrow cellswere cultured with MCSF for 3 d to promote osteoclastprecursor proliferation (d 1—d 3), and then treated withRANKL and MCSF for 3 d to induce osteoclast differen-

tiation (d 4—d 6) (Fig. 1A). HDAC7mRNA was decreased during MCSF-mediated proliferation (d 1—d 3), andfurther reduced by RANKL treatment(d 4) but then gradually returned (Fig.1B). HDAC7 protein showed a similarexpression pattern (Fig. 1C). Further-more, HDAC7 mRNA was signifi-cantly down-regulated after 24 h ofRANKL treatment, and further sup-pressed by the cotreatment withrosiglitazone, an agonist of the nuclearreceptor PPAR� that has been shownto accelerate osteoclastogenesis (16,35) (Fig. 1D). These results suggest thatHDAC7 may be a negative regulator ofosteoclast differentiation.

HDAC7 overexpression inhibitsosteoclast differentiation

To investigate the roles of HDAC7in osteoclastogenesis, we first exam-ined the effects of HDAC7 gain offunction. Bone marrow cells fromwild-type mice were transfected with aHDAC7 expression plasmid or a vec-tor control before osteoclast differenti-

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FIG. 1. HDAC7 expression during osteoclast differentiation. A, A schematic diagram of theex vivo bone marrow osteoclast differentiation assay. B, HDAC7 mRNA expression on eachday of osteoclast differentiation (n � 3). C, HDAC7 protein expression on each day ofosteoclast differentiation (n � 3). D, HDAC7 mRNA expression 24 h after RANKL treatment inthe absence or presence of rosiglitazone (Rosi). R, RANKL; V, vehicle. Statistical analyses wereperformed with Student’s t test and are shown as mean � SD; *, P � 0.05; ***, P � 0.005.


ation. HDAC7 expression was effectively elevated at bothmRNA and protein levels (Fig. 2, A and E). As a result,osteoclast differentiation was suppressed, illustrated bythe reduced expression of RANKL-induced and rosiglita-zone-stimulated differentiation marker genes such asNFATc1, c-fos, and TRAP (Fig. 2B), as well as resorptiveactivity marker genes such as Ctsk, CAR2, MMP9,Atp6v0d2, and Clcn7 (Fig. 2C), leading to decreasednumber and size of TRAP� multinucleated mature oste-oclasts (Fig. 2D).

To explore the mechanisms for the antiosteoclasto-genic effects of HDAC7 overexpression, we examined thelevels of �-catenin and cyclin D1, the down-regulation ofwhich upon RANKL treatment is required for osteoclast

differentiation (16). HDAC7 overexpression preventedthe down-regulation of �-catenin and cyclin D1 proteinsin whole cells, and more dramatically in the nucleus, lead-ing to their constitutive expression (Fig. 2E). This indi-cates that HDAC7 gain of function inhibits osteoclastdifferentiation by sustaining �-catenin and cyclin D1levels.

HDAC7 deletion enhances osteoclastdifferentiation

We next determined the effects of HDAC7 loss of func-tion on osteoclastogenesis. To delete HDAC7 in the mac-rophage/osteoclast lineage, we crossed HDAC7 flox mice(29) with lysozyme-cre (Ly-cre) mice (32) to generate Ly-

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FIG. 2. HDAC7 overexpression inhibits osteoclast differentiation. Bone marrow cells from wild-type (WT) mice were transfected with HDAC7 orvector control before osteoclast differentiation with RANKL (R), with or without rosiglitazone (Rosi). A, HDAC7 mRNA expression was increased byHDAC7 transfection (n � 3). B, RANKL-induced and Rosi-stimulated expression of osteoclast transcription factors (NFATc1 and c-fos) anddifferentiation marker (TRAP) were attenuated by HDAC7 overexpression (n � 3). C, RANKL-induced and Rosi-stimulated expression of osteoclastresorptive activity markers were decreased by HDAC7 overexpression (n � 3). D, Osteoclast formation was suppressed by HDAC7 overexpression.Mature osteoclasts were identified as multinucleated TRAP� (purple) cells (n � 3). Scale bar, 25�m. E, RANKL-mediated down-regulation of �-catenin and cyclin D1 proteins was abolished by HDAC7 overexpression. Whole-cell extract or nuclear extract were isolated from thedifferentiation cultures 3, 5, 7, or 9 d after RANKL stimulation, and immunoblotted with antibodies for �-catenin, cyclin D1, HDAC7, or �-actin.Statistical analyses were performed with Student’s t test and are shown as mean � SD; *, P � 0.05; **, P � 0.01; ***, P � 0.005; ****, P �0.001. V, Vehicle.


HDAC7 (HDAC7flox/flox; Ly-cre�) conditional knockoutmice. Osteoclast differentiation from the bone marrow ofLy-HDAC7 mice was enhanced compared with the litter-mate HDAC7flox/flox controls, illustrated by more andlarger TRAP� multinucleated mature osteoclasts in cul-ture (Fig. 3A) and higher expression of osteoclast differ-entiation and activity marker genes (Fig. 3, B and C).

Upon RANKL treatment, HDAC7 deletion resulted ina more rapid down-regulation of �-catenin and cyclin D1mRNAs (Fig. 3D), along with an accelerated reduction in�-catenin and cyclin D1 proteins in whole cells, and moreprofoundly in the nucleus (Fig. 3E). Western blot analysisshowed that HDAC7 protein expression was severelyblunted in the Ly-HDAC7 differentiation cultures, con-firming efficient HDAC7 gene deletion by Ly-cre (Fig.

3E). Together, these results show that HDAC7 loss offunction stimulates osteoclast differentiation by acceler-ating RANKL-mediated �-catenin and cyclin D1down-regulation.

HDAC7 deletion promotes osteoclast precursorproliferation

We next examined the effect of HDAC7 deletion onMCSF-mediated osteoclast precursor proliferation,which requires the up-regulation of �-catenin and cyclinD1 (16). BrdU incorporation was significantly increasedin the bone marrow cultures from Ly-HDAC7 mice com-pared with littermate control mice after 1 d or 3 d ofMCSF treatment (Fig. 4A). Consistent with this observa-tion, nuclear �-catenin and cyclin D1 protein levels were

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FIG. 3. HDAC7 deletion enhances osteoclast differentiation. Bone marrow cells from Ly-HDAC7 knockout mice or littermate controls weredifferentiated into osteoclasts with MCSF and RANKL in the absence or presence of rosiglitazone (Rosi). A, RANKL-induced and Rosi-stimulatedosteoclast formation was enhanced by HDAC7 deletion. Mature osteoclasts were multinucleated TRAP� (purple) cells (n � 3). Scale bar, 25 �m. B,RANKL-induced and Rosi-stimulated expression of osteoclast transcription factors (NFATc1 and c-fos) and differentiation marker (TRAP) wereincreased by HDAC7 deletion (n � 3). C, RANKL-induced and Rosi-stimulated expression of osteoclast resorptive activity markers were increased byHDAC7 deletion (n � 3). D and E, RANKL-mediated down-regulation of �-catenin and cyclin D1 mRNA (D) and protein (E) were accelerated byHDAC7 deletion (n � 3). RNA, whole-cell extract, or nuclear extract were isolated from the differentiation cultures 3, 5, 7, or 9 d after RANKLstimulation and immunoblotted with antibodies for �-catenin, cyclin D1, HDAC7, or �-actin. Statistical analyses were performed with Student’s ttest and are shown as mean � SD; *, P � 0.05; **, P � 0.01; ***, P � 0.005; ****, P � 0.001. R, RANKL, V, vehicle; WT, wild type.


elevated in the Ly-HDAC7 proliferation cultures (Fig.4B). This was opposite from the decreased �-catenin andcyclin D1 protein levels in the Ly-HDAC7 differentiationcultures (Fig. 3E). Our previous study showed thatRANKL functions as a �-catenin switch so that �-cateninis increased before RANKL treatment but decreased uponRANKL stimulation (16). Our current results suggest thatHDAC7 also differentially regulates �-catenin functionso that it inhibits �-catenin before RANKL treatment butpromotes �-catenin upon RANKL stimulation.

HDAC7 reverses RANKL-mediated �-catenin switchby suppressing NFATc1

To further investigate the mechanisms for RANKL-dependent regulation of �-catenin by HDAC7, we nextexamined how HDAC7 and RANKL-induced transcrip-tion factors regulate �-catenin activity using a transienttransfection assay of the TOP-flash luciferase reporter ina heterologous cell line. Although HEK293 cells represent

a different cellular context from oste-oclasts, this cell line is widely used fortransient transfection and reporteranalysis to dissect the functions of in-dividual transcription factor. First, weidentified NFATc1 as the transcriptionfactor that mediates the RANKLswitch, because �-catenin activity wasspecifically suppressed by NFATc1(Fig. 4C), but not other RANKL-in-duced transcription factors such as c-fos (data not shown). Second, the ef-fects of HDAC7 on �-catenin activitywas NFATc1 dependent because�-catenin activity was inhibited byHDAC7 in the absence of NFATc1,but stimulated by HDAC7 in the pres-ence of NFATc1 (Fig. 4C). Third, atransient transfection assay of theNFATc1-luciferase reporter revealedthat HDAC7 dose-dependently inhib-ited NFATc1 activity (Fig. 4D). To-gether with the HDAC7 suppression ofNFATc1 expression during osteoclastdifferentiation (Figs. 2B and 3B), theseresults show that HDAC7 attenuatesNFATc1 function and abolishesNFATc1 suppression of �-catenin inthe presence of RANKL, consequentlyblocking osteoclast differentiation. Onthe other hand, in the absence ofRANKL, HDAC7 inhibits �-cateninexpression and activity (Fig. 4, B andC), thereby decreasing osteoclast pre-

cursor proliferation. Therefore, the combined suppres-sion of precursor proliferation and osteoclast differentia-tion by HDAC7 would predict that HDAC7 deletion inthe osteoclast lineage increases bone resorption and de-creases bone mass in vivo.

Osteoclastic HDAC7 deletion in mice decreasesbone mass by increasing bone resorption

To determine the in vivo consequences of osteoclasticHDAC7 deletion, we next analyzed the bone phenotypein Ly-HDAC7 mice. Micro-computed tomography(�CT) analysis revealed that Ly-HDAC7 mice exhibited alower bone mass manifested as a significantly decreasedbone volume/tissue volume ratio (BV/TV, �26%), bonesurface (BS, �29%), and trabecular number (�25%),along with a significantly increased trabecular separation(�47%), leading to weaker bones with a higher structuremodel index (�53%) and a lower connectivity density

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FIG. 4. HDAC7 Reverses RANKL-mediated �-catenin switch by suppressing NFATc1. A,Osteoclast precursor proliferation was increased by HDAC7 deletion (n � 10). BrdUincorporation was compared between bone marrow cultures from Ly-HDAC7 knockout miceor littermate controls 1 or 3 d after MCSF treatment. B, �-Catenin and cyclin D1 protein levelsduring precursor proliferation were elevated by HDAC7 deletion. Whole-cell extract or nuclearextract was isolated from the bone marrow cultures 1 or 3 d after MCSF treatment, andimmunoblotted with antibodies for �-catenin, cyclin D1, or �-actin. C, �-Catenin activity wasinhibited by HDAC7 in the absence of NFATc1, but stimulated by HDAC7 in the presence ofNFATc1 in a transient transfection assay. TOP-flash activity was normalized by FOP-flashactivity control (n � 6). *, HDAC7 compared with vector control; �, �NFATc1 compared with�NFATc1. D, NFATc1 activity was suppressed in a dose-dependent manner by HDAC7 in atransient transfection assay (n � 6). Statistical analyses were performed with Student’s t testand are shown as mean � SD; *, P � 0.05; **** or ��, P � 0.001. WT, Wild type.


(�48%) (Fig. 5A-B). ELISA analyses of serum markersshowed that this low-bone mass phenotype was specifi-cally caused by an increased bone resorption because thebone resorption marker CTX-1 (C-terminal telopeptidesof Type I collagen) was 102% higher (Fig. 5C), whereasthe bone formation marker P1NP was unchanged (Fig.5D). Consistent with these observations, histomorphom-etry analysis showed that osteoclast number and surfacewere significantly increased in the Ly-HDAC7 knockoutmice compared with littermate control mice (Fig. 5, E andF). These findings indicate that HDAC7 deletion in the

macrophage/osteoclast population results in osteopeniadue to elevated bone resorption.

To further investigate the in vivo effects of HDAC7deletion in the entire osteoclast lineage, we decided toexamine another cre driver that can target early osteoclastprogenitor cells. Our previous study used Tie2cre for thispurpose (35); however, Tie2cre-mediated HDAC7 dele-tion results in early embryonic lethality (29). We haverecently shown that osteoclast progenitors reside inPPAR�� hematopoietic bone marrow cells; and PPAR�-tTA;TRE-cre (PT-cre) driver can efficiently target the entire

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20

30***

Oc.

N/B

.S (%

)

WT Ly-HDAC70

2

4

6***

Oc.

S/B

.S (%

)

WT Ly-HDAC70

4

8

12

16 ****

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FIG. 5. Ly-HDAC7 knockout mice have low bone mass due to high bone resorption. A and B, Ly-HDAC7 mice displayed a low-bone massphenotype. Tibias from Ly-HDAC7 mice or wild-type (WT) littermate controls (3-month-old males, n � 4) were analyzed by �CT. A, Representativeimages of the trabecular bone of the tibial metaphysis (top) (scale bar, 10 �m) and the entire proximal tibia (bottom) (scale bar, 1mm). B,Quantification of trabecular bone volume and architecture. Tb.N, Trabecular number; Tb.Sp, trabecular separation; SMI, structure model index;Conn.D., connectivity density. C, Serum CTX-1 was increased (3-month-old males, n � 6). D, Serum P1NP was unaltered (3-month-old males, n �6). E and F, Bone histomorphometry analysis shows increased osteoclasts in the Ly-HDAC7 mice compared with littermate controls (3-month-oldmales, n �4). E, Representative images of TRAP-stained femoral sections. Osteoclasts were identified as multinucleated TRAP� (purple) cells. Scale bar,100 �m. F, Quantification of osteoclast surface and osteoclast number. Oc.S, osteoclast surface; B.Ar, bone area. Statistical analyses were performed withStudent’s t test and are shown as mean � SD; *, P � 0.05; **, P � 0.01; ***, P � 0.005; ****, P � 0.001; n.s., nonsignificant. , P � 0.05.


osteoclast lineage (16, 31). Thus, we bred HDAC7flox/flox

mice with PT-cre mice to generate PT-HDAC7(HDAC7flox/flox; PT-cre�) conditional knockout mice.�CT analysis showed that the PT-HDAC7 mice had asimilar low-bone-mass phenotype as the Ly-HDAC7 micewith reduced BV/TV, bone surface, trabecular number,and connectivity density, along with increased bone sur-face/bone volume (BS/BV) ratio and trabecular separation(Fig. 6, A and B). ELISA analyses of serum markersshowed that the PT-HDAC7 mice had increased boneresorption (Fig. 6C) but unaltered bone formation (Fig.6D). Consistently, osteoclast differentiation from thebone marrow of PT-HDAC7 mice was enhanced com-pared with the littermate HDAC7flox/flox controls (Fig.6E). These data further confirm the in vivo antiosteoclas-togenic role of HDAC7. Together, the findings from twoosteoclastic HDAC7 knockout mouse models convinc-ingly demonstrated that HDAC7 is a physiologically rel-evant negative regulator of bone resorption that exertsprofound consequences on skeletal mass.

Discussion

In this study, using both in vitro and in vivo strategies, wehave identified HDAC7 as an important and physiologi-cally relevant negative regulator of osteoclastogenesis andbone resorption. Mechanistic studies reveal that HDAC7suppresses both osteoclast precursor proliferation and os-teoclast differentiation via a dual regulation of �-cateninand NFATc1. In the absence of RANKL, HDAC7 inhibitsprecursor proliferation by attenuating �-catenin and cy-clin D1 expression as well as �-catenin activity. UponRANKL stimulation, HDAC7 inhibits osteoclast differ-entiation by suppressing NFATc1 expression and activityand preventing the down-regulation of �-catenin and cy-clin D1. Therefore, HDAC7 impedes osteoclastogenesisby a novel mechanism of reversing the RANKL/NFATc1-mediated �-catenin switch (Fig. 7). Consequently,HDAC7 deletion in the osteoclast lineage results in higherbone resorption and lower bone mass in two mouse ge-netic models. This is the first in vivo study that reports a

WT PT-HDAC7

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BV/

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150

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6

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FIG. 6. PT-HDAC7 knockout mice have low bone mass due to high bone resorption. A and B, PT-HDAC7 mice displayed a low-bone massphenotype. Tibias from PT-HDAC7 mice or littermate controls (3-month-old males, n � 4) were analyzed by �CT. A, Representative images of thetrabecular bone of the tibial metaphysis (top) (scale bar, 10 �m) and the entire proximal tibia (bottom) (scale bar, 1 mm). B, Quantification oftrabecular bone volume and architecture. Tb.N, Trabecular number; Tb.Sp, trabecular separation; Conn.D., connectivity density. C, Serum CTX-1was increased (3-month-old males, n � 4). D, Serum P1NP was unaltered (3-month-old males, n � 4). E, RANKL-induced and rosi-stimulatedexpression of osteoclast transcription factors (NFATc1 and c-fos) and functional gene (TRAP) were increased in PT-HDAC7 bone marrowdifferentiation culture (n � 3). R, RANKL; Rosi, rosiglitazone; V, vehicle. Statistical analyses were performed with Student’s t test and are shown asmean � SD; *, P � 0.05; **, P � 0.01; ***, P � 0.005; n.s., nonsignificant. , P � 0.05.


critical physiological function of an individual HDAC inosteoclastogenesis and bone resorption.

Future studies are required to elucidate the detailedmolecular mechanisms for how HDAC7 regulates the ex-pression and activity of �-catenin, NFATc1, and cyclinD1. Because both histone and nonhistone proteins can bemodified by acetylation, HDAC7 may function via mul-tiple targets to alter chromatin structure as well as tomodulate the activity, stability, recruitment, and/or sub-cellular localization of these transcription factors. An-other important question is which histone acetyltrans-ferases oppose the function of HDAC7 duringosteoclastogenesis to balance the acetylation switches.

Numerous HDAC inhibitors are currently in clinicaltrials for the treatment of a bevy of diseases including

cancer and neurodegeneration (25,26). Interestingly, inhibition of class IIaHDACs (HDAC 4, 5, and 7) has alsobeen recently implicated as a potentialtherapeutic strategy for amelioratingtype 2 diabetes (37, 38). In light of thebone loss side effects of several currentdiabetes and cancer drugs such asrosiglitazone and exemestane (39–41),it is important to understand the func-tional roles of each HDAC in bonephysiology and the effects of HDACinhibition on skeletal health. On theone hand, some HDAC inhibitors maybe beneficial to the skeleton and repre-sent potential therapy for bone dis-eases. On the other hand, some HDACinhibitors may cause bone fragility,and combined treatment with bone-boosting drugs such as bisphospho-nates or denosumab may be beneficial(42).

Although in vitro studies have re-ported that broad-spectrum HDAC in-hibitors suppress osteoclast differenti-ation (20–24), our in vivo study hasunexpectedly revealed that the specificdeletion of HDAC7 in mice promotesosteoclastogenesis and bone resorp-tion. The physiological roles of otherHDACs, including other class IIaHDAC members, in osteoclasts are stillunknown. Hence, the dissection of thespecific regulation by each individualHDAC using mouse genetics will be thekey to distinguish the friends and foesof bone and to facilitate the develop-

ment of new HDAC inhibitors with maximum therapeu-tic benefits but less bone loss side effects. To this end, invitro studies suggest that HDAC7 (a class II HDAC) andHDAC3 (a class I HDAC) may exert opposite effects onosteoclast differentiation (28). Initiated by our identifica-tion of HDAC7 as the first antiosteoclastogenic HDAC invivo, future studies will promise to elucidate the manymore versatile functions of the HDAC family in skeletalphysiology.

Acknowledgments

We thank Drs. Eric Olson and Rhonda Bassel-Duby at Univer-sity of Texas Southwestern for providing HDAC7 flox mice andHDAC7 expression plasmid.

RANKHDAC7

Cyclin D1β-catenin

OsteoclastDifferentiation

NFATc1

PrecursorProliferation

RANKL

RANK

Osteoclast Osteoblast

Bone Resorption Bone Mass

Cyclin D1β-catenin

↓

↓

FIG. 7. A simplified model for how HDAC7 suppresses osteoclastogenesis. In the absence ofRANKL, HDAC7 inhibits osteoclast precursor proliferation by attenuating �-catenin functionand cyclin D1 expression. Upon RANKL stimulation, HDAC7 inhibits osteoclast differentiationby suppressing NFATc1 and prevents RANKL/NFATc1-mediated down-regulation of �-cateninand cyclin D1. Consequently, HDAC7 decreases osteoclastogenesis and bone resorption,thereby increasing bone mass.


Address all correspondence and requests for reprints to:Yihong Wan, Ph.D., University of Texas Southwestern MedicalCenter, Department of Pharmacology, 6001 Forest Park Road,Room ND8.502B, Dallas, Texas 75390-9041. E-mail:[email protected].

This work was supported by grants from National Institutesof Health (R01 DK089113, to Y.W.), Cancer Prevention Re-search Institute of Texas (CPRIT) (RP100841, to Y.W.), TheWelch Foundation (I-1751, to Y.W.), and University of TexasSouthwestern Endowed Scholar Startup Fund (to Y.W.). Y.W. isa Virginia Murchison Linthicum Scholar in Medical Researchand a recipient of the Basil O’Connor Scholar Award.

Disclosure Summary: The authors have nothing to disclose.

References

1. Bruzzaniti A, Baron R 2006 Molecular regulation of osteoclastactivity. Rev Endocr Metab Disord 7:123–139

2. Novack DV, Teitelbaum SL 2008 The osteoclast: friend or foe?Annu Rev Pathol 3:457–484

3. Wan Y 2010 PPAR� in bone homeostasis. Trends EndocrinolMetab 21:722–728

4. Grigoriadis AE, Wang ZQ, Cecchini MG, Hofstetter W, Felix R,Fleisch HA, Wagner EF 1994 c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science266:443–448

5. Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H,Saiura A, Isobe M, Yokochi T, Inoue J, Wagner EF, Mak TW,Kodama T, Taniguchi T 2002 Induction and activation of the tran-scription factor NFATc1 (NFAT2) integrate RANKL signaling interminal differentiation of osteoclasts. Dev Cell 3:889–901

6. Diarra D, Stolina M, Polzer K, Zwerina J, Ominsky MS, Dwyer D,Korb A, Smolen J, Hoffmann M, Scheinecker C, van der Heide D,Landewe R, Lacey D, Richards WG, Schett G 2007 Dickkopf-1 is amaster regulator of joint remodeling. Nat Med 13:156–163

7. Fulciniti M, Tassone P, Hideshima T, Vallet S, Nanjappa P, Etten-berg SA, Shen Z, Patel N, Tai YT, Chauhan D, Mitsiades C, Prab-hala R, Raje N, Anderson KC, Stover DR, Munshi NC 2009 Anti-DKK1 mAb (BHQ880) as a potential therapeutic agent for multiplemyeloma. Blood 114:371–379

8. Li X, Ominsky MS, Warmington KS, Morony S, Gong J, Cao J, GaoY, Shalhoub V, Tipton B, Haldankar R, Chen Q, Winters A, BooneT, Geng Z, Niu QT, Ke HZ, Kostenuik PJ, Simonet WS, Lacey DL,Paszty C 2009 Sclerostin antibody treatment increases bone forma-tion, bone mass, and bone strength in a rat model of postmeno-pausal osteoporosis. J Bone Miner Res 24:578–588

9. Ominsky MS, Vlasseros F, Jolette J, Smith SY, Stouch B, DoellgastG, Gong J, Gao Y, Cao J, Graham K, Tipton B, Cai J, Deshpande R,Zhou L, Hale MD, Lightwood DJ, Henry AJ, Popplewell AG,Moore AR, Robinson MK, Lacey DL, Simonet WS, Paszty C 2010Two doses of sclerostin antibody in cynomolgus monkeys increasesbone formation, bone mineral density, and bone strength. J BoneMiner Res 25:948–959

10. Poole KE, van Bezooijen RL, Loveridge N, Hamersma H, Papapou-los SE, Lowik CW, Reeve J 2005 Sclerostin is a delayed secretedproduct of osteocytes that inhibits bone formation. FASEB J 19:1842–1844

11. Tian E, Zhan F, Walker R, Rasmussen E, Ma Y, Barlogie B,Shaughnessy Jr JD 2003 The role of the Wnt-signaling antagonistDKK1 in the development of osteolytic lesions in multiple my-eloma. N Engl J Med 349:2483–2494.

12. Day TF, Guo X, Garrett-Beal L, Yang Y 2005 Wnt/�-catenin sig-naling in mesenchymal progenitors controls osteoblast and chon-

drocyte differentiation during vertebrate skeletogenesis. Dev Cell8:739–750

13. Glass II DA, Bialek P, Ahn JD, Starbuck M, Patel MS, Clevers H,Taketo MM, Long F, McMahon AP, Lang RA, Karsenty G 2005Canonical Wnt signaling in differentiated osteoblasts controls os-teoclast differentiation. Dev Cell 8:751–764

14. Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C 2005Canonical Wnt/�-catenin signaling prevents osteoblasts from dif-ferentiating into chondrocytes. Dev Cell 8:727–738

15. Holmen SL, Zylstra CR, Mukherjee A, Sigler RE, Faugere MC,Bouxsein ML, Deng L, Clemens TL, Williams BO 2005 Essentialrole of �-catenin in postnatal bone acquisition. J Biol Chem 280:21162–21168

16. Wei W, Zeve D, Suh JM, Wang X, Du Y, Zerwekh JE, Dechow PC,Graff JM, Wan Y 2011 Biphasic and dosage-dependent regulationof osteoclastogenesis by �-catenin. Mol Cell Biol 31:4706–4719

17. Otero K, Shinohara M, Zhao H, Cella M, Gilfillan S, Colucci A,Faccio R, Ross FP, Teitelbaum SL, Takayanagi H, Colonna M 2012TREM2 and �-catenin regulate bone homeostasis by controllingthe rate of osteoclastogenesis. J Immunol 188:2612–2621

18. Haberland M, Montgomery RL, Olson EN 2009 The many roles ofhistone deacetylases in development and physiology: implicationsfor disease and therapy. Nat Rev Genet 10:32–42

19. McGee-Lawrence ME, Westendorf JJ 2011 Histone deacetylases inskeletal development and bone mass maintenance. Gene 474:1–11

20. Cantley MD, Fairlie DP, Bartold PM, Rainsford KD, Le GT, LuckeAJ, Holding CA, Haynes DR 2011 Inhibitors of histone deacety-lases in class I and class II suppress human osteoclasts in vitro. J CellPhysiol 226:3233–3241

21. Kim HN, Lee JH, Jin WJ, Ko S, Jung K, Ha H, Lee ZH 2012MS-275, a benzamide histone deacetylase inhibitor, prevents oste-oclastogenesis by down-regulating c-Fos expression and suppressesbone loss in mice. Eur J Pharmacol 691:69–76

22. Nakamura T, Kukita T, Shobuike T, Nagata K, Wu Z, Ogawa K,Hotokebuchi T, Kohashi O, Kukita A 2005 Inhibition of histonedeacetylase suppresses osteoclastogenesis and bone destruction byinducing IFN-� production. J Immunol 175:5809–5816

23. Rahman MM, Kukita A, Kukita T, Shobuike T, Nakamura T,Kohashi O 2003 Two histone deacetylase inhibitors, trichostatin Aand sodium butyrate, suppress differentiation into osteoclasts butnot into macrophages. Blood 101:3451–3459

24. Williams PJ, Nishu K, Rahman MM 2011 HDAC inhibitor tricho-statin A suppresses osteoclastogenesis by upregulating the expres-sion of C/EBP-� and MKP-1. Ann NY Acad Sci 1240:18–25

25. Johnstone RW 2002 Histone-deacetylase inhibitors: novel drugsfor the treatment of cancer. Nat Rev Drug Discov 1:287–299

26. Kazantsev AG, Thompson LM 2008 Therapeutic application ofhistone deacetylase inhibitors for central nervous system disorders.Nat Rev Drug Discov 7:854–868

27. Jensen ED, Schroeder TM, Bailey J, Gopalakrishnan R, WestendorfJJ 2008 Histone deacetylase 7 associates with Runx2 and repressesits activity during osteoblast maturation in a deacetylation-inde-pendent manner. J Bone Miner Res 23:361–372

28. Pham L, Kaiser B, Romsa A, Schwarz T, Gopalakrishnan R, JensenED, Mansky KC 2011 HDAC3 and HDAC7 have opposite effectson osteoclast differentiation. J Biol Chem 286:12056–12065

29. Chang S, Young BD, Li S, Qi X, Richardson JA, Olson EN 2006Histone deacetylase 7 maintains vascular integrity by repressingmatrix metalloproteinase 10. Cell 126:321–334

30. Tang W, Zeve D, Suh JM, Bosnakovski D, Kyba M, Hammer RE,Tallquist MD, Graff JM 2008 White fat progenitor cells reside inthe adipose vasculature. Science 322:583–586

31. Wei W, Zeve D, Wang X, Du Y, Tang W, Dechow PC, Graff JM,Wan Y 2011 Osteoclast progenitors reside in the peroxisome pro-liferator-activated receptor �-expressing bone marrow cell popula-tion. Mol Cell Biol 31:4692–4705

32. Clausen BE, Burkhardt C, Reith W, Renkawitz R, Forster I 1999


Conditional gene targeting in macrophages and granulocytes usingLysMcre mice. Transgenic Res 8:265–277

33. Wei W, Dutchak PA, Wang X, Ding X, Wang X, Bookout AL,Goetz R, Mohammadi M, Gerard RD, Dechow PC, MangelsdorfDJ, Kliewer SA, Wan Y 2012 Fibroblast growth factor 21 promotesbone loss by potentiating the effects of peroxisome proliferator-activated receptor �. Proc Natl Acad Sci USA 109:3143–3148

34. Wei W, Wang X, Yang M, Smith LC, Dechow PC, Sonoda J, EvansRM, Wan Y 2010 PGC1� mediates PPAR� activation of osteoclas-togenesis and rosiglitazone-induced bone loss. Cell Metab 11:503–516

35. Wan Y, Chong LW, Evans RM 2007 PPAR-� regulates osteoclas-togenesis in mice. Nat Med 13:1496–1503

36. Bai S, Kopan R, Zou W, Hilton MJ, Ong CT, Long F, Ross FP,Teitelbaum SL 2008 NOTCH1 regulates osteoclastogenesis di-rectly in osteoclast precursors and indirectly via osteoblast lineagecells. J Biol Chem 283:6509–6518

37. Mihaylova MM, Vasquez DS, Ravnskjaer K, Denechaud PD, YuRT, Alvarez JG, Downes M, Evans RM, Montminy M, Shaw RJ2011 Class IIa histone deacetylases are hormone-activated regula-tors of FOXO and mammalian glucose homeostasis. Cell 145:607–621

38. Wang B, Moya N, Niessen S, Hoover H, Mihaylova MM, Shaw RJ,Yates III JR, Fischer WH, Thomas JB, Montminy M 2011 A hor-mone-dependent module regulating energy balance. Cell 145:596–606

39. Cheung AM, Tile L, Cardew S, Pruthi S, Robbins J, Tomlinson G,Kapral MK, Khosla S, Majumdar S, Erlandson M, Scher J, Hu H,Demaras A, Lickley L, Bordeleau L, Elser C, Ingle J, Richardson H,Goss PE 2012 Bone density and structure in healthy postmeno-pausal women treated with exemestane for the primary preventionof breast cancer: a nested substudy of the MAP. 3 randomisedcontrolled trial. Lancet Oncol 13:275–284

40. Home PD, Pocock SJ, Beck-Nielsen H, Curtis PS, Gomis R,Hanefeld M, Jones NP, Komajda M, McMurray JJ 2009 Rosiglita-zone evaluated for cardiovascular outcomes in oral agent combina-tion therapy for type 2 diabetes (RECORD): a multicentre, ran-domised, open-label trial. Lancet 373:2125–2135

41. Kahn SE, Haffner SM, Heise MA, Herman WH, Holman RR, JonesNP, Kravitz BG, Lachin JM, O’Neill MC, Zinman B, Viberti G2006 Glycemic durability of rosiglitazone, metformin, or glyburidemonotherapy. N Engl J Med 355:2427–2443

42. Baron R, Ferrari S, Russell RG 2011 Denosumab and bisphospho-nates: different mechanisms of action and effects. Bone 48:677–692

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HDAC7 Inhibits Osteoclastogenesis by Reversing RANKL-Triggered β-Catenin Switch