HMGB1 Regulates RANKL-Induced Osteoclastogenesis in a MannerDependent on RAGE

Zheng Zhou,1 Jun-Yan Han,2 Cai-Xia Xi,1 Jian-Xin Xie,1 Xu Feng,3 Cong-Yi Wang,2 Lin Mei,1 and Wen-Cheng Xiong1

ABSTRACT: High-mobility group box 1 (HMGB1), a nonhistone nuclear protein, is released by macrophagesinto the extracellular milieu consequent to cellular activation. Extracellular HMGB1 has properties of apro-inflammatory cytokine through its interaction with receptor for advanced glycation endproducts (RAGE)and/or toll-like receptors (TLR2 and TLR4). Although HMGB1 is highly expressed in macrophages anddifferentiating osteoclasts, its role in osteoclastogenesis remains largely unknown. In this report, we presentevidence for a function of HMGB1 in this event. HMGB1 is released from macrophages in response toRANKL stimulation and is required for RANKL-induced osteoclastogenesis in vitro and in vivo. In addition,HMGB1, like other osteoclastogenic cytokines (e.g., TNF�), enhances RANKL-induced osteoclastogenesis invivo and in vitro at subthreshold concentrations of RANKL, which alone would be insufficient. The role ofHMGB1 in osteoclastogenesis is mediated, in large part, by its interaction with RAGE, an immunoglobindomain containing family receptor that plays an important role in osteoclast terminal differentiation andactivation. HMGB1-RAGE signaling seems to be important in regulating actin cytoskeleton reorganization,thereby participating in RANKL-induced and integrin-dependent osteoclastogenesis. Taken together, theseobservations show a novel function of HMGB1 in osteoclastogenesis and provide a new link between inflam-matory mechanisms and bone resorption.J Bone Miner Res 2008;23:1084–1096. Published online on February 25, 2008; doi: 10.1359/JBMR.080234

Key words: high-mobility group box 1, receptor for advanced glycation endproducts, osteoclastogenesis,RANKL

INTRODUCTION

INFLAMMATORY OSTEOLYSIS, A MAJOR complication ofrheumatoid arthritis, periodontal disease, and orthopedic

implant destabilization, results in large part from accelerat-ed bone resorption triggered by pro-inflammatory cyto-kines. Osteoclasts (OCs), multinucleated and terminallydifferentiated cells derived from hematopoietic monocyte/macrophage precursors, are responsible for bone resorp-tion. OC differentiation is regulated by multiple factors,including macrophage-colony stimulating factor (M-CSF)and RANKL (also known as ODF or TRANCE).(1–5) M-CSF and its receptor, c-Fms (a receptor tyrosine kinaseexpressed on the cell surface), drive early lineage develop-ment from bone marrow macrophages (BMMs) and areessential for OC survival.(2) RANKL is present on themembrane of osteoblast progenitors and also exists in asoluble form in the bone microenvironment.(6) The essen-tial role of RANKL in OC differentiation is established bythe following observations. First, RANKL-deficient micelack OCs and develop severe osteopetrosis, in addition toimmunologic defects.(1–5) Second, systemic administrationof RANKL increases bone resorption.(1–5) Third, in vitro,RANKL has all the attributes of a bona fide OC differen-

tiation factor: it favors OC differentiation in conjunctionwith M-CSF; it bypasses the need for stromal cells and vi-tamin D3 to induce OC differentiation; and it activates ma-ture OCs to resorb mineralized bone.(1–5) RANKL bindingto the RANK receptor, a member of the TNF� receptorfamily localized to BMMs and OCs, promotes OC differ-entiation and activation.(1–5)

In addition to M-CSF and RANKL, several pro-inflammatory cytokines, also called osteoclastogenic cyto-kines, have been implicated in OC differentiation. Amongthese, TNF�, interleukin (IL)-1, and lipopolysaccharide(LPS) seem to contribute to OC differentiation/activationassociated with inflammatory osteolysis.(7–9) Furthermore,integrin engagement by cell adhesion to the bone matrix,TREM2 (triggering receptors expressed by myeloid cells), areceptor of the immunoglobulin super family, and DC-STAMP, a putative seven transmembrane receptor, havebeen implicated in regulating OC terminal differentiationand activation.(10–15) The latter observations suggest theinvolvement of other ligands/osteoclastogenic factors in ad-dition to M-CSF, RANKL, and TNF�. The identity of theseother ligands/osteoclastogenic factors is currently unknown.

High-mobility group box 1 (HMGB1), also called am-photerin or HMG1, is a 30-kDa abundant nonhistonenuclear protein expressed in all eukaryotic cells and subjectto release by activated macrophages or injured cells.(16,17)The authors state that they have no conflicts of interest.

1Institute of Molecular Medicine and Genomics and Department of Neurology, Medical College of Georgia, Augusta, Georgia, USA;2Center for Biotechnology and Genomic Medicine, Medical College of Georgia, Augusta, Georgia, USA; 3Department of Pathology,University of Alabama at Birmingham, Birmingham, Alabama, USA.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 23, Number 7, 2008Published online on February 25, 2008; doi: 10.1359/JBMR.080234© 2008 American Society for Bone and Mineral Research

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Once in the extracellular space, HMGB1 displays proper-ties of a pro-inflammatory cytokine.(16,17) ExtracellularHMGB1 has an essential role in innate immunity, as well asdendritic cell maturation and function, and these functionsare mediated, at least in part, by receptor for advancedglycation endproduct (RAGE).(18,19) RAGE, a member ofthe immunoglobulin super family of cell surface molecules,binds to multiple ligands, in addition to HMGB1.(20,21) It isinvolved in the pathogenesis of multiple inflammation-associated disorders, including diabetic complications,(22)

Alzheimer’s disease,(21) and inflammatory conditions.(23) Inaddition to RAGE, TLR2/4, receptors for LPS that areessential for innate immunity, are also implicated as recep-tors for HMGB1.(24,25) Recently, we showed a role ofRAGE in OC terminal differentiation and activation.(26)

Such a function seems to be mediated by RAGE regulationof osteoclastic actin cytoskeleton organization and integrinsignaling.(26) However, which RAGE ligands are involvedin osteoclastogenesis remains unclear.

Herein, we present evidence for a role of extracellularHMGB1, acting as an autocrine ligand of RAGE, inRANKL-induced osteoclastogenesis in vitro and in vivo. Inaddition to TNF� and LPS, RANKL stimulates HMGB1release from macrophages. Such extracellular HMGB1seems to be needed for RANKL-induced OC differentia-tion. Analogous to TNF� and IL-1, extracellular HMGB1stimulates differentiation of OC precursors in the presenceof subthreshold levels of RANKL in vitro and in vivo. Thelatter is blocked in RAGE-deficient mice, suggesting a cen-tral role for HMGB1–RAGE interaction. Finally, weshowed that extracellular HMGB1, through RAGE, regu-lates macrophage polarization and spreading and osteoclas-tic actin ring formation, suggesting a mechanism underlyingits role on osteoclastogenesis. Taken together, our resultsare consistent with the hypothesis that extracellularHMGB1 is a physiologically/pathologically relevant osteo-clastogenic cytokine, contributing to osteoclastogenesis,thereby participating in focal/inflammatory osteolysis.

MATERIALS AND METHODS

Reagents and animals

Antibody specific to phospho-PYK2 (PY402) was pur-chased from Transduction Laboratories (Lexington, KY,USA), and antibodies to phospho-Erk1/2 (p-Erk1/2) andErk1/2 were from Cell Signaling (Beverly, MA, USA).Monoclonal RAGE and PYK2 antibody were purchasedfrom Chemicon (Temecula, CA, USA) and BD Transduc-tion Laboratory (San Jose, CA, USA), respectively. Thec-fos polyclonal antibody was from Santa Cruz Biotechnol-ogy (Santa Cruz, CA, USA). Polyclonal anti-HMGB1 an-tibody was purchased from BD Pharmingen (San Diego,CA, USA) or generated using the recombinant HMGB1protein as antigen (Cocalico Biologicals, Reamstown, PA,USA). Antiserum to HMGB1 was purified using a proteinA–based affinity column/elution buffer system according tothe manufacturer’s instructions (Pierce, Rockford, IL,USA) and was repurified using a polymyxin B affinity col-umn to remove contaminating LPS. The neutralizing activ-

ity of anti-HMGB-1 antibody was confirmed in macro-phage cultures exposed to recombinant HMGB-1 andassayed for TNF release.

Murine M-CSF was obtained from R&D Systems (Min-neapolis, MN, USA). Nicotine was purchased from Sigma(St Louis, MO, USA). Recombinant glutathione-S-transferase (GST)-RANKL and GST-HMGB1 proteinswere generated and purified as previously described.(27,28)

These recombinant proteins were repurified using a poly-myxin B affinity column to remove contaminating LPS.However, the LPS content in purified HMGB1 was ∼90pg/�g of protein, measured by the chromogenic LimulusAmebocyte Lysate (LAL) assay (Cambrex). Thus, in ourstimulation experiments, polymyxin B was added to cellculture medium at ∼6 units/pg of LPS, which was confirmedto be able to neutralize the maximal amount of contami-nating LPS/endotoxin.

RAGE−/− mice have been crossed into a C57BL/6 ge-netic background, as described.(26,29) Age-matchedC57BL/6 wildtype (WT) mice were used as controls.RAGE mutation was confirmed by expression of greenfluorescent protein (GFP), an indicator of Cre expression inthe mice,(29) RT-PCR for expression of RAGE mRNA,and Western blot analysis. TLR4−/− (C.C3-Tlr4Lps-d/J) andcontrol mice were purchased from The Jackson Laboratory(Bar Harbor, ME, USA). All experimental procedureswere approved by the Animal Subjects Committee at theMedical College of Georgia, according to U.S. NIH guide-lines.

In vivo osteoclastogenesis and bonehistomorphometric analysis

Three-month-old wildtype and mutant mice were sub-jected to daily intraperitoneal or supracalvarial administra-tion of PBS, HMGB1 (10 �g/mouse/d), RANKL (2 �g/mouse/d) with or without anti-HMGB1 antibody (3 �g/mouse/d) for 5 days. Animals were killed, and the calvarialbones were fixed overnight in 10% buffered formalin, de-calcified in 14% EDTA, embedded in paraffin, sectioned,and stained for TRACP. The percentage of osteoclasticerosion surfaces was measured.

In vitro osteoclastogenesis and resorption assay

Mouse BMMs, perfusion osteoclasts (pre-OCs), and os-teoclasts (OCs) were generated as described.(26) In brief,whole bone marrow cells were flushed from long bones of4- to 6-wk-old WT or RAGE−/− mice and plated on 100-mmtissue culture plates in �MEM containing 10% FBS and 10ng/ml recombinant M-CSF (R&D Systems). Cells were in-cubated at 37°C with 5% CO2 overnight. Nonadherent cellswere harvested and subjected to Ficoll-Hypaque gradientcentrifugation for purification of BMMs. Isolated BMMswere cultured in �-MEM containing 10% FBS plus 10 ng/ml recombinant M-CSF. To generate pre-OCs, BMMs werecultured in �MEM containing 10% FBS in the presence of10 ng/ml recombinant M-CSF and 100 ng/ml recombinantRANKL for 2–3 days. To generate mature OCs, 5 × 104

BMMs were plated in 1 well of a 24-well plate in �MEMcontaining 10% FBS in the presence of 10 ng/ml recombi-nant M-CSF and 100 ng/ml recombinant GST-RANKL.

HMGB1/RAGE REGULATION OF OSTEOCLASTOGENESIS 1085

The HMGB1-induced osteoclasts were generated by re-placing RANKL with indicated concentrations of HMGB1at day 3 and continued to culture for over 3 days. Matureosteoclasts (multinucleated, large, spread cells) began toform at day 4 of culture. The identity of osteoclasts wasconfirmed by TRACP staining. RAW 264.7 cells were cul-tured with DMEM with 4 �M L-glutamine, 1.5 g/liter sodiumbicarbonate, and 4.5 g/liter glucose, containing 10% FBS.

For in vitro osteoclastic resorption activity, 5 × 104

BMMs were replated on BD BioCoat Osteologic coverslips(BD Biosciences) in 24-well plates, in the presence ofM-CSF (1%), RANKL (100 ng/ml), and HMGB1 of indi-cated concentrations (2–10 �g/ml). The BD BioCoat Os-teologic coverslips were coated with inorganic hydroxyapa-tite matrix. After 9 days, cells were washed and stained with5% of silver nitrate exposed to UV light for 1 h. Resorptionpits were analyzed using NIH Image J software.

Immunofluorescence staining

BMMs and osteoclasts cultured on glass were starved andstimulated with 100 ng/ml RANKL for the indicated timeand fixed with 4% paraformaldehyde and stained with anti-HMGB1 with FITC-conjugated second antibodies, and Al-exa Fluor 488-phalloidin as described.(26) Images were cap-tured with a Zeiss confocal microscope.

Western blot analysis

BMMs and pre-OCs were cultured and starved overnightin serum-free media. Cells were stimulated with 2.5 �g/mlHMGB1 for the indicated times, followed by lysis in RIPAbuffer. For integrin signaling experiments, pre-OCs werelifted from the growth substrate, starved for 2 h, and re-plated on vitronectin (VN)-coated cell culture dishes withor without further stimulation with HMGB1. For HMGB1release and translocation experiments, cultured media werecollected. Cells were harvested and isolated into nuclearand cytoplasmic fractions with the CelLytic NuCLEAR Ex-traction Kit per manufacturer’s instruction (Sigma, StLouis, MO, USA). Lysates were subjected to SDS-PAGEand immunoblotting analysis using the indicated antibodies.

RT-PCR analysis

Total RNA was prepared from BMMs, pre-OCs, andosteoclasts using Trizol (Invitrogen). These cells were gen-erated as described (see In vitro osteoclastogenesis usingpurified BMMs) at day 0 (BMMs), day 2 (pre-OCs), andday 5 (osteoclasts) in culture in the presence of M-CSF,RANKL, or HMGB1. cDNAs were synthesized from 2 �gof total RNA using SuperScript III First Strand SynthesisSystem (Invitrogen) in a volume of 20 �l. The reactionmixture was adjusted to 100 �l with distilled water. Onemicroliter of cDNA was amplified with the specific primersindicated by PCR. The PCR product was separated on a1.5% agarose gel and visualized by ethidium bromidestaining. The following primers were used: integrin �3,5�-TTACCCCGTGGACATCTACTA-3� and 5�-AGTC-TTCCATCCAGGGCAATA-3�; calcitonin receptor,5�-CATTCCTGTACTTGGTTGGC-3� and 5�-AGCA-ATCGACAAGGAGTGAC-3�; MMP9, 5�-CCTGTGT-

GTTCCCGTTCATCT-3� and 5�-CGCTGGAATGATC-TAAGCCCA-3�; TRACP, 5�-ACAGCCCCCCACTC-CCACCCT-3� and 3�-TCAGGGTCTGGGTCTCCTT-GG-5�; cathepsin K, 5�-GGAAGAAGACTCACC-AGAAGC-3� and 3�-GTCATATAGCCGCCTCCA-CAG -5�; and GAPDH, 5�-TGAA GGTCGGTGTGAAC-GGATTTGGC-3� and 3�-CATGTAGGCCATGAGGTC-CACCAC-5�.

Statistical analysis

Data were analyzed using an unpaired two-tailed Stu-dent’s t-test and are expressed as the mean ± SE; p < 0.05was considered significant.

RESULTS

RANKL stimulation of HMGB1 translocation andrelease from macrophages and OCs

Our previous work has shown impaired in vitro osteo-clastogenesis in RAGE−/− BMMs, thereby suggesting thepossibility that HMGB1, a known RAGE ligand highly ex-pressed in macrophages and osteoclasts, may be involved inthis event.(25,30,31) To test this concept, we examined theeffects of RANKL and M-CSF, two essential cytokines forOC differentiation and function, on HMGB1 release.RAW264.7 macrophages were treated with RANKL or M-CSF for different times. Western blotting of culture mediaand cell lysates with antibodies against HMGB1 was per-formed to assess levels of extracellular/released HMGB1and cytoplasmic and nuclear HMGB1, respectively.Whereas M-CSF had no effect on HMGB1 release (datanot shown), RANKL increased levels of HMGB1 in media,but not in cell lysates, consistent with stimulation ofHMGB1 release (Fig. 1A). The latter response occurredafter a lag (i.e., HMGB1 in the medium increased only after6 h of stimulation; Fig. 1A), although it was dose dependent(maximal response at 20 ng/ml; Fig. 1B). In addition tothese observations in the RAW264.7 macrophage cell line,the same effect of RANKL, but not M-CSF, on HMGB1release was observed in BMMs (Figs. 1C and 1D). We fur-ther examined the subcellular distribution of HMGB1 byWestern blotting of nuclear and cytoplasmic fractions ofBMMs treated with RANKL. On exposure to RANKL for>6 h, HMGB1 in BMMs seemed to translocate significantlyfrom the nuclear to the cytoplasmic compartment (Fig. 1C).Consistent with these data, immunostaining analysisshowed that, although most HMGB1 was detected in thenuclei of quiescent BMMs, treatment with RANKL for 18h increased cytosolic staining of HMGB1, where it seemedto colocalize with F-actin (Fig. 1E). Taken together, theseobservations suggest that RANKL stimulates HMGB1translocation and release from macrophages. The specific-ity of this event is underscored by the absence of a similareffect with M-CSF.

In addition to BMMs, we also examined the subcellulardistribution of HMGB1 in OCs stimulated with RANKL.In quiescent, serum-starved OCs, HMGB1 was primarilylocalized to nuclei (Fig. 2A). After stimulation withRANKL (∼30 min), translocation of HMGB1 to the cyto-

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plasm occurred (Fig. 2A). Cytosolic HMGB1 seemed tocolocalize with actin rings labeled by phalloidin (Fig. 2A).A similar effect was observed on stimulation with TNF�but not M-CSF (Fig. 2B). Taken together, these results sug-gest that RANKL increases HMGB1 release and translo-cation in both macrophages and OCs.

Requirement of extracellular HMGB1 forosteoclastic actin ring formation

HMGB1 co-localization with actin rings of OCs led us tospeculate a role for HMGB1 in osteoclastic actin cytoskel-eton remodeling. To test this hypothesis, we first examinedthe effects of nicotine on osteoclastic F-actin structures.Nicotine, an inhibitor of LPS-induced release ofHMGB1,(32,33) was added to the culture medium of OCsdifferentiated in vitro in the presence of M-CSF/RANKL.Nicotine (at concentrations of ∼10 �M) significantly re-duced RANKL-induced HMGB1 release (Fig. 3A). In par-allel, treatment with the same range of nicotine concentra-tions resulted in a significantly reduced number of actin-ring containing cells compared with control cultures (Figs.3B and 3C), implicating HMGB1 release in osteoclastic ac-tin ring formation. In view of previous observations indi-cating that nicotine also decreases release of other cyto-kines, such as TNF�, in addition to HMGB1,(33) it wasnecessary to further assess the specificity of the inhibitory

effect of nicotine. To this end, neutralizing antibody toHMGB1 was used. Indeed, the number of actin-ringcontaining cells was significantly reduced in the presence ofanti-HMGB1 antibody (Figs. 3B and 3C). Taken together,these results suggest that HMGB1 release, as well as extra-cellular HMGB1, seems to be necessary for osteoclasticactin ring formation.

Requirement of extracellular HMGB1 forRANKL-induced osteoclastogenesis in vitroand in vivo

We next tested the hypothesis that HMGB1 might con-tribute to RANKL-induced osteoclastogenesis, because ac-tin cytoskeleton organization is important for this event.First, we examined the effects of nicotine and/or anti-HMGB1 antibodies on RANKL-dependent in vitro osteo-clastogenesis. In the presence of either nicotine (at concen-trations of ∼10 �M) or anti-HMGB1 antibody, significantattenuation of RANKL-induced osteoclastogenesis was ob-served, as indicated by TRACP staining (Figs. 4A and 4B).Coordinately, RANKL-induced osteoclastic resorptive ac-tivity was also significantly reduced in the presence of anti-HMGB1 antibody (Figs. 4C and 4D). Together, these ob-servations suggest a requirement of HMGB1 release, aswell as extracellular HMGB1, in RANKL-induced osteo-clastogenesis and osteoclast activation in vitro.

FIG. 1. RANKL stimulationof HMGB1 translocation andrelease from macrophages in atime-dependent manner. West-ern blot analysis of HMGB1 inmedia and total cell lysatesfrom cultures of RAW264.7macrophages (A and B) andbone marrow macrophages(BMMs) (C and D) exposed toRANKL (A–C) or M-CSF (D)for the indicated times or con-centrations. In C, BMMs werealso harvested and isolatedinto nuclear and cytoplasmicfractions with the CelLyticNuCLEAR Extraction Kit(Sigma, St Louis, MO, USA).Lysates were subjected toWestern blot analysis. (E) Im-munostaining of HMGB1 inBMMs exposed to RANKLfor the indicated times. BMMswere fixed and immunostainedus ing ant ibodies aga ins tHMGB1 (rabbit polyclonal an-tibody, green), phalloidin to la-bel actin filaments (red), andDAPI to label nuclei (blue).Marker bar, 10 �m.

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We examined whether there was a requirement forHMGB1 in RANKL-induced osteoclastogenesis in vivo.For these experiments, RANKL (2 �g/mouse/d) or vehicle,with or without antibodies to HMGB1 (3 �g/mouse/d), wasinjected supracalvarially daily for 5 days into WT mice. Atthe time of death, TRACP-stained sections of calvariaewere prepared to determine OC activity histomorphometri-cally. RANKL increased osteoclastogenesis in WT mice(Figs. 4E and 4F), as previously reported.(3,4) However, thiseffect was substantially attenuated when RANKL was co-injected with antibodies to HMGB1 (Figs. 4E and 4F);

there was an inhibition of ∼67% in the presence of anti-HMGB1 antibodies. This is similar to the degree of inhibi-tion of osteoclastogenesis induced by RANKL in the pres-ence of antibodies to HMGB1 in vitro (Fig. 4B). Thus,extracellular HMGB1 seems to contribute importantly toRANKL-induced osteoclastogenesis in vivo.

HMGB1 induction of osteoclastogenesis in vitroand in vivo in a manner dependent on RAGE

Having established a role for HMGB1 in RANKL-induced OC formation, it was important to determine

FIG. 2. RANKL, but not M-CSF, regulation of HMGB1translocation in OCs. (A) Timecourse of HMGB1 transloca-tion in OCs in response toRANKL. Polyclonal rabbita n t i - H M G B 1 a n t i b o d y(green), phalloidin (red to la-bel F-actin), and DAPI (blue)were used for immunostainingof OCs differentiated fromBMMs. (B) Immunostaininganalysis of HMGB1 in OCs ex-posed to M-CSF, RANKL, andTNF� for 30 min. Marker bars,25 �m.

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whether HMGB1 itself was sufficient to induce osteoclas-togenesis. To address this issue, vehicle (PBS) or HMGB1(10 �g/mouse) was injected intraperitoneally daily for 5days into WT mice. Mice were killed on day 6, and OCnumber was determined by histomorphometric analysis ofTRACP-stained sections of calvariae (as above). HMGB1substantially increased osteoclastogenesis in WT mice(Figs. 5A and 5D; ∼4-fold; p < 0.01), suggesting the suffi-ciency of HMGB1 for induction of osteoclastogenesis invivo.

RAGE and TLR2/4 have been proposed as HMGB1 re-ceptors mediating HMGB1-induced cell signaling andchanges in cellular behavior.(34) To determine whetherRAGE and/or TLR4 were involved in mediating HMGB1function(s) in vivo, we took advantage of mice deficient inRAGE or TLR4. HMGB1 induction of osteoclastogenesis

in vivo seemed to be largely dependent on RAGE, becauseHMGB1 failed to have a stimulatory effect on osteoclasto-genesis in RAGE−/− mice (Figs. 5B and 5D). In contrast,the role of TLR4 in this context seemed to be less, becauseHMGB1 induction of osteoclastogenesis was preserved, al-though slightly reduced, in TLR4−/− mice (Figs. 5C and5D). Taken together, these observations suggest thatRAGE has an integral role in the HMGB1 induction ofosteoclastogenesis in vivo.

In the complex in vivo milieu, HMGB1, like most osteo-clastogenic agents, may exert its effects through receptorson several cell types, for example, stromal, osteoblastic cellsand/or OCs. To determine whether HMGB1 directly pro-motes differentiation of OC precursors, in vitro osteoclas-togenesis with purified BMMs was analyzed. First, isolatedBMMs were cultured with increasing amounts of the

FIG. 3. Requirement of ex-t r a c e l l u l a r H M G B 1 f o rRANKL-induced actin ringformation in OCs in vitro. (A)RANKL-stimulated HMGB1release in BMMs, as describedin Fig. 1, was inhibited in thepresence of nicotine (10 �M).(B) Immunostaining analysisof ta l in , phal lo id in , andHMGB1 in OCs exposed toRANKL in the presence or ab-sence of nicotine or anti-HMGB1 antibodies. Markerbars, 25 �m. (C) Quantitativeanalysis of data from B. Datawere presented as percentageof OCs with actin ring forma-tion (mean ± SE). **p < 0.01,significant difference from con-trol (treatment with RANKLalone; t-test).

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HMGB1 in the presence/absence of a subosteoclastogenic(permissive) concentration of RANKL (10 ng/ml).HMGB1 alone increased TRACP staining in BMMs. How-ever, under these conditions, HMGB1 did not induce for-mation of terminally differentiated multinucleated osteo-clasts (Figs. 6A and 6B). This experiment was repeatedusing cultured pre-OCs (i.e., perfusion OCs generated fromBMMs pre-exposed to M-CSF and RANKL for 3 days).Addition of increasing amounts of HMGB1 to culture me-dia of pre-OCs, in the presence of a permissive concentra-tion of RANKL, resulted in a dose-dependent increase inOC formation, based on TRACP staining (Figs. 6C and6E). These HMGB1-induced OCs appeared to be smallerin size and have reduced numbers of nuclei (∼10 nuclei/OC)compared with RANKL-induced OCs (∼20 nuclei/OC).

However, HMGB1-induced OCs expressed markers in-cluding calcitonin R (CTR), cathepsin K (Cath K), MMP9,TRACP, and integrin �3 associated with differentiated OCs(Fig. 6G), contained intact actin ring structures (Fig. 7C),and exhibited comparable bone resorptive activity as OCsformed in response to RANKL (Figs. 6H and 6I). Theseobservations suggest that HMGB1 may be sufficient to in-duce osteoclastogenesis (i.e., formation of multinucleatedcells with intact actin ring formation and function) frompre-OCs but not BMMs. Thus, as an osteoclastogenic agent,HMGB1 has properties analogous to TNF� or IL-1, in thatformation of mature OCs requires precursor cells previ-ously exposed to RANKL, and therefore, committed to theOC pathway.(35,36)

To determine the role of RAGE in HMGB1-induced

FIG. 4. Requirement of HMGB1 for RANKL-induced osteoclastogenesis in vitro and in vivo. (A) HMGB1 is required for RANKL-induced osteoclastogenesis in vitro. Purified BMMs from WT mice were incubated with M-CSF (10%) for 2–3 days and cultured in thepresence of RANKL (100 ng/ml) and M-CSF (1%), with or without anti-HMGB1 antibody (0.3 �g/ml) or nicotine (10 �M) for 4 days.The arrows denote a representative TRACP+ cell in each panel. (B) Quantitative analysis of TRACP+ multinuclei cells (MNCs) basedon data from A. Data were presented as percentage of total number of BMMs (mean ± SE). **p < 0.01, significant difference fromcontrol (treatment with RANKL alone; t-test). (C) Representative resorption caused by RANKL-induced OCs in the absence orpresence of anti-HMGB1 antibodies (0.3 �g/ml) was visualized by von Kossa staining. The resorption assay was performed by culturingOCs on coverslips coated with calcium phosphate matrix for 9 days and staining coverslips with von Kossa to display resorption pits(white). (D) Quantitative analysis of the average resorption area based on data from C. (E) HMGB1 is required for RANKL-inducedosteoclastogenesis in vivo. C57/BL6 mice (3 mo old) were supracalvarially injected daily for 5 days with vehicle (PBS), RANKL (2�g/mouse/d) + nonspecific (NS) IgG (3 �g/mouse/d), or RANKL (2 �g/mouse/d) + anti-HMGB1 IgG (3 �g/mouse/d). Animals werekilled on day 6, and histological sections of calvariae were stained for TRACP and analyzed histomorphometrically for osteoclasterosion surface. Arrows denote an area displaying TRACP+ cells in each panel. (F) Histomorphometric analysis of TRACP+ OCscovered surface area based on data from E. **p < 0.01 significant difference from RANKL alone (t-test).

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osteoclastogenesis in vitro, we repeated the above experi-ments using macrophages and pre-OCs derived fromRAGE−/− mice. In BMMs from WT or RAGE−/− mice,there was no detectable difference in terms of TRACP in-duction in response to HMGB1, suggesting a minimal/negligible effect of RAGE in this event (Figs. 6B and 6E),but implicating RAGE-independent receptors (e.g., TLR2/4/9) to be involved in this event. However, HMGB1 induc-tion of the formation of multinucleated actin ring contain-ing cells was inhibited in RAGE−/− pre-OCs, indicating arole for the HMGB1–RAGE interaction (Figs. 6D and 6F).Taken together, these results showed that HMGB1 in-creases TRACP activity in BMMs and enhances in vitroosteoclastogenesis from pre-OCs in the presence of permis-sive concentrations of RANKL. However, only the latterseemed to be dependent on RAGE.

HMGB1 regulation of actin cytoskeletonreorganization and cross-talk with integrin in amanner dependent on RAGE

In light of the observations that both extracellularHMGB1 and RAGE are needed for osteoclastic actin ringformation (Fig. 3),(26) we speculated that HMGB1, through

RAGE, plays an important role in actin cytoskeleton re-modeling in BMMs/OCs. To test this speculation, we firstre-examined RAGE expression and distribution in differ-entiating OCs. RAGE protein was detected in wildtype, butnot RAGE−/−, BMMs, indicating a specificity of the anti-body (Fig. 7A). In addition, RAGE was highly expressed inpre-OCs and mature OCs (Fig. 7A) and mainly distributedalong actin ring structures of OCs (Fig. 7B), where it co-localized with HMGB1, supporting the hypothesis. We nextexamined F-actin structures in HMGB1-induced OCs thatare derived from WT and RAGE−/− mice. HMGB1 induc-tion of the formation of multinucleated actin ring–containing cells was inhibited in RAGE−/− pre-OCs (Fig.7C). Together, these results are in line with a role ofHMGB1-RAGE signaling in actin ring formation of OCs.

We assessed the possibility of cross-talk between signal-ing induced by HMGB1 and integrin engagement, becauseRAGE, acting as a regulator of integrin signaling, partici-pates in OC maturation and activation(26) and HMGB1-induced recruitment of neutrophil.(37) To this end, pre-OCsgenerated from wildtype or RAGE−/− mice were lifted andreplated on VN-coated dishes in the absence of serum. Af-ter treatment with HMGB1 at different times, adherent

FIG. 5. HMGB1 induction ofosteoclastogenesis in vivo in amanner dependent on RAGEC57/BL6, RAGE − / − , andTLR4−/− mice (3 mo old) wereintraperitoneally injected dailyfor 5 days with vehicle (PBS)and purified recombinantHMGB1 (10 �g/mouse/d).Animals were killed on day 6,and histologic sections of cal-v a r i a e w e r e s t a i n e d f o rTRACP and analyzed histo-morphometrically for osteo-clast erosion surface. Repre-sentative images from WT (A),RAGE−/− (B), and TLR4−/−

(C) mice. (D) Histomorpho-metric analysis of TRACP+ os-teoclast erosion surface. (**p <0.01 vs. PBS control, Student’st-test). Arrows in A–C denotean area of displaying TRACP+

cells.

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FIG. 6. HMGB1 increases TRACP+ cells from BMMs but stimulates osteoclastogenesis from pre-OCs. BMMs of WT (A) andRAGE−/− (B) were cultured in the presence of vehicle (control) or the indicated concentrations of HMGB1 or RANKL. Whereindicated, a permissive concentration of RANKL (10 ng/ml) was added. TRACP staining was performed. Arrow denotes a region withTRACP+ cells (insets show this area at higher power in the first three panels). Pre-OCs of wildtype (C) and RAGE−/− (D) were culturedin the presence of M-CSF (10 ng/ml) and HMGB1 at the indicated concentrations or vehicle alone. TRACP (C and D) staining wasperformed on day 5. Quantitative analysis of TRACP+ BMMs (E) and TRACP+ multinuclei cells (MNCs) (F) based on data from A–D.Data were presented as percentage of total number of BMMs (mean ± SE). **p < 0.01, significant difference from the control (t-test).#p < 0.01, significant difference from wildtype. (G) RT-PCR analysis to assess expression of transcripts for cathepsin K (Cath K),calcitonin R (CTR), MMP9, TRACP, and integrin �3 in BMMs, pre-OCs, and OCs formed in response to HMGB1 at indicatedconcentrations for 4 days. (H) Representative resorption caused by control (CTL; pre-OCs incubated with buffer for 9 days) OCsformed in response to indicated concentrations of HMGB1 or RANKL was visualized by von Kossa staining. The resorption assay wasperformed as described in the legend of Fig. 4C. (I) Quantitative analysis of the average resorption area based on data from H.

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cells were lysed, and phosphorylation of PYK2 and Erk1/2and c-fos activation/induction were examined. Under theseconditions, phosphorylation of PYK2 and Erk1/2 and c-fosactivation were induced by HMGB1 in WT pre-OCs (Fig.7D), which exhibit different time courses (e.g., earlier on-set) compared with pre-OCs treated with VN alone orHMGB1 alone (data not shown). However, activation ofthese proteins was reduced or abolished in RAGE−/− pre-OCs (Fig. 7D). These observations suggest a convergenceof signaling pathways activated by integrin engagement andHMGB1 in a RAGE-dependent manner.

DISCUSSION

HMGB1, a nuclear protein released from activated mac-rophages or injured cells, displays pro-inflammatory cyto-kine-like properties once it enters the extracellular space.As a delayed mediator of inflammation, it has been impli-cated in lethal sepsis, innate immunity, arthritis, and tumor-associated inflammation, and recently, in endochondral os-sification.(16,25,38) However, its role in osteoclastogenesisand molecular mechanisms underlying its functions remain

to be elucidated. Our work provides evidence thatHMGB1, through RAGE, regulates actin cytoskeleton or-ganization and integrin signaling in BMMs and participatesin osteoclastogenesis in vitro and in vivo, which leads to theworking model depicted in Fig. 8. According to this model,RANKL stimulates HMGB1 release from the nuclear com-partment of BMMs. Such extracellular HMGB1 is neces-sary for RANKL-induced and integrin activation–dependent actin ring formation and osteoclastogenesis invitro and in vivo. Whereas TLR2/4/9 and RAGE have beenimplicated as receptors for HMGB1, RAGE seems to par-ticipate in HMGB1-induced actin ring formation and OCmaturation in vitro and in vivo. TLR2/4/9 may also be in-volved in HMGB1-induced osteoclastogenesis; however,the exact role of these receptors in this event remains to befurther studied. Our observations are in line with previousreports that HMGB1 is released from bone cells (both os-teoblasts and osteoclasts),(30,31) provides insight into a func-tion of HMGB1 in regulating osteoclastogenesis, and reveala novel signaling mechanism underlying RANKL regula-tion of OC differentiation. Our work highlights propertiesof HMGB1 as a new member of the group of osteoclasto-

FIG. 7. HMGB1 regulation of actin cytoskeleton reorganization in BMMs in a manner dependent on RAGE. (A) RAGE expressionin BMMs and in differentiating OCs derived from wildtype and RAGE−/− mice was examined by Western blot analysis. (B) Co-immunostaining analysis of RAGE, HMGB1, and F-actin (by phalloidin) distribution in mature OCs. (C) Immunostaining analysis ofF-actin structures in OCs derived from wildtype and RAGE−/− mice. DAPI was used to label nuclei, and GFP was a marker forRAGE−/− cells.(26) (D) Defective HMGB1 signaling in RAGE−/− BMMs cultured onto VN-coated dishes. Pre-OCs from WT andRAGE−/− mice were lifted and replated on VN-coated dishes in the absence of serum. Adherent cells were treated with HMGB1 atthe indicated doses for the indicated times and lysed. Equal amounts of total proteins were immunoblotted with the indicatedantibodies.

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genic cytokines participating in focal osteolysis. In this con-text, because release of HMGB1 is elicited by lipopolysac-charide and multiple inflammatory mediators (IL-1, TNF�,interferon-� and -�), we propose that the HMGB1–RAGEinteraction could represent a final common pathway in in-flammation-associated bone resorption.(9,35,39)

HMGB1, a nuclear nonhistone protein, contains 215amino acids with two DNA-binding domains (HMG boxesA and B) and a negatively charged C terminus. Release ofnuclear HMGB1 from activated macrophages/monocytesor injured cells undergoing necrosis is a prerequisite for itsfunction as a pro-inflammatory cytokine and/or participa-tion in innate immunity.(16) In this context, LPS and TNF�have been found to stimulate HMGB1 release from mac-rophages.(25,40) We provide evidence that RANKL alsostimulates HMGB1 release from macrophages. Althoughthe molecular mechanisms by which RANKL regulatesHMGB1 release and translocation from the nuclear com-partment to the cytoplasm in macrophages are unclear,there are likely to be parallels with the response of HMGB1to these other pro-inflammatory stimuli. LPS and TNF�induce HMGB1 translocation from the nuclear compart-ment to the cytoplasm after acetylation of HMGB1.(16,25,40)

We speculate that RANKL may increase HMGB1 translo-cation by enhancing acetylation of HMGB1 as well. Cyto-plasmic HMGB1 may then be released to the extracellularspace through unconventional vesicles or injured mem-branes in activated macrophages induced by long-termtreatment with cytokines.

Once in the extracellular space, HMGB1 seems to be animportant participant in RANKL-induced osteoclastogen-esis in vitro and in vivo. In support of this view, both block-ing HMGB1 antibody and nicotine inhibited RANKL in-duction of in vitro osteoclastogenesis. Antibody to HMGB1is an efficient and specific inhibitor of extracellularHMGB1 that has been shown to reverse the lethality ofestablished sepsis.(16,25) Nicotine, a cholinergic agonist, hasanti-inflammatory properties because of inhibition of cyto-kine release, including export of HMGB1 from activatedmacrophages.(33) This property of nicotine may underlie itsability to enhance survival in experimental models of severesepsis.(33) Further support for an important role of extra-cellular HMGB1 in RANKL induction of osteoclastogen-

esis is the observation that co-injection of HMGB1 anti-body along with RANKL in vivo attenuated RANKL-induced osteoclastogenesis. The co-participatory roles ofRANKL and HMGB1 in osteoclastogenesis is consistentwith the observation that HMGB1 induction of osteoclastformation requires that precursor cells are pre-exposed to(i.e., primed by) permissive levels of RANKL. These prop-erties of extracellular HMGB1 remarkably resemble char-acteristics of other inflammatory cytokines, such as TNF�and IL-1.(35,39) Whether extracellular HMGB1 is involvedin the osteoclastogenic function of TNF� or LPS is un-known but is certainly a possibility.

In the complex in vivo milieu, HMGB1 may behave simi-larly to other osteoclastogenic cytokines (e.g., TNF�), ex-erting its effects through receptors on stromal cells, osteo-blasts, and/or osteoclasts. We have shown that HMGB1 issufficient to induce in vitro osteoclastogenesis from pre-OCs. These data suggest the possibility that cells of theosteoclast lineage can both release and respond to HMGB1in vivo. However, this does not exclude the potential in-volvement of osteoblasts in the in vivo effects of HMGB1.Indeed, RAGE is expressed on both osteoblasts and OCs.HMGB1-induced in vivo osteoclastogenesis seems to be amore striking effect than suggested by our in vitro studiesassessing its osteoclastogenic activity. The latter observa-tion supports the potential contribution of additional cellsin vivo in supporting HMGB1-induced osteoclastogenesis.

The molecular mechanisms underlying HMGB1 functionare largely unknown. RAGE and TLR2/4/9 have been im-plicated as receptors for HMGB1. For BMMs (and cells ofthe innate immune response in general), it seems that TLR4is critical for HMGB1 early signaling events (e.g., activationof Erk and NF-�B; data not shown), whereas RAGE mayhave a modulatory role in later events that sustain/propagate the host response (data not shown). The lattercould occur independently of TLR2/4, but coordinatelywith integrin activation and actin cytoskeleton organiza-tion. In pre-OCs, RAGE is necessary for integrin signalingand HMGB1 cross-talk to integrin signaling. Such an effect(regulating integrin engagement-induced signaling and ac-tin ring formation) underlies HMGB1 induction of osteo-clastogenesis from pre-OCs in vitro and in vivo. However,mechanisms through which RAGE couples to integrins andregulates actin cytoskeleton remodeling will require furtherstudy to elucidate. Interestingly, HMGB1 has been recentlyfound to induce polarized co-distribution of RAGE with �2integrin in neutrophils.(37) RAGE associates with �2 inte-grins not only in trans (endothelial RAGE interacts withneutrophil �2 integrins),(41) but also in cis (neutrophilRAGE binds to neutrophil �2 integrins).(37) Such interac-tions seem to be needed for inflammation-associated neu-trophil recruitment.(37,41) In this context, we speculate thatRAGE may function as do other immunoglobulin super-family molecules, such as platelet endothelial cell adhesionmolecule 1 (PECAM-1), in modulating the activation stateof integrins(42) through its interaction with �2 integrin and/or actin cytoskeleton reorganization.

In summary, our results support the concept thatHMGB1 has properties of an osteoclastogenic cytokineparticipating in osteoclastogenesis in vitro and in vivo. The

FIG. 8. A working hypothesis for HMGB1 regulation of osteo-clastogenesis. HMGB1 is released from BMMs in response toRANKL stimulation. The extracellular HMGB1, through RAGEin large, regulates osteoclastic actin cytoskeleton remodeling, dif-ferentiation, and function.

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Fig 8 live 4/C

phlogogenic properties of HMGB1, along with its osteo-clastogenic activity, are likely to be highly relevant to thepathogenesis of chronic inflammatory disorders, includingdiabetes, atherosclerosis, and rheumatoid arthritis. Thepro-inflammatory nature of these disorders results in anenvironment in which secretion and/or passive release (i.e.,from necrotic cells) of HMGB1 is likely to be enhanced.HMGB1, through RAGE, may have a synergistic effectwith RANKL or other osteoclastogenic cytokines, regulat-ing actin cytoskeleton reorganization and integrin signaling,thereby driving osteoclastogenesis in these inflammatorydisorders and leading to OC-induced bone loss or inflam-matory osteolysis. Thus, our results suggest that HMGB1-RAGE signaling may provide a pathophysiologically rel-evant pathway for the design of therapeutic approaches totreat disorders associated with inflammation-associatedbone loss.

ACKNOWLEDGMENTS

We are grateful to Drs Bernd Arnold, Peter Nawroth,Angelika Bierhaus, Huan Yang, DJ Ferguson, and DavidStern for reagents. This study was supported by NIH GrantAR048120 to W-CX. ZZ designed, performed, and analyzedthe experiments and wrote the paper. Y-JH, XF, C-YW,and LM provided reagents. C-XX performed some experi-ments. W-CX designed experiments and wrote the paper.

REFERENCES

1. Boyle WJ, Simonet WS, Lacey DL 2003 Osteoclast differen-tiation and activation. Nature 423:337–342.

2. Tanaka S, Takahashi N, Udagawa N, Tamura T, Akatsu T,Stanley ER, Kurokawa T, Suda T 1993 Macrophage colony-stimulating factor is indispensable for both proliferation anddifferentiation of osteoclast progenitors. J Clin Invest 91:257–263.

3. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M,Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A,Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N,Suda T 1998 Osteoclast differentiation factor is a ligand forosteoprotegerin/osteoclastogenesis-inhibitory factor and isidentical to TRANCE/RANKL. Proc Natl Acad Sci USA95:3597–3602.

4. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Bur-gess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H,Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX,Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, DelaneyJ, Boyle WJ 1998 Osteoprotegerin ligand is a cytokine thatregulates osteoclast differentiation and activation. Cell 93:165–176.

5. Teitelbaum SL 2000 Osteoclasts, integrins, and osteoporosis. JBone Miner Metab 18:344–349.

6. Takayanagi H 2005 Mechanistic insight into osteoclast differ-entiation in osteoimmunology. J Mol Med 83:170–179.

7. Kim N, Kadono Y, Takami M, Lee J, Lee SH, Okada F, KimJH, Kobayashi T, Odgren PR, Nakano H, Yeh WC, Lee SK,Lorenzo JA, Choi Y 2005 Osteoclast differentiation indepen-dent of the TRANCE-RANK-TRAF6 axis. J Exp Med202:589–595.

8. Ye H, Arron JR, Lamothe B, Cirilli M, Kobayashi T, ShevdeNK, Segal D, Dzivenu OK, Vologodskaia M, Yim M, Du K,Singh S, Pike JW, Darnay BG, Choi Y, Wu H 2002 Distinctmolecular mechanism for initiating TRAF6 signalling. Nature418:443–447.

9. Lam J, Takeshita S, Barker JE, Kanagawa O, Ross FP, Teitel-baum SL 2000 TNF-alpha induces osteoclastogenesis by direct

stimulation of macrophages exposed to permissive levels ofRANK ligand. J Clin Invest 106:1481–1488.

10. Koga T, Inui M, Inoue K, Kim S, Suematsu A, Kobayashi E,Iwata T, Ohnishi H, Matozaki T, Kodama T, Taniguchi T,Takayanagi H, Takai T 2004 Costimulatory signals mediatedby the ITAM motif cooperate with RANKL for bone homeo-stasis. Nature 428:758–763.

11. Cella M, Buonsanti C, Strader C, Kondo T, Salmaggi A, Col-onna M 2003 Impaired differentiation of osteoclasts in TREM-2-deficient individuals. J Exp Med 198:645–651.

12. Paloneva J, Mandelin J, Kiialainen A, Bohling T, Prudlo J,Hakola P, Haltia M, Konttinen YT, Peltonen L 2003 DAP12/TREM2 deficiency results in impaired osteoclast differentia-tion and osteoporotic features. J Exp Med 198:669–675.

13. Colonna M 2003 TREMs in the immune system and beyond.Nat Rev Immunol 3:445–453.

14. Yagi M, Miyamoto T, Sawatani Y, Iwamoto K, Hosogane N,Fujita N, Morita K, Ninomiya K, Suzuki T, Miyamoto K, OikeY, Takeya M, Toyama Y, Suda T 2005 DC-STAMP is essentialfor cell-cell fusion in osteoclasts and foreign body giant cells. JExp Med 202:345–351.

15. Kim N, Takami M, Rho J, Josien R, Choi Y 2002 A novelmember of the leukocyte receptor complex regulates osteoclastdifferentiation. J Exp Med 195:201–209.

16. Lotze MT, Tracey KJ 2005 High-mobility group box 1 protein(HMGB1): Nuclear weapon in the immune arsenal. Nat RevImmunol 5:331–342.

17. Scaffidi P, Misteli T, Bianchi ME 2002 Release of chromatinprotein HMGB1 by necrotic cells triggers inflammation. Na-ture 418:191–195.

18. Dumitriu IE, Baruah P, Valentinis B, Voll RE, Herrmann M,Nawroth PP, Arnold B, Bianchi ME, Manfredi AA, Rovere-Querini P 2005 Release of high mobility group box 1 by den-dritic cells controls T cell activation via the receptor for ad-vanced glycation end products. J Immunol 174:7506–7515.

19. Dumitriu IE, Baruah P, Bianchi ME, Manfredi AA, Rovere-Querini P 2005 Requirement of HMGB1 and RAGE for thematuration of human plasmacytoid dendritic cells. Eur J Im-munol 35:2184–2190.

20. Nawroth P, Bierhaus A, Marrero M, Yamamoto H, Stern DM2005 Atherosclerosis and restenosis: Is there a role for RAGE?Curr Diab Rep 5:11–16.

21. Lue LF, Yan SD, Stern DM, Walker DG 2005 Preventing ac-tivation of receptor for advanced glycation endproducts in Alz-heimer’s disease. Curr Drug Targets CNS Neurol Disord4:249–266.

22. Schmidt AM, Yan SD, Yan SF, Stern DM 2001 The multili-gand receptor RAGE as a progression factor amplifying im-mune and inflammatory responses. J Clin Invest 108:949–955.

23. Bierhaus A, Humpert PM, Stern DM, Arnold B, Nawroth PP2005 Advanced glycation end product receptor-mediated cel-lular dysfunction. Ann N Y Acad Sci 1043:676–680.

24. Wang H, Ward MF, Fan XG, Sama AE, Li W 2006 Potentialrole of high mobility group box 1 in viral infectious diseases.Viral Immunol 19:3–9.

25. Dumitriu IE, Baruah P, Manfredi AA, Bianchi ME, Rovere-Querini P 2005 HMGB1: Guiding immunity from within.Trends Immunol 26:381–387.

26. Zhou Z, Immel D, Xi CX, Bierhaus A, Feng X, Mei L,Nawroth P, Stern DM, Xiong WC 2006 Regulation of osteo-clast function and bone mass by RAGE. J Exp Med 203:1067–1080.

27. Li J, Wang H, Mason JM, Levine J, Yu M, Ulloa L, Czura CJ,Tracey KJ, Yang H 2004 Recombinant HMGB1 with cytokine-stimulating activity. J Immunol Methods 289:211–223.

28. McHugh KP, Hodivala-Dilke K, Zheng MH, Namba N, Lam J,Novack D, Feng X, Ross FP, Hynes RO, Teitelbaum SL 2000Mice lacking beta3 integrins are osteosclerotic because of dys-functional osteoclasts. J Clin Invest 105:433–440.

29. Constien R, Forde A, Liliensiek B, Grone HJ, Nawroth P,Hammerling G, Arnold B 2001 Characterization of a novelEGFP reporter mouse to monitor Cre recombination as dem-onstrated by a Tie2 Cre mouse line. Genesis 30:36–44.

HMGB1/RAGE REGULATION OF OSTEOCLASTOGENESIS 1095

30. Yang J, Shah R, Robling AG, Templeton E, Yang H, TraceyKJ, Bidwell JP 2007 HMGB1 is a bone-active cytokine. J CellPhysiol 14:730–739.

31. Charoonpatrapong K, Shah R, Robling AG, Alvarez M, ClappDW, Chen S, Kopp RP, Pavalko FM, Yu J, Bidwell JP 2006HMGB1 expression and release by bone cells. J Cell Physiol207:480–490.

32. Matthay MA, Ware LB 2004 Can nicotine treat sepsis? NatMed 10:1161–1162.

33. Wang H, Liao H, Ochani M, Justiniani M, Lin X, Yang L,Al-Abed Y, Metz C, Miller EJ, Tracey KJ, Ulloa L 2004 Cho-linergic agonists inhibit HMGB1 release and improve survivalin experimental sepsis. Nat Med 10:1216–1221.

34. Park JS, Svetkauskaite D, He Q, Kim JY, Strassheim D, Ish-izaka A, Abraham E 2004 Involvement of toll-like receptors 2and 4 in cellular activation by high mobility group box 1 pro-tein. J Biol Chem 279:7370–7377.

35. Wei S, Kitaura H, Zhou P, Ross FP, Teitelbaum SL 2005 IL-1mediates TNF-induced osteoclastogenesis. J Clin Invest115:282–290.

36. Kitaura H, Zhou P, Kim HJ, Novack DV, Ross FP, TeitelbaumSL 2005 M-CSF mediates TNF-induced inflammatory osteoly-sis. J Clin Invest 115:3418–3427.

37. Orlova VV, Choi EY, Xie C, Chavakis E, Bierhaus A, IhanusE, Ballantyne CM, Gahmberg CG, Bianchi ME, Nawroth PP,Chavakis T 2007 A novel pathway of HMGB1-mediated in-flammatory cell recruitment that requires Mac-1-integrin.EMBO J 26:1129–1139.

38. Taniguchi N, Yoshida K, Ito T, Tsuda M, Mishima Y, Furu-matsu T, Ronfani L, Abeyama K, Kawahara K, Komiya S,Maruyama I, Lotz M, Bianchi ME, Asahara H 2007 Stage-

specific secretion of HMGB1 in cartilage regulates endochon-dral ossification. Mol Cell Biol 27:5650–5663.

39. Boyce BF, Li P, Yao Z, Zhang Q, Badell IR, Schwarz EM,O’Keefe RJ, Xing L 2005 TNF-alpha and pathologic bone re-sorption. Keio J Med 54:127–131.

40. Bonaldi T, Talamo F, Scaffidi P, Ferrera D, Porto A, Bachi A,Rubartelli A, Agresti A, Bianchi ME 2003 Monocytic cellshyperacetylate chromatin protein HMGB1 to redirect it to-wards secretion. EMBO J 22:5551–5560.

41. Chavakis T, Bierhaus A, Al-Fakhri N, Schneider D, Witte S,Linn T, Nagashima M, Morser J, Arnold B, Preissner KT,Nawroth PP 2003 The pattern recognition receptor (RAGE) isa counterreceptor for leukocyte integrins: A novel pathway forinflammatory cell recruitment. J Exp Med 198:1507–1515.

42. Wee JL, Jackson DE 2005 The Ig-ITIM superfamily memberPECAM-1 regulates the “outside-in” signaling properties ofintegrin alpha(IIb)beta3 in platelets. Blood 106:3816–3823.

Address reprint requests to:Wen-Cheng Xiong, PhD

Institute of Molecular Medicine and GenomicsDepartment of Neurology

Medical College of GeorgiaAugusta, GA 30912, USAE-mail: wxiong@mcg.edu

Received in original form August 15, 2007; revised form February 21,2008; accepted February, 22, 2008.

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