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M2:03836, revised Regulation of Receptor Activator of NF-κB Ligand-induced Osteoclastogenesis by Endogenous Interferon-ß (IFN-ß) and Suppressors of Cytokine Signaling (SOCS): The Possible Counteracting Role of SOCSs in IFN-ß-inhibited Osteoclast Formation* Toshikichi Hayashi‡§**, Toshio Kaneda‡² **, Yoshiaki Toyama§, Masayoshi Kumegawa‡, and Yoshiyuki Hakeda‡¶ From the ‡Department of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan; §Department of Orthopaedic Surgery, Keio University School of Medicine, Shinanomachi, Tokyo 160-8582, Japan; and ²Department of Pathophysiology, Faculty of Pharmaceutical Sciences, Hoshi University, Ebara, Tokyo 142-8501, Japan Running title: Roles of Endogenous IFN-ß and SOCSs in Osteoclastogenesis ¶To whom correspondence should be addressed: Yoshiyuki Hakeda, Ph.D., Department of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama 350-0283, Japan. Tel: +81-492-79-2769; Fax: +81-492-71-3523; E-mail: [email protected] 1 Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc. JBC Papers in Press. Published on May 22, 2002 as Manuscript M203836200 by guest on March 17, 2018 http://www.jbc.org/ Downloaded from

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Page 1: M2:03836, revised Regulation of Receptor Activator of NF-κB Ligand

M2:03836, revised

Regulation of Receptor Activator of NF-κB Ligand-induced Osteoclastogenesis by Endogenous

Interferon-ß (IFN-ß) and Suppressors of Cytokine Signaling (SOCS):

The Possible Counteracting Role of SOCSs in IFN-ß-inhibited Osteoclast Formation*

Toshikichi Hayashi‡§**, Toshio Kaneda‡†**, Yoshiaki Toyama§, Masayoshi Kumegawa‡, and

Yoshiyuki Hakeda‡¶

From the ‡Department of Oral Anatomy, Meikai University School of Dentistry, Sakado,

Saitama 350-0283, Japan; §Department of Orthopaedic Surgery, Keio University School of

Medicine, Shinanomachi, Tokyo 160-8582, Japan; and †Department of Pathophysiology,

Faculty of Pharmaceutical Sciences, Hoshi University, Ebara,

Tokyo 142-8501, Japan

Running title: Roles of Endogenous IFN-ß and SOCSs in Osteoclastogenesis

¶To whom correspondence should be addressed: Yoshiyuki Hakeda, Ph.D.,

Department of Oral Anatomy, Meikai University School of Dentistry,

Sakado, Saitama 350-0283, Japan.

Tel: +81-492-79-2769;

Fax: +81-492-71-3523;

E-mail: [email protected]

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Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 22, 2002 as Manuscript M203836200 by guest on M

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SUMMARY

Bone resorption and the immune system are correlated with each other, and both are controlled

by a variety of common cytokines produced in the bone microenvironments. Among these

immune mediators, the involvement of type-I interferons (IFNs) in osteoclastic bone resorption

remains unknown. In this study, we investigated the participation of IFN-ß and suppressors of

cytokine signaling (SOCS)-1 and –3 in osteoclastogenesis. Addition of exogenous IFN-ß to

osteoclast progenitors (bone-derived monocytes/macrophages) inhibited their differentiation

toward osteoclasts induced by receptor activator of NF-κB ligand (RANKL) and M-CSF

with/without TGF-ß, which inhibition was associated with down-regulation of the gene

expressions of molecules related to osteoclast differentiation. In addition, RANKL induced the

expression of IFN-ß; and, furthermore, neutralizing antibody against type-I IFNs accelerated the

osteoclast formation, indicating type-I IFNs as potential intrinsic inhibitors. On the other hand,

RANKL also induced the expression of SOCS-1 and –3, suppressors of the IFN signaling.

Pretreatment with RANKL for sufficient time for the induction of SOCSs attenuated

phosphorylation of STAT-1 in response to IFN-ß in osteoclast progenitors, causing a decrease in

the binding activity of nuclear extracts toward the interferon stimulated response element (ISRE).

mRNA levels of STAT-1, STAT-2, and IFN-stimulated gene factor (ISGF)-3γ, comprising

ISGF-3, were not altered by RANKL. Thus, although the inhibitory cytokine such as IFN-ß is

produced in response to RANKL, the inhibition of osteoclastogenesis may be rescued by the

induction of signaling suppressors such as SOCSs.

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INTRODUCTION

Osteoclasts, the cells primarily responsible for bone resorption, are of hemopoietic stem cell

origin. Precursors of osteoclasts have been demonstrated to share common properties with those

of the monocyte/macrophage (M/MØ)1 cell lineage (1, 2). Many systemic hormones and local

cytokines participate in regulating osteoclast differentiation (3, 4). Receptor activator of NF-κB

(RANK) ligand (RANKL) has been identified as the most important and critical molecule for

osteoclast development in cooperation with macrophage colony-stimulating factor (M-CSF),

and the RANKL/RANK system produces an essential signal for osteoclast differentiation in the

interaction between stromal cells and cells of the osteoclast lineage (5- 7). More recently, we

demonstrated that endogenous production of transforming growth factor (TGF-ß) was essential

for osteoclastogenesis as a cofactor with RANKL and M-CSF (8). Most of the osteotropic

factors regulating osteoclast differentiation also affect immune system as well, suggesting a

strong relationship between the two systems. Such immune mediators can be divided into 2

groups regarding their effects on osteoclast differentiation: one comprising stimulatory cytokines

such as interleukin (IL)-1, IL-6, IL-8, IL-11, IL-17, and TNF-α; and the other, inhibitory

cytokines including IL-4, IL-10, IL-12, IL-13, IL-18, and interferon-γ (IFN- γ) (9-19).

Furthermore, the cytokines belonging to each group can also be separated into 2 subclasses based

on their direct or indirect action via stromal cells on cells of the osteoclast lineage.

Among the above inhibitory cytokines, one well-known immune mediator is the T-cell product

IFN-γ, a type-II IFN (20). IFN-γ has been demonstrated from in vivo and in vitro observations

to strongly inhibit osteoclast differentiation and function (21). On the other hand, involvement of

type-I IFNs (IFNα/ß) in osteoclastogenesis remains to be investigated, although the inhibitory

effect has most recently been reported(22). Type-I IFNs are produced by a variety of cell types

in response to viral, bacterial, and protozoan infections, representing a critical link between innate

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and acquired immunity through their multiple effects on natural killer cells and T cells (23-25).

In addition, evidence indicating their actions on other cell types such as monocytes, macrophages

and dendritic cells is accumulating (26-30). Osteoclasts, macrophage, and dendritic cells are

derived from common mononuclear hematopoietic progenitors (1, 2, 31). Furthermore, the

osteoclast formation induced by RANKL requires activation of NF-κB and AP-1 (32, 33), and

the expression of IFN-ß in macrophages also depends on the activation of the transcription factor

followed by the binding to NF-κB and AP-1 sites in the promoter of IFN-ß gene (34-36).

Taken together, the available data suggest that type-I IFNs may potentially influence

osteoclastogenesis in an autocrine manner.

In this study, we found that bone marrow-derived M/MØ as osteoclast progenitors

constitutively expressed type-I IFN and that the expression of IFN-ß was particularly up-

regulated by RANKL. IFN-ß intrinsically inhibited the differentiation of osteoclasts. However,

RANKL simultaneously induced the expression of suppressors of cytokine signaling (SOCS)-1

and SOCS-3 (37), which were earlier shown to block the signaling of IFNs by inhibiting

phosphorylation of STATs by Janus kinases (JAK, 38), thus causing a decrease in IFN-

dependent transcription factor complex [IFN-stimulated gene factor-3 (ISGF-3)] formation

(39). Thus, although the inhibitory cytokines such as type-I IFNs are produced in response to

RANKL, the inhibition of osteoclastogenesis may be rescued by inducing the production of

signaling suppressors such as SOCSs. The results presented here indicate the intimate cross talk

between inhibitory and stimulatory signalings in osteoclast formation.

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EXPERIMENTAL PROCEDURES

Reagents-----Recombinant human M-CSF and recombinant mouse soluble RANKL (sRANKL)

were kindly provided by Morinaga Milk Industry Co. (Tokyo, Japan) and Snow Brand Milk Industry

Co. (Tochigi, Japan). Recombinant human TGF-ß1 was obtained from Genzyme/Techne (Cambridge,

MA). Recombinant mouse IFN-ß were purchased from PBL Biomedical Laboratories (New

Brunswick, NJ). The IFN-ß was titrated with the use of the cytopathic effect inhibition assay (40). In

this antiviral assay, 1 unit/ml of IFN-ß was the quantity necessary to produce a cytopathic effect of

50%. Sheep neutralizing antiserum against mouse IFN-α/ß was purchased from Biosource

International (Camarillo, CA). Anti-STAT-1 and -STAT-2 antibodies were from Santa Cruz

Biotechnologies, Inc. (San Diego, CA). Anti- phospho-STAT-1 antibody was obtained from Cell

Signaling Technology (Beverly, MA).

Isolation of M/MØ-Like Hemopoietic Cells from Mouse Bone Marrow Cells-----Femora and tibiae were obtained

from 4 - 5-week-old ICR mice (Shizuoka Laboratories Animal Center, Shizuoka, Japan), and the connective soft

tissues were removed from the bones. Bone marrow cells were flushed out from the bone marrow cavity, suspended

in α-MEM (ICN Biomedicals, Aurora, OH) supplemented with 10% FBS (Intergen, Purchase,

NY), M-CSF (100ng/ml) and 100 U/ml of penicillin, and cultured in petri-dishes in a humidified

atmosphere of 5% CO2. During 3 days in culture, the cells remaining on the bottom of the dishes

consisted of a large population of adherent M/MØ-like cells expressing MØ-specific antigens

such as Mac-1 and F4/80 and exhibiting phagocytotic activity (8), and only a small population of

non-adherent cells and adherent stromal cells. After removal of nonadherent cells and stromal

cells by washing the dishes with PBS and by subsequent incubation for 5 min in 0.25%

trypsin/0.05% EDTA, respectively, the M/MØ-like hemopoietic cells were harvested in PBS by

vigorously pipetting.

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Osteoclastogenesis from M/MØ -Like Hemopoietic Cells-----Isolated M/MØ-like hemopoietic

cells were seeded at an initial density of 2.5×104/cm2 and cultured in α-MEM/10% FBS/M-CSF (20

ng/ml) with or without sRANKL (50 ng/ml) and/or other cytokines or agents. The culture medium was

exchanged every 2 days. After a culture period of the desired length, the cells were fixed in 10%

formalin and stained for tartrate-resistant acid phosphatase (TRAP) activity with a leukocyte acid

phosphatase kit (Sigma). The total number of cells and numbers of TRAP-positive multinucleate cells

(MNCs) with more than 3 nuclei were counted under a microscope. The TRAP-MNCs were

considered to be osteoclastic cells. Thereafter, nuclei of these cells were stained with propidium

iodide (50 µg/ml) in 0.1% sodium citrate, and the numbers of nuclei in the total cell population, and in

the TRAP-positive MNCs in the culture were counted under a fluorescence microscope. The total

number of nuclei was considered to represent the rate of cell division. A fusion index was calculated

as the percentage of the number of nuclei in TRAP-positive MNCs per those in total cells; and this

value was considered to be the percent of cells that participated in cell fusion, indicating osteoclast

maturation.

Reverse Transcription-Polymerase Chain Reaction (PCR)-----Total RNA (1 µg) extracted from

cells in the culture was used as a template for cDNA synthesis. cDNA was prepared by use of a

Superscript II preamplification system (Life Technologies, Gaithersburg, MD). Primers were

synthesized on the basis of the reported mouse cDNA sequences for TRAP, integrin αv, integrin ß3,

calcitonin receptor, cathepsin K, CD14, interferon-α and -ß, SOCS-1, SOCS-3, STAT-1, STAT-2,

and ISGF-3γ. Sequences of the primers used for PCR were as follows: TRAP forward, 5-

CACGATGCCAGCGACAAGAG-3; TRAP reverse, 5-TGACCCCGTATGTGGCTAAC-3;

integrin αv forward, 5-GCCAGCCCATTGAGTTTGATT-3; integrin αv reverse, 5-

GCTACCAGGACCACCGAGAAG-3; integrin ß3 forward, 5-

TTACCCCGTGGACATCTACTA-3; integrin ß3 reverse, 5-

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AGTCTTCCATCCAGGGCAATA-3; cathepsin K forward, 5-

GGAAGAAGACTCACCAGAAGC-3; cathepsin K reverse, 5-

GTCATATAGCCGCCTCCACAG-3; calcitonin receptor forward, 5-

ACCGACGAGCAACGCCTACGC-3; calcitonin receptor reverse, 5-

GCCTTCACAGCCTTCAGGTAC-3; CD14 forward, 5-AAGTTCCCGACCCTCCAAGTT-3;

CD14 reverse, 5-CTGCCTTTCTTTCCTTACATC-3; IFN-ß forward, 5-

CTTCTCCACCACAGCCCTCTC-3; IFN-ß reverse, 5-CCCACGTCAATCTTTCCTCTT-3;

IFN-α (including consensus sequence of IFN-αs (41)) forward, 5-

ATGGCTAGRCTCTGTGCTTTCCT-3; IFN-α reverse, 5-

AGGGCTCTCCAGAYTTCTGCTCTG-3; SOCS-1 forward, 5-

AGGATGGTAGCACGCAACCAGGT-3; SOCS-1 reverse, 5-

GATCTGGAAGGGGAAGGAACTCAGGTA-3; SOCS-3 forward, 5-

GCCATGGTCACCCACAGCAAGTT-3; SOCS-3 reverse, 5-

AAGTGGAGCATCATACTGATCCAGGA-3; STAT-1 forward, 5-

CGAAGAGCGACCAAAAACAG-3; STAT-1 reverse, 5-TGCTGGAAGAGGAGGAAGGT-

3; STAT-2 forward, 5-TTTGCTGGGACCTGTGCTCA-3; STAT-2 reverse, 5-

TGGGTTCTCGGGGATGTTCT-3; ISGF-3γ forward, 5-GAACCCTCCCTAACCAACCA-3;

ISGF-3γ reverse, 5-AGGTGAGCAGCAGCGAGTAG-3; ß-actin forward, 5-

TCACCCACACTGTGCCCATCTAC-3’; ß-actin reverse, 5-

GAGTACTTGCGCTCAGGAGGAGC-3’. Amplification was conducted for 22-32 cycles, each

of 94oC for 30s, 58oC for 30s, and 72 oC for 1 min in a 25-µl reaction mixture containing 0.5 µl

of each cDNA, 25 pmol of each primer, 0.2 mM dNTP, and 1 U of Taq DNA polymerase

(Qiagen, Valencia, CA). After amplification, 15 µl of each reaction mixture was analyzed by

1.5% agarose gel electrophoresis, and the bands were then visualized by ethidium bromide

staining.

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Western blot analysis-----After various treatments, the cells were washed with PBS, scraped into a

solution consisting of 10 mM sodium phosphate (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium

deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM aminoethyl-benzenesulfonyl fluoride, 10 µg/ml

leupeptin, and 10 µg/ml aprotinin, and sonicated for 15 seconds. The protein concentration in the cell

lysate was measured with a bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL).

Each sample containing equal amounts of protein was subjected to 10% SDS-polyacrylamide gel

electrophoresis (PAGE), and the proteins separated in the gel were subsequently electrotransferred

onto a polyvinylidene difluoride membrane. After having been blocked with 5% skim milk, the

membrane was incubated with anti-STAT1 and –phospho-STAT1 antibodies, and subsequently with

peroxidase-conjugated anti- mouse or anti-rabbit IgG antibody. Immunoreactive proteins were

visualized with Western blot chemiluminescence reagents (Amersham Co., Arlington Heights, IL)

following the manufacturer’s instructions.

Electrophoretic mobility shift assay (EMSA)----- After M/MØ-like hemopoietic cells had been

incubated with or without sRANKL for 8 h, the cells were treated with IFN-ß for the indicated

times, then washed twice with ice-cold PBS, incubated for 10 min on ice in 1 ml of ice-cold

buffer A [10 mM Hepes (pH 7.4), 10 mM KCl, 0.1 mM EDTA, 0.1% NP-40, 1 mM DTT, 1 mM

p-ABSF, 2 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µg/ml leupeptin], and scraped into the buffer.

The cell lysates were incubated further for 10 min on ice and then transferred to tubes. The

nuclei obtained by centrifugation for 1 min at 5000 X g were extracted by a 30-min incubation in

ice-cold buffer C, consisting of 50 mM Hepes (pH 7.5), 420 mM KCl, 0.1 mM EDTA, 5 mM

MgCl2, 20 % glycerol, 1 mM DTT, 1 mM p-ABSF, 2 µg/ml aprotinin, 2 µg/ml pepstatin, and 2

µg/ml leupeptin. Then, the extracts were centrifuged at 14,000 X g for 30 min, and the

supernatants were used for the EMSA. Double-stranded oligonucleotides containing an IFN-

stimulated response element (ISRE, 5’-

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GATCCATGCCTCGGGAAAGGGAAACCGAAACTGAAGCC-3’, element underlined (42)) were end-radiolab

polynucleotide kinase (Promega, Madison, WI) according to the manufacturers instruction, and

combined and incubated with 1 µg of nuclear extract in binding buffer [10 mM Tris-HCl (pH

7.5), 4% glycerol, 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol] for 30

min at room temperature. The specificity of the reaction was confirmed by competition with a

50-fold molar excess of nonlabeled oligonucleotides. The protein-DNA complexes were

resolved by 7.2% PAGE in 0.5×TBE buffer and visualized by autoradiography. In addition, the

nuclear extracts were incubated with anti-STAT-1 and anti-STAT2 for 30 min on ice after

binding to the oligonucleotides, and then were subjected to PAGE.

RESULTS

Exogenous IFN-ß Suppresses Osteoclastogenesis Induced by M-CSF and RANKL----When bone marrow-

derived M/MØ were incubated with M-CSF and sRANKL, and particularly when TGF-ß was also present, most of

the cells differentiated into cells of the osteoclast lineage and became mature osteoclasts, as described previously (8).

Addition of exogenous IFN-ß reduced number of osteoclastic TRAP-positive MNCs formed in these cultures in a

dose-related manner (Fig. 1), whereas the total nuclear number was not changed (data not shown). The fusion index

of TRAP-positive MNCs, defined as the percent of cells participating in the fusion, was suppressed by IFN-ß in

parallel with the inhibition of the formation of TRAP-positive MNCs. Semiquantitative PCR analysis revealed that

treatment with M-CSF and sRANKL for 6 days or with M-CSF, TGF-ß and sRANKL for 4 days induced an

increase in the levels of mRNA for TRAP, cathepsin K, calcitonin receptor, and integrinαv and ß3, all of which

are abundantly expressed in osteoclasts and considered as marker molecules for osteoclast

differentiation (Fig. 2). IFN-ß decreased the induced levels of mRNA for these molecules (Fig.

2). These results indicate that exogenous IFN-ß strongly suppressed the process of osteoclast

differentiation from the progenitors, bone marrow-derived M/MØ cells.

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Inhibition of Osteoclastogenesis by Endogenous Type-I IFNs----Type-I IFNs were

demonstrated earlier to be produced by a variety of cell types including M/MØ cells (27, 30),

suggesting the potential ability of osteoclast progenitors to produce type-I IFNs in osteoclast

progenitors. Thus, we examined the expression of type-I IFNs in osteoclast progenitors in

response to sRANKL. The messenger RNA level of IFN-αs was did not change consistently.

(data not shown).However, the level of IFN-ß mRNA significantly increased as early as 1 h after

exposure to sRANKL (Fig. 3A). The increase reached a maximum after 5 h of treatment, and the

increase was not affected by adding cycloheximide (Fig. 3B), indicating that sRANKL strongly

induced the expression of IFN-ß in osteoclast progenitors and that the induction was not

mediated by any other RANKL-induced proteins but occurred via the direct action of RANKL.

Thus, we next examined how the endogenous type-I IFNs influenced osteoclastogenesis from

osteoclast precursors. When anti-IFNα/ß neutralizing antibody was added to cultures of bone

marrow-derived M/MØ cells in the presence of M-CSF and sRANKL with or without TGF-ß,

formation of osteoclastic TRAP-positive MNCs in the cultures was accelerated compared with

that in the absence of the antibody (Fig. 4A). The acceleration depended on the dose of the

neutralizing antibodies (Fig. 4B and 4C), suggesting that the osteoclast progenitors endogenously

produced type-I IFNs depending on the signal of RANKL and that these cytokines inhibited the

osteoclastogenesis induced by RANKL.

Induction of SOCS-1 and –3 by sRANKL in Osteoclastogenesis----These above results

highlight the discrepancy between the induction of osteoclast differentiation and the production

of the inhibitory cytokines such as type-I IFNs, both of which were mediated by RANKL

signaling, thus leading to a possibility that there may be a mechanism for negating the inhibitory

action of endogenous IFNα/ß on osteoclastogenesis. So, next we focused on SOCSs as negative

regulators of the action of IFNα/ß. Eight isoforms of SOCS were earlier identified as a family of

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intracellular proteins controlling the magnitude and/or duration of signals propagated by diverse

cytokine receptors by suppressing their signal transduction process (37). We examined effect of

sRANKL on the expression of SOCS-1 and –3 mRNAs in osteoclast progenitors by

semiquantitative RT-PCR. Messenger RNA levels of SOCSs in bone marrow-derived M/MØ

cells as osteoclast progenitors were time-dependently increased by treatment with sRANKL.

The magnitude of the induction of SOCS-3 mRNA was greater than that of SOCS-1; and the

increase was observed as early as 1 h after adding sRANKL to the cultures, reached a maximum

at 5 h, and continued until 22 h, although the levels gradually decreased after 8 h oftreatment

(Fig. 5A). RANKL-induced SOCS-1 expression was delayed slightly compared with that of

SOCS-3. In addition, the inductive effect of sRANKL was not reduced by the addition of

cycloheximide (Fig. 5B), indicating the direct induction by sRANKL.

RANKL-Induced SOCSs Counteract Inhibitory Effects of IFNα/ß on Osteoclastogenesis----

The signal of type-I IFNs is transmitted through the IFN-α/ß receptor (IFNAR) expressed on the

plasma membrane of target cells into an intracellular transcription complex (ISGF-3) composed

of STAT-1, STAT-2, and ISGF-3γ; and SOCSs inhibit the signaling (39). Thus, we examined

whether the RANKL-induced SOCSs would reduce the IFNα/ß signaling in osteoclast

progenitors. IFN-ß stimulated the binding of nuclear proteins to an oligonucleotide sequence

containing the interferon-stimulated response element (ISRE), the increase being significant as

early as 20 min after the addition of IFN-ß (Fig. 6A). When the probe and the nuclear extracts

were incubated with anti-STAT-1 and anti-STAT-2 antibodies, the complex was further shifted

to the upper position and decreased, respectively, in the gel (Fig. 6B). However, when the

progenitors were pretreated for 8 h with sRANKL prior to the treatment with IFN-ß, the IFN-ß-

induced complex with the nuclear extracts and ISRE probe was significantly reduced, suggesting

a possible mechanism for suppression of the IFN-ß signaling. The duration of the pretreatment

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was enough for induction of SOCS expression by RANKL as demonstrated in Fig. 5. As SOCS-

1 and SOCS-3 have been demonstrated to inhibit the phosphorylation of STAT-1 and STAT-2

by JAKs, which is essential for formation of the ISGF-3 complex (39), we examined the effect of

sRANKL on the phosphorylation of STAT-1 in osteoclast progenitors. IFN-ß increased the

level of phosphorylated STAT-1 in the progenitors, the increase being significant as early as 15

min after exposure to IFN-ß, with a maximal stimulation at 45 min (Fig. 7). The time-dependent

profile was consistent with that of formation of the complex between nuclear extracts and ISRE

probe. Pretreatment with sRANKL for 8 h reduced the levels of the phosphorylated STAT-1,

whereas the total protein level of STAT-1 was not changed (Fig. 7). Further, the levels of

STAT-2 and ISGF-3γ mRNAs were also unaltered by exposure to sRANKL (Fig. 8). These

results suggest that the IFN-ß signaling is reduced by the blocking of the phosphorylation of

STATs due to the increase in SOCSs induced by RANKL. Thus, RANKL-induced SOCSs

appear to counteract the inhibitory effects of IFNα/ß on osteoclastogenesis.

DISCUSSION

In this study, we demonstrated that IFN-ß endogenously produced in osteoclast progenitors

potentially depressed the differentiation toward mature osteoclasts induced by RANKL and M-

CSF. However, expression of SOCSs, particularly that of SOCS-3, was simultaneously induced

by RANKL. When the progenitors were pretreated with RANKL for a period of time sufficient

to induce SOCS expression, the signaling of IFN-ß was significantly suppressed, the suppression

being mediated by the inhibition of phosphorylation of STAT-1, which resulted in impairment of

the ISGF-3 complex formation responsible for the signal transduction of type-I IFNs. Thus, the

isolated osteoclast progenitors expressed the immune mediators and their suppressors, in the

balance of which the osteoclast differentiation could progressively proceed.

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The expression of type-I IFNs is stimulated by a variety of extracellular stimuli such as viral

and bacterial infections and cytokines involved in host defense. Various hemopoietic cells

including cells belonging to the monocyte and macrophage lineage express type-I IFNs

according to immune system, and their proliferation and differentiation are widely regulated by

type-I IFNs (26-29, 43). A potent inducer of type-I IFN production in macrophages is bacterial

lipopolysaccharide (LPS, 44-46). LPS has been demonstrated to induce the expression of IFN-ß

in macrophages, mediating the induction of inducible nitric-oxide synthase (iNOS; 45, 46). The

LPS action is mediated through a Toll-like receptor-dependent signal pathway finally by

activation of NF-κB and AP-1(47-49), suggesting each or both transcription factors-dependent

type-I IFN expression. Similarly to the above case of macrophages, Takayanagi et al. (22) have

most recently demonstrated the induction of IFN-ß by RANKL through c-Fos pathway in

osteoclast precursors, although they did not define the contribution of NF-κB to the induction.

Their observations are consistent with our findings presented in this study. In fact, promoter of

the IFN-ß gene contains AP-1 and NF-κB binding sites, which may mediate the RANKL-

induced IFN-ß expression (22, 34-36).

Studies on the involvement of IFNs in osteoclastogenesis have been restricted to the action of

IFN-γ produced by T cells, indicating the direct and inhibitory effect on osteoclast recruitment

and function (21, 50-54). Mice lacking IFN-γ receptor (IFN-γR) showed a notable exacerbation

of osteoclast formation and severe bone destruction when LPS was locally microinjected into

calvariae of the knockout mice (21). On the other hand, the involvement of type-I IFNs in

osteoclastogenesis has so far been less studied. However, mice lacking IFNAR1, a receptor of

IFN-a/ß, or IFN-ß showed a nobable increase in osteoclast formation and a reduction in

trabecular bone mass, respresenting osteopenia (22). Consistently, we also demonstrated in this

study the direct inhibition by endogenous IFN-ß of osteoclast differentiation in vitro. It should

be noted that the bone mass in IFN-γR knockout homozygotes was the same level as that of the

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heterozygotes and that the nobable destruction of bone mass in the mutant mice was T-cell-

dependent (21). On the other hand, the reduction in bone mass in IFNAR1-/- or IFN-ß-/- mice

was observed without any stimuli (22). Taken together, these results suggest that type-I IFNs are

involved in physiological process of osteoclastogenesis and that IFN-γ may pathologically

participate in the process.

RANKL mediates an essential signal to osteoclast progenitors for their differentiation into

mature and functional osteoclasts (7). As shown in this study, however, RANKL simultaneously

induces the production of inhibitory cytokines such as type-I IFNs in the osteoclast progenitors.

Such endogenous actions of IFNs may represent a feedback regulation of osteoclastogenesis as

demonstrated in a most recent study (22). If so, the differentiation from the progenitors would be

blocked by the endogenous inhibitory cytokines. Therefore, we assume that there would be a

mechanism to avoid such possible feedback inhibition in process of osteoclast differentiation.

Signals of type-I IFNs are mediated through JAK/STAT systems following the binding of the

ligands to their receptors (IFNARs, 55). Several mechanisms of inhibition have been

characterized that negatively regulate the JAK/STAT activation pathway. These include the

family of protein inhibitors of activated STATs (PIAS), of which there are 4 isoforms that bind to

activated STATs and inhibit DNA binding (56). However, as presented here, phosphorylation of

STAT-1 was inhibited by pretreatment with RANKL, meaning a blockade of activation of

STATs, simultaneously with a reduction in the binding of nuclear extracts to ISRE probes. These

results imply a PIAS-independent mechanism. SOCS, also called JAK-binding protein (JAB)

and CIS (cytokine inducible SH2-protein), was originally discovered as the product of an

immediate early gene induced by stimulation with IL-3 and erythropoietin; and so far 8 isoforms

have been cloned (37). Among these SOCSs, SOCS-1 and SOCS-3 have been extensively

studies for their actions, and are considered to be potent negative feedback regulators of a number

of cytokines and growth factors whose signals are mediated by the JAK/STAT system (38).

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These SOCSs block the phosphorylation of STATs by inhibiting the kinase activity of JAKs,

causing a blockade of STAT activation (57, 58). In this study, we showed that RANKL induced

the expression of SOCS-1 and SOCS-3 in osteoclast progenitors. Pretreatment with RANKL for

a period sufficient for the induction of SOCSs attenuated phosphorylation of STAT-1 in response

to IFN-ß in the osteoclast progenitors. In parallel with the inhibition of STAT-1

phosphorylation, the pretreatment with RANKL reduced the binding of nuclear extracts to ISRE

sites. Although we have not ruled out the possibility that dephosphorylation of STAT1 by SH2

domain-containing tyrosine phosphatase 1 is involved in the inhibition of JAK/STAT signaling

(59), the RANKL-induced SOCS expression would be one of the mechanisms for the blockade

of the signaling in osteoclastogenesis. To our knowledge, this is the first report indicating the

involvement of SOCSs in osteoclast recruitment. However, we should note that the inhibition of

IFN signals by the suppressors was not complete, because the neutralization by anti-type-I

antibody still had an effect on the formation of osteoclasts.

Expression of SOCS genes is controlled at the transcriptional level, with the STAT family of

transcription factors being the major mechanism of SOCS induction (60, 61), representing a

classical negative feedback mechanism. On the other hand, the JAK/STAT-independent SOCS

gene activation has also been demonstrated. For instance, LPS and CpG-DNA stimulated the

expression of the SOCS gene in macrophages, and thereby STAT-1 activation in response to

IFN-γ was suppressed (62, 63). Similarly, IL-1ß induction of SOCS-3 has been demonstrated

to mediate growth hormone resistance in primary rat liver hepatocytes (64). We found that the

RANKL induction of SOCS gene expression is caused by the direct action of RANKL, not

mediated by newly synthesized protein in osteoclastogenesis. The signal of RANKL in

osteoclast progenitors is mediated by the activation of NF-κB and AP-1 as occurs with LPS and

IL-1ß do so, and the promoter region of SOCS-3 gene contains NF-κB and AP-1 binding sites

in addition to STAT response elements (60). Taken together, the data suggest that the induction

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of SOCS expression by RANKL in osteoclast progenitors presented here may result from an NF-

κB-dependent mechanism and not from a STAT-dependent one.

In conclusion, although the inhibitory cytokine such as type-I IFNs is produced in response to

RANKL, the potential inhibition of osteoclastogenesis may be rescued by the induction of

signaling suppressors such as SOCSs, thus allowing the differentiation of osteoclasts to proceed.

These results presented here indicate the intimate cross talk between inhibitory and stimulatory

cytokines in osteoclast formation.

Acknowledgment----We thank Drs. K. Yamaguchi (Snow Brand Milk Industry Co.) and M.

Yamada (Morinaga Milk Industry Co.) for their generous gifts of recombinant sRANKL and

recombinant M-CSF, respectively.

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FOOTNOTES

*This work was supported by a grant-in-aid for scientific research from the Japanese Ministry of Education,

Science, Sports, and Culture (12671780 and 1247038).

**Both authors contributed equally to this work.

1The abbreviations used are : M/MØ, monocyte/macrophage; RANK, receptor activator of NF-

κB; RANKL, RANK ligand; M-CSF, macrophage colony-stimulating factor; TGF-ß, transforming

growth factor; IL, interleukin; IFN, interferon; SOCS, suppressor of cytokine signaling; ISGF-3,

IFN-stimulated gene factor-3; TRAP, tartrate-resistant acid phosphatase; MNCs, multinucleate

cells; ISRE, interferon-stimulated response element; IFNAR, IFN-α receptor; TNF-α, tumor

necrosis factor-α.

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FIGURE LEGENDS

FIG. 1. Inhibitory effect of exogenous IFN-ß on formation of osteoclasts from their precursors.

The osteoclast precursors (M/MØ cells) was isolated after 3-day treatment of bone marrow cells

with M-CSF (100 ng/ml), and the isolated M/MØ (5 x 104 cells/each well of 24-wells plate)

were cultured with various concentrations of recombinant IFN-ß in the presence of M-CSF (20

ng/ml) and sRANKL (50 ng/ml) for 7 days (A, C), or in the presence (B, D) of M-CSF (20

ng/ml), sRANKL (50 ng/ml), and TGF-ß1 (1 ng/ml) for 5 days. At the end of the culture period,

the cells were stained for nuclei and TRAP activity. Number of TRAP-positive MNCs per well

(A, B), and total number of nuclei and number of TRAP-positive MNCs were counted. Fusion

index (C, D) indicates the rate of TRAP-positive osteoclast precursors that participated in cell

fusion resulting in mature osteoclasts. The experiments were performed four times and the

reproducibility was confirmed. Values are means + SE for 3 cultures in a representative

experiment.

FIG. 2. Semiquantitative RT-PCR analysis for the expression of various mRNAs. Isolated

M/MØ cells were treated for 6 days with M-CSF (20 ng/ml, M), M-CSF + sRANKL (50 ng/ml.

MR), M-CSF + sRANKL + IFN-ß (10 U/ml, MRI), and M-CSF + IFN-ß (MI), or for 4 days

with M-CSF+ TGF-ß (1 ng/ml, MT), M-CSF + TGF-ß + sRANKL (MTR), M-CSF + TGF-ß

+ sRANKL + IFN-ß (MTRI), and M-CSF + TGF-ß + IFN-ß (MTI), and then the total RNA in

each culture was extracted. Primers used were designed for mouse genes of TRAP, cathepsin K,

calcitonin receptor, integrins αv and ß3 and ß-actin. Each cDNA was amplified for the number

of PCR cycles indicated in parentheses.

FIG. 3. Alteration in mRNA of type-I IFNs expressed in osteoclast progenitors in response to

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sRANKL. Isolated M/MØ cells were pretreated with M-CSF (20 ng/ml) for 1 day prior to

incubation with (R) or without (C) sRANKL (50 ng/ml) for the indicated times (A), and

thereafter the total RNA in each culture was extracted. Primers used were designed for mouse

genes of mouse IFN-ß, and ß-actin. B, cycloheximide (3 µg/ml) was added or not to the

cultures at 3 h prior to 5h-treatment with sRANKL. Numbers in parentheses indicate the

number of PCR cycles.

FIG. 4. Effect of neutralizing anti-IFNα/ß antibody on osteoclast generation. A, Osteoclast

precursors (M/MØ cells) were isolated after a 3-day treatment of bone marrow cells with M-

CSF (100 ng/ml), and the isolated M/MØ (5 x 104 cells/each well of 24-wells plate) were

cultured for the indicated periods with neutralizing anti-IFN α /ß antiserum [closed symbols, 40

neutralization units (N.U)/ml] or the same concentration of normal sheep serum (open symbols)

in the presence(triangles) of M-CSF (20 ng/ml) and sRANKL (50 ng/ml), or in the presence

(circles) of M-CSF (20 ng/ml), sRANKL (50 ng/ml) and TGF-ß (1 ng/ml). One N.U is defined

as the amount of antiserum required to neutralize 50% of the activity of 1 unit of mouse IFN-α/ß.

B and C, the isolated M/MØ cells were cultured with various concentrations of neutralizing anti-

IFN-α/ß antiserum (shaded bars) or normal sheep serum (open bars) in the presence(B) of M-

CSF (20 ng/ml) and sRANKL (50 ng/ml) for 6 days, or in the presence (C) of M-CSF (20

ng/ml), sRANKL (50 ng/ml), and TGF-ß (1 ng/ml) for 4 days. At the end of the culture period,

the cells were stained for TRAP activity; and the number of TRAP-positive MNCs per well was

then counted. The experiments were performed three times and the reproducibility was

confirmed. Values are means + SE for 3 cultures in a representative experiment.

FIG. 5. Increase in mRNA of SOCS-1 and -3 expressed in osteoclast progenitors in response

to sRANKL. Isolated M/MØ cells were pretreated with M-CSF (20 ng/ml) for 1 day prior to

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incubation with (R) or without (C) sRANKL (50 ng/ml) for the indicated times (A), and

thereafter total RNA in each culture was extracted. Primers used were designed for mouse genes

of mouse SOCS-1, SOCS-3 and ß-actin. B, cycloheximide (3 µg/ml) was added or not to the

cultures at 3 h prior to 5h-treatment with sRANKL. Numbers in parentheses indicated the

number of PCR cycles.

FIG. 6. Electrophoretic mobility shift assay targeting ISRE site. Isolated M/MØ cells were

supported in M-CSF (20 ng/ml) for 1 day, and then were pretreated or not with sRANKL (50

ng/ml) for 8 h. Thereafter, the cells were treated with IFN-α/ß (10 U/ml) for the indicated times

(A). At the end of the treatment, nuclear extracts were prepared. The extracts were incubated

with 32P-labeled oligonucleotide probes for ISRE, and the mixtures were then subjected to

PAGE (6% gel). B, The extract from the cells treated for 60 min with sRANKL without the

pretreatment with sRANKL (lane 4 from left) were incubated or not with the labeled IRSE probes

in the presence or absence of anti-STAT-1 or anti-STAT-2 antibody.

FIG. 7. Phosphorylation of STAT-1 in response to IFN-ß in osteoclast progenitors. Isolated

M/MØ cells were supported in M-CSF (20 ng/ml) for 1 day, and then were pretreated or not with

sRANKL (50 ng/ml) for 8 h. Thereafter, the cells were treated with IFN-α/ß (10 U/ml) for the

indicated times. At the end of the treatment, the cells were extracted. The cell extracts were

subjected to Western blotting analysis with anti-phospho-STAT-1 (upper panel) and anti-

STAT-1 antibodies (lower panel).

FIG. 8. Expression of mRNA of STAT-1, STAT-2 and ISGF-3γ in osteoclast progenitors in

response to sRANKL. Isolated M/MØ cells were pretreated with M-CSF (20 ng/ml) for 1 day

prior to incubation with (R) or without (C) sRANKL (50 ng/ml) for indicated times, and

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thereafter total RNA in each culture was extracted. Primers used were designed for mouse genes

of mouse STAT-1, STAT-2, ISGF-3γ and ß-actin. Numbers in parentheses indicate the

number of PCR cycles.

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Yoshiyuki HakedaToshikichi Hayashi, Toshio Kaneda, Yoshiaki Toyama, Masayoshi Kumegawa and

counteracting role of SOCSs in IFN-ß inhibited osteoclast formationendogenous interferon-ß and suppressors of cytokine signaling (SOCS): The possible

Regulation of receptor activator of NF-kB ligand-induced osteoclastogenesis by

published online May 22, 2002J. Biol. Chem. 

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