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Endocrinology Vol. 145, No. 6 2721-2729
Copyright © 2004 by The Endocrine Society

Lipopolysaccharide-Induced Osteoclastogenesis in Src Homology 2-Domain Phosphatase-1-Deficient Viable Motheaten Mice

Shin-Ichi Hayashi, Motokazu Tsuneto, Takayuki Yamada, Michinari Nose, Miya Yoshino, Leonard D. Shultz and Hidetoshi Yamazaki

Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science (S.I.H., M.T., T.Y., M.N., M.Y., H.Y.); Division of Orthopedic Surgery, Department of Medicine of Sensory and Motor Organs (M.N.), Faculty of Medicine, Tottori University, and Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science (H.Y.), Yonago, Tottori 683-8503, Japan; and The Jackson Laboratory (L.D.S.), Bar Harbor, Maine 04609

Address all correspondence and requests for reprints to: Dr. Shin-Ichi Hayashi, Division of Immunology, Department of Molecular and Cellular Biology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-Machi, Yonago, Tottori 683-8503, Japan. E-mail: shayashi{at}grape.med.tottori-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteoclasts are hemopoietic cells that participate in bone resorption and remodeling. Receptor activator of nuclear factor-{kappa}B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) are critical for development of osteoclasts. The Toll-like receptor (TLR) family shares some of the downstream signaling with RANK. The TLR4 ligand, lipopolysaccharide (LPS), is reported to accelerate bone lysis; however, signaling via TLRs has never been reported to induce osteoclastogenesis without RANKL. In this study we showed that significant numbers of mature osteoclasts were generated from protein tyrosine phosphatase Src homology 2-domain phosphatase-1-defective Hcphme-v/Hcphme-v (mev/mev) bone marrow cells in the presence of M-CSF and LPS without addition of RANKL in culture. This M-CSF plus LPS-induced osteoclastogenesis was not inhibited by an anti-TNF{alpha} antagonistic antibody or by osteoprotegerin, a decoy receptor for RANKL. The replacement of RANKL by TLR ligands only occurred with LPS. Other ligands, a peptidoglycan for TLR2 or an unmethylated CpG oligonucleotide for TLR9, did not support osteoclast generation. The osteoclast precursors as well as RANKL-responsive osteoclast precursors were present in the Kit-positive cell-enriched fraction of bone marrow cells. Although mev/mev bone marrow cells required a comparable concentration of RANKL or TNF{alpha} as wild-type cells for the initiation of osteoclastogenesis, the numbers of multinucleated osteoclasts in mev/mev bone marrow cultures were significantly increased by the equivalent dose of RANKL or TNF{alpha} in the presence of M-CSF. These results indicate that a defect of Src homology 2-domain phosphatase-1 function not only accelerates physiological osteoclast development by RANKL/RANK, but also acquires a novel pathway for osteoclastogenesis by LPS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SIGNALING VIA MACROPHAGE-colony-stimulating factor (M-CSF) and receptor activator of nuclear factor- {kappa}B (NF-{kappa}B) ligand (RANKL) plays an essential role for the development of osteoclast precursors (OCPs) into tartrate- resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) that resorb and remodel bones (1, 2, 3, 4). Functional M-CSF-deficient Csf1op/Csf1op (op/op) mice lack mature osteoclasts, resulting in severe osteopetrosis (5, 6). The op/op mice carrying the Bcl2 transgene are cured of the disease, and aged op/op mice show spontaneous reversal of osteopetrosis (7, 8). Therefore, signaling through the M-CSF receptor (Fms) is thought to function as a cell survival signal, and vascular endothelial growth factor partially replaces its function (9). Viable motheaten (mev/mev) mice mutated at the Src homology 2-domain phosphatase-1 (SHP-1) protein tyrosine phosphatase (Hcph) locus (10, 11) show accelerated osteoclastogenesis in vitro and in vivo (12, 13). Especially, numbers of TRAP+ MNCs were significantly increased (12, 13). In mice that are doubly homozygous for mutations at the Csf1 and Hcph loci (op/op mev/mev mice) (12), partial, but significant, bone marrow (BM) formation was observed, indicating that the signaling via the receptor tyrosine kinases is regulated negatively by SHP-1 (10, 11).

RANK and its ligand, RANKL, are members of TNF receptor and TNF superfamilies, respectively (14, 15). Targeted mutation in either gene prevents the development of osteoclasts, resulting in the development of osteopetrosis (16, 17). Therefore, RANK/RANKL signaling is thought to be essential for osteoclastogenesis. However, several recent studies using RANK-knockout (RANK-KO) mice showed that osteoclastogenesis was induced without RANK/RANKL signaling (18, 19). Although it is still not clear whether RANK/RANKL signaling is totally absent (20), mouse TNF{alpha} was reported to induce osteoclastogenesis in vivo and in vitro in the presence of M-CSF (18, 21).

Signaling via members of the Toll-like receptor (TLR) superfamily shares some of the downstream pathways, such as TNF receptor-associated factor 6 (TRAF6), NF-{kappa}B, and MAPK, with RANK (22, 23, 24). Recently, SHP-1 was reported to interact with TRAF6 (25). Although lipopolysaccharide (LPS) is known to accelerate the bone lysis (26, 27) and promote the survival of osteoclasts (28), signaling via TLRs has never been reported to induce mature osteoclasts without RANKL (4). We reported that in vivo LPS injection increased the generation of BM osteoclast precursors, but the maturation from even these treated cells into TRAP+cells required RANKL and M-CSF (29). Moreover, a cloned macrophage-like cell that lacks the p53 gene differentiates into mature osteoclasts without M-CSF; however, RANKL is essential for its maturation (29, 30, 31). Therefore, signaling via TLRs may be insufficient as a substitute for RANK signaling.

In this study we showed that SHP-1 deficiency enabled BM cells treated with LPS plus M-CSF in the absence of RANKL to give rise to mature osteoclasts that resorb bone. The osteoclastogenesis does not depend on the production of RANKL or TNF{alpha}. These results indicate that SHP-1 regulates downstream signaling for osteoclastogenesis through not only receptor tyrosine kinases, but also members of the TNF receptor superfamily.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
C57BL/6 (B6) mice were purchased from Japan-Clea (Yokohama, Japan). C57BL/6J-Hcphme-v/Hcphme-v homozygotes (mev/mev) and their littermates (+/?) were raised at The Jackson Laboratory (Bar Harbor, ME). All mice were used at 7–16 wk of age.

Cell preparation and cultures
Mice were killed by cervical dislocation under ether anesthesia. BM cells were collected by flushing femoral shafts using a 26-gauge sterile needle. Cells from the peritoneal cavity (PECs) were obtained by injecting 4–8 ml ice-cold {alpha}MEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; BioWhittaker, Walkersville, MD), 50 U/ml streptomycin, and 50 µg/ml penicillin (Meiji Chemical Co. Ltd., Tokyo, Japan).

To induce osteoclast differentiation, BM cells (1–2 x 104/well) and PECs (2–10 x 104/well) were cultured in 24-well plates (Corning Costar, Corning, NY) with 1 ml {alpha}MEM-supplemented 10% FBS and antibiotics in the presence of 50 ng/ml human M-CSF (a gift from Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan) and/or 25 or 50 ng/ml recombinant human soluble RANKL (PeproTech EC Ltd., London, UK), 50 ng/ml recombinant mouse TNF{alpha} (R&D Systems, Inc., Place, NE), or TLR ligands for 6 d. According to the manufacturer, RANKL was produced by Escherichia coli, but the endotoxin level in the RANKL is less than 0.1 ng/µg. Cultures were fed every 3 d by replacing spent medium with fresh medium.

The number of multinucleated TRAP+cells (TRAP+ MNCs) was expressed as the mean ± SD of triplicate cultures (32). In some experiments, 20 ng/ml recombinant human IL-1{alpha} (a gift from Dr. S. Ono, Osaka University, Osaka, Japan) and 400 ng/ml human osteoprotegerin (OPG), a decoy receptor for RANKL (PeproTech EC Ltd.) were added to the cultures.

Pit formation assay
BM cells (5 x 103/well) were cultured on dentine slices (a gift from Dr. N. Udagawa, Matsumoto Dental University, Nagano, Japan) in 0.2 ml {alpha}MEM containing 10% FBS and 50 ng/ml M-CSF with 50 ng/ml soluble RANKL or 50 ng/ml LPS for 20 d in 96-well plates. After removal of the cells with 2 N NaOH, the slices were stained with 2% Coomassie Brilliant Blue R250 in methanol to visualize resorption pits (14).

TLR ligands
LPS from Salmonella minnesota Re 595 (Sigma-Aldrich Corp., St. Louis, MO) or E. coli 055 B5 (Difco, Detroit, MI) were used for in vitro and in vivo experiments. As both LPS preparations induce similar responses, most experiments used S. minnesota R595 LPS unless otherwise indicated. Peptidoglycan (PGN) from Staphylococcus aureus (Fluka Chemie, Buchs, Switzerland), used to stimulate TLR2, was dissolved in water, sonicated, and sterilized in a hot water bath. A phosphorothioated oligonucleotide (ODN; 5'-TCC ATG ACG TTC CTG ATG CT-3'; CpG), used as an unmethylated CpG ODN to stimulate TLR9, and a control phosphorothioated ODN (5'-GCT TGA TGA CTC AGC CGG AA-3') were purchased from Hokkaido System Science (Hokkaido, Japan) (33).

Antibodies (Abs)
An antagonistic rat antimouse TNF{alpha} monoclonal Ab (XT3) was purchased from Endogen (Woburn, MA) and used at 5 µg/ml for inhibition of TNF{alpha} activity. A nonantagonistic rat antimouse Kit Ab (ACK4) (34) was used as a control.

For flow cytometric analysis, monoclonal Abs against Fms (AFS98, biotinylated) (35), Kit [ACK2, phycoerythrin (PE)-conjugated [(34), or Mac-1 (CD11b, FITC-conjugated; M1/70, BD PharMingen, San Diego, CA) were used in Hanks’ medium containing 2% BSA (fraction V, Sigma-Aldrich Corp.) and 0.05% NaN3. The stained cells were analyzed using an EPICS-XL flow cytometer (Coulter Electronics, Hialeah, FL). Magnetic cell sorting for Kit-positive cell separation from BM cells was performed using the Mini-MACS column with PE-conjugated ACK2 and anti-PE antibody-conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).

RT-PCR
To determine the expression of TNF{alpha} (encoded by Tnf), RANKL (Tnfsf11), IL-1 receptor-associated kinase (IRAK) (Irak1), IRAK-M (Irak3), and hypoxanthine phosphoribosyl transferase (Hprt) genes, RT-PCR was performed. Total RNA was isolated using Isogen (Nippon Gene, Toyama, Japan) and was reverse transcribed using Reverse TraAce (Toyobo, Osaka, Japan). The DNA fragments were amplified from the mouse cDNAs by PCR. Hot-lid PCR amplification of cDNA equivalent to 20, 2, and 0.2 ng total RNA was carried out in 1x PCR buffer (1.5 mM MgCl2) containing 0.2 mM deoxy-NTPs (Takara, Shiga, Japan), 0.75 U rTaq DNA polymerase (Toyobo), and primers (used at 1.2 µM). Amplifications were carried out on DNA thermal cyclers (MJ Research, Inc., Watertown, MA). After an initial 3-min denaturing step (94 C), each PCR cycle consisted of 45-sec denaturing (94 C), 1-min annealing (55 or 60 C), and 1.5-min elongation (72 C). After the final cycle, the reaction was held for 3 min at 72 C. The PCR products were then separated on a 2% agarose gel, stained with ethidium bromide, and photographed. The primers used here were as follows: Tnf, 5'-CAC GCT CTT CTG TCT ACT GAA CTT CG-3' and 5'-GGC TGG GTA GAG AAT GGA TGA ACA CC-3'; Tnfsf11, 5'-CAG CAC TCA CTG CTT TTA TAG AAT CC-3' and 5'-AGC TGA AGA TAG TCT GTA GGT ACG C-3'; Irak1, 5'-GCC AGT GGA AAG TGA TGA GAG TG-3' and 5'-GAA AAA GCC TGA TGA CAG CAG TTG-3'; Irak3, 5'-TCC TTC AGG TGT CCT TCT CCA CTG-3' and 5'-CCT CTT CTC CAT TGG CTT GCT C-3'; and Hprt, 5'-AAT GAT CAG TCA ACG GGG GAC A-3' and 5'-CCA GCA AGC TTG CA ACCT TAA CCA-3'.

MAPK inhibitors
Inhibitors of the MAPK signaling pathway, PD098059 (2'-amino-3' methoxyflavone; Wako Pure Industry, Kyoto, Japan) for MAPK kinase (ERK1/2), SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; Wako] for p38, and SP600125 [anthrax(1-9-cd)pyrazol-6(2H)-one; Tocris Cookson Ltd., Avonmouth, UK] for c-Jun N-terminal kinase, were dissolved to 20 mM in dimethylsulfoxide and used at 20 µM.

Statistical analysis
Data are presented as the mean ± SD. Statistical significance was assessed by t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPS induces osteoclastogenesis from mev/mev BM cells
To induce osteoclastogenesis, BM cells from SHP-1- deficient mev/mev mice and their wild-type littermates (+/?) were cultured with M-CSF and RANKL for 6 d. mev/mev BM cells developed 1.5–4 times higher numbers of multinucleated TRAP+ cells (TRAP+ MNCs) than their +/? littermates (Fig. 1AGo, left) (12, 13). The TLR family shares some of the downstream signaling via RANK (22, 23, 24). However, signaling via TLRs has never been reported to induce osteoclastogenesis without RANKL-RANK signaling (4). We cultured BM cells from mev/mev and +/? littermates with LPS plus M-CSF for 6 d. Although TRAP+ mononuclear cells were generated, few TRAP+ MNCs were detected in the wild-type littermate cultures even in the presence of up to 200 ng/ml LPS (Fig. 1AGo, right, and Fig. 1BGo). In the presence of LPS and M-CSF, mev/mev BM cells gave rise to significantly higher numbers of TRAP+ mononuclear cells and TRAP+ MNCs than normal littermates (Fig. 1CGo). The number of TRAP+ MNCs induced by LPS (20 ng/ml) and M-CSF was 1/10th to 1/3rd of those induced with RANKL (50 ng/ml) and M-CSF in mev/mev BM cultures (Fig. 1AGo, right). Osteoclastogenesis was increased in a dose-dependent manner for the LPS added (Fig. 1BGo). Osteoclasts from mev/mev BM cells induced by M-CSF and LPS had bone-resorbing activity on dentine slices, although the activity of pit formation was lower than that induced by M-CSF and RANKL (Fig. 1DGo).



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  		FIG. 1. Osteoclastogenesis of mev/mev BM cells induced by M-CSF and LPS. BM cells from mev/mev mice ({blacksquare}) and their wild-type littermates (+/?; {square}) were cultured with RANKL (A, left) or LPS in the presence of M-CSF for 6 d. LPS was added at 20 ng/ml (A, right; C and D), and varying doses (B; 0–200 ng/ml). A, Numbers of TRAP+ MNCs from three representative experiments. C, Numbers of total, mononuclear, and multinuclear TRAP+ cells. D, Photomicrographs of TRAP-stained cultures (d 6; magnification, x100) and pit formation on dentine slices (d 20; magnification, x200) of +/? (left) and mev/mev (right) BM cells. Significant differences compared with the responses of +/? littermates are indicated by an asterisk (P < 0.05). In all experiments, no TRAP+cells were observed without M-CSF.

 
One receptor for LPS activation is known to be a TLR4, a member of the TLR superfamily (36). To assess whether the inductive effect on osteoclastogenesis is specific for LPS-TLR4 signaling or is a general characteristic of signaling via TLR superfamily members, we examined the effects of ligands for other TLRs, PGN for TLR2 (37) and unmethylated CpG for TLR9 (33), on osteoclastogenesis in the presence of M-CSF. PGN or CpG showed little inductive effect on osteoclastogenesis in either mev/mev or +/? mice (Fig. 2AGo, left). As recently reported, CpG inhibited osteoclast development of BM cells from both mev/mev and +/? control mice induced by RANKL and M-CSF (Fig. 2AGo, right) (29, 38). This indicates that LPS, but not other TLR ligands tested here, induces osteoclastogenesis of SHP-1-deficient BM cells in the presence of M-CSF.



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  		FIG. 2. LPS induces osteoclastogenesis from mev/mev BM cells. A, BM cells (2 x 104) from mev/mev mice ({blacksquare}) and their wild-type littermates ({square}) were cultured with or without 20 ng/ml LPS, 1 µg/ml PGN, 0.1 µM control ODN, or 0.1 µM CpG-ODN in the presence of 50 ng/ml M-CSF (left) or M-CSF plus RANKL (right) for 6 d. B, BM cells cultured with or without 20 µM MAPK inhibitors or the vehicle control dimethylsulfoxide (0.1%, vol/vol) in the presence of M-CSF and RANKL (left) or M-CSF and LPS (right) for 6 d. Significant differences compared with the responses of untreated cultures are indicated by an asterisk (P < 0.05). In all experiments, no TRAP+ cells were observed without M-CSF.

 
We also compared the effects of inhibitors of MAPK signaling on RANKL- and LPS-induced osteoclastogenesis. Inhibitors (20 µM) of the MAPK signaling pathway, PD098059 for MEK (ERK1/2), SB203580 for p38, and SP600125 for c-Jun N-terminal kinase, were added to the cultures for 6 d (Fig. 2BGo). SB203580 and SP600125 completely inhibited osteoclastogenesis in BM cells cultured with RANKL plus M-CSF and with LPS plus M-CSF. Although PD098059 did not completely inhibit osteoclastogenesis in either culture condition, this MAPK inhibitor reduced levels of osteoclastogenesis equivalently in these two culture system.

LPS-induced osteoclast development of mev/mev BM cells is not accounted for by the production of RANKL or TNF{alpha}
A recent report showed that TNF{alpha} mimicked the function of RANKL for stimulating in vitro osteoclastogenesis of BM cells (21). BM cells from mev/mev and +/? mice were cultured with 50 ng/ml TNF{alpha} or 50 ng/ml RANKL in the presence of M-CSF for 6 d, and comparable numbers of osteoclasts in the cultures with TNF{alpha} or RANKL were observed in the BM cell cultures from each mouse stain (Fig. 3AGo). We also incubated the cultures with M-CSF and IL-1{alpha}, but osteoclasts were not generated from either mev/mev or their littermate BM cells (Fig. 3AGo).



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  		FIG. 3. Effects of LPS, TNF{alpha}, and IL-1{alpha} on osteoclastogenesis. A, BM cells (2 x 104) from +/? littermates ({square}) or mev/mev mice ({blacksquare}) were cultured with M-CSF and RANKL, 20 ng/ml LPS, TNF{alpha}, or IL-1{alpha} for 6 d. The mean number and SD of TRAP+ MNCs per well from the simultaneous experiments are shown. B, cDNA from BM cells of +/? littermates and mev/mev mice cultured with M-CSF or M-CSF plus LPS for 3 d were prepared, and RT-PCRs for Tnf, Tnfsf11, Irak1, and Irak4 were performed. Sequential doses of cDNA (equivalent to 20, 2, and 0.2 ng total RNA) were used as a template. Control responses were indicated by using primers for Hprt. C, Osteoclastogenesis induced by serial doses of RANKL (left) or TNF{alpha} (right) in the presence of M-CSF. In all experiments, no TRAP+ cells were observed without M-CSF.

 
As LPS is known to induce TNF{alpha} production (28), we examined whether LPS-induced osteoclastogenesis was related to TNF{alpha} or RANKL production. If BM cells of mev/mev mice may produce high levels of TNF{alpha} or RANKL, osteoclastogenesis should occur in mev/mev BM cells. Therefore, BM cells from mev/mev and +/? mice were cultured with M-CSF, or M-CSF plus LPS for 3 d. Although LPS addition increased the expression of the TNF{alpha} (Tnf) gene transcript and induced a small amount of transcript of the RANKL (Tnfsf11) gene, doses of transcripts of Tnf and Tnfsf11 genes were comparable in both strains of mice (Fig. 3BGo).

Recently, Kobayashi et al. (36, 37, 38, 39) reported that IRAK (encoded by Irak1) associated with MyD88 and IRAK-M (encoded by Irak3) regulate the signaling via TLRs. We assessed the expression of Irak1 and Irak3 in BM cells cultured with M-CSF or M-CSF plus LPS. Irak3 gene expression was increased in the presence of LPS as reported; however, no significant difference was detected between mev/mev and +/? cultured cells (Fig. 3BGo). An alternative possibility was that mev/mev BM cells may require a smaller amount of TNF{alpha} or RANKL to induce osteoclast development than wild-type BM cells, and LPS induces sufficient production of TNF{alpha} or RANKL to support osteoclastogenesis only in mev/mev BM cells. Thus, minimal requirements of TNF{alpha} or RANKL to induce TRAP+ MNCs in cultures were assessed. More than 1 ng/ml TNF{alpha} or RANKL (Fig. 3CGo) was needed to induce osteoclastogenesis from both mev/mev and wild-type littermate BM cells.

To further confirm these observations, mev/mev BM cells were cultured with anti-TNF{alpha} antagonistic antibody (XT3) or OPG in the presence of M-CSF and LPS, and the inhibitory effect on LPS-induced osteoclastogenesis in mev/mev BM cells was observed. OPG and XT3 completely inhibited M-CSF and RANKL (50 ng/ml)-induced and M-CSF and TNF{alpha} (50 ng/ml)-induced osteoclastogenesis, respectively; however, neither reagent inhibited M-CSF plus LPS-induced osteoclastogenesis (Fig. 4Go). Although less than 1 ng/ml RANKL or TNF{alpha} might be produced by BM cells in the cultures, these results suggest that SHP-1 regulates the magnitude of osteoclastogenesis, but not the minimal requirements of RANKL or TNF{alpha}, and indicate that osteoclast development by mev/mev BM cells induced by M-CSF plus LPS may not be directly related to the production of RANKL or TNF{alpha}.



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  		FIG. 4. LPS-induced osteoclastogenesis is not inhibited by OPG or anti-TNF{alpha} antibody. BM cells (2 x 104) from +/? littermates (top) or mev/mev (bottom) were cultured with M-CSF and 50 ng/ml RANKL (left), 50 ng/ml TNF{alpha} (center), or 20 ng/ml LPS (right) for 6 d in the presence or absence of OPG, XT3 (anti-TNF{alpha} antagonistic antibody), or control antibody (ACK4) for 6 d. The mean number and SD of TRAP+ MNCs per well from the simultaneous experiments are shown. Significant differences compared with the responses of untreated cultures are indicated by an asterisk (P < 0.05). In all experiments, no TRAP+ cells were observed without M-CSF.

 
LPS-responsive OCPs in mev/mev BM cells are enriched in the Kit-positive population
It has been reported that OCPs in the BM are enriched in the Kit+ cell fraction when osteoclastogenesis was induced with stromal cells (40) or with M-CSF and RANKL (41). To assess the phenotypic characterization of LPS-responsive OCPs in BM cells, we enriched the Kit+ cells using magnetic cell sorting (Fig. 5AGo) and induced osteoclastogenesis by addition of M-CSF plus RANKL or M-CSF plus LPS. In +/? BM cells, OCPs were enriched in the bound fraction of anti-Kit antibody and magnetic beads. Although a few TRAP+ MNCs were induced from unfractionated +/? BM cells, the Kit+ cell-enriched fraction gave rise to significant numbers of TRAP+ MNCs in cultures. A few TRAP+ mononuclear cells (data not shown), but few TRAP+ MNCs, were observed in the Kit– cell fraction (Fig. 5BGo).



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  		FIG. 5. Osteoclastogenesis from the Kit+ BM cell-enriched population. The mev/mev and +/? littermate BM cells were incubated with PE-labeled anti-Kit antibody (ACK2) and subsequently with anti-PE antibody-conjugated beads (pre-column cells). The cells were applied to the magnetic bead columns, and nonbound cells (passed) and cells bound to the columns (bound) were recovered. A, One aliquot of cells was analyzed by flow cytometry. Cells stained with PE-streptavidin were used as a negative control (shadowed). B, Other aliquots of cells ({blacksquare}, 2 x 104/well; , 5 x 103/well) were cultured with 50 ng/ml M-CSF and 50 ng/ml RANKL or with M-CSF and 20 ng/ml LPS. On d 6 of culture, the number of TRAP+ MNCs was counted. Addition of M-CSF alone induced no TRAP+ MNCs or fewer than seven TRAP+ mononuclear cells from either mouse strain.

 
Kit+ cell-enriched populations from mev/mev BM cells contained more cells that expressed high levels of Kit than those from +/? BM cells (Fig. 5AGo). When the same numbers (2 x 104/well) of Kit+ cells enriched from mev/mev BM cells as +/? BM cells were cultured with M-CSF and RANKL or M-CSF and LPS, the numbers of TRAP+ MNCs in unfractionated and Kit+ cell-enriched BM cells were comparable, although those from the cell fraction passed through the magnetic bead column were dramatically reduced (Fig. 5BGo). It is possible that macrophage-like cells overgrew in the wells of the Kit+ cell-enriched fraction, resulting in saturation of osteoclastogenesis. Because Kit+ cells proliferated at high levels in the presence of M-CSF, and when 25% of mev/mev BM cells were cultured, M-CSF plus RANKL-responding and M-CSF plus LPS-responding OCPs were significantly enriched in the column-bound fraction (Fig. 5BGo). Therefore, the majority of OCPs in mev/mev BM cells might also be present in the Kit+ cell-enriched fraction.

Moreover, to confirm whether LPS-responsive OCPs express Kit, we precultured BM cells with M-CSF for 3 d and dish-adherent cells were harvested. A majority (+/?; 87.4%, and mev/mev; 88.6%) of the precultured cells expressed Mac-1, and half (+/?; 52.2%, and mev/mev; 50.2%) of the cells were also Fms+; however, few (+/?; 0.32%, and mev/mev; 0.12%) of the precultured cells expressed Kit. The harvested cells were further cultured with M-CSF and RANKL, LPS, or RANKL plus LPS for 6 d. In the presence of M-CSF and RANKL, TRAP+ MNCs were generated from both mev/mev and +/? precultured BM cells, and mev/mev cells gave rise to significantly higher numbers of TRAP+ MNCs than +/? cells (Fig. 6BGo). In contrast, few of precultured BM cells gave rise to TRAP+ MNCs in the presence of M-CSF and LPS, and addition of LPS inhibited osteoclastogenesis induced by M-CSF and RANKL (Fig. 6Go, B and C) (29). These results indicate that BM cells precultured with M-CSF lose Kit-expression, and these cells from mev/mev mice lose the potential of LPS-responsive differentiation into osteoclasts.



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  		FIG. 6. Few precultured BM cells with M-CSF give rise to osteoclasts in the presence of M-CSF and LPS. A, Freshly prepared BM cells (2 x 104/well) were cultured with () or without ({square}) LPS in the presence of M-CSF and RANKL for 6 d. B, BM cells (5 x 106/dish) were cultured with 50 ng/ml M-CSF. On d 3, the harvested cells (4 x 103/well) were cultured with () or without ({square}) LPS in the presence of M-CSF and RANKL for 6 d. C, Freshly prepared (2 x 104/well) or precultured (4 x 103/well) BM cells from mev/mev mice ({blacksquare}) or +/? littermates ({square}) were cultured with LPS and M-CSF for 6 d. The number of TRAP+ MNCs in a well were counted. Significant differences compared with the responses (B) without LPS or those of +/? littermates (C) are indicated by an asterisk (P < 0.05).

 
Reduction of osteoclastogenesis from mev/mev PECs
Recently, we reported that OCPs in BM and extramedullary organs, such as peritoneal cavity expressed distinct characteristics (29). To assess the influence of SHP-1 deficiency on OCPs in peritoneal cavities, we cultured PECs from mev/mev mice and their +/? littermates with M-CSF plus RANKL for 6 d. In the presence of M-CSF and RANKL, the number of TRAP+ MNCs generated from mev/mev PECs was significantly reduced compared with that from +/? littermates (Fig. 7AGo). The mev/mev BM cells cultured in corresponding experiments (experiments 1–3 in Fig. 1AGo) generated higher numbers of TRAP+ MNCs than the wild-type controls. The addition of LPS, TNF{alpha}, or IL-1{alpha} did not induce osteoclastogenesis in the presence of M-CSF (Fig. 7BGo). LPS and TNF{alpha} inhibited osteoclastogenesis induced by M-CSF and RANKL in control PECs as reported previously (29). IL-1{alpha} increased slightly the number of TRAP+ MNCs from wild-type PECs by M-CSF and RANKL. In contrast, osteoclast development from mev/mev PECs was absent or extremely low in all conditions tested (Fig. 7BGo).



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  		FIG. 7. Osteoclastogenesis from mev/mev PECs. A, PECs (10 x 104/well) from mev/mev mice ({blacksquare}) or +/? littermates ({square}) were cultured with M-CSF and RANKL for 6 d. The number of TRAP+ MNCs from three representative experiments in corresponding experiments in Fig. 1AGo were demonstrated. B, PECs were cultured with RANKL and/or TNF{alpha}, LPS, or IL-1{alpha} in the presence of M-CSF. C, PECs (5 x 104/well) from B6 mice were cultured without () or with those from mev/mev ({blacksquare}) or +/? littermates ({square}) in the presence of M-CSF and RANKL. In all experiments, no TRAP+ cells were observed without M-CSF.

 
In flow cytometric analysis, Mac-1+ cells were 22.6% and 21.9%, and Mac-1+ Fms+ cells were 12.6% and 9.6% of the cell fraction in +/? and mev/mev PECs, respectively. Therefore, the contents of the cell lineage in both strains of mice were similar. The mev/mev PECs may contain cells that inhibited osteoclast maturation, resulting in suppression of osteoclast development from mev/mev PECs, although cells with the potential to differentiate into mature osteoclasts in the mev/mev peritoneal cavity may be present. To examine this possibility, we performed mixing experiments with PECs from B6 mice. The number of TRAP+ MNCs in mixed cultures was almost additive to that in each PEC from B6 and mev/mev, and B6 and +/? mice (Fig. 7CGo). This indicates that mev/mev PECs may lack OCPs, or the OCPs in mev/mev PECs may lose differentiative potential, rather than be inhibited by cells in their peritoneal cavity.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we showed that BM cells from mev/mev mice defective in SHP-1 gave rise to mature osteoclasts in the presence of M-CSF and LPS without exogenous RANKL or TNF{alpha} in culture. It has been reported that the multinucleation and bone resorption of mev/mev osteoclasts induced by M-CSF and RANKL or by coculturing with stromal cells are accelerated (12, 13). Therefore, the total numbers of TRAP+-cells (mononuclear and multinuclear cells) were relatively comparable, but the numbers of TRAP+ MNCs in mev/mev cultures were significantly higher than those in +/? littermates (12, 13) (Hayashi, S.-I., unpublished observation). Wild-type BM cells gave rise to TRAP+ mononuclear cells in the presence of M-CSF and LPS; however, none or only a few multinucleated cells were observed in culture. Moreover, the numbers of TRAP+ mononuclear cells in +/? control mice were also significantly lower than those in SHP-1-deficient mev/mev BM cells.

Previously, we reported that OCPs in the normal BM were enriched in Kit+ cells (40, 42). In the current study OCPs in +/? BM responding to M-CSF plus RANKL were enriched in the Kit+ cell-enriched population. Almost all TRAP+ mononuclear cells induced by M-CSF and LPS were derived from this fraction. OCPs responding to both RANKL and LPS in mev/mev BM were also enriched in the magnetic bead column-bound fraction. Kit+ cell-enriched populations from mev/mev BM cells expressed a higher level of Kit per cell than those from +/? cells. Few BM cells precultured with M-CSF for 3 d expressed Kit. In the presence of M-CSF and RANKL, osteoclasts were generated from both mev/mev and +/? precultured BM cells. In contrast, precultured BM cells gave rise to few TRAP+ MNCs in the presence of M-CSF and LPS. Moreover, LPS inhibited osteoclastogenesis induced by M-CSF and RANKL (29). Corresponding to losing Kit expression of BM cells precultured with M-CSF, these cells from mev/mev mice lose the potential of LPS-responsive differentiation into osteoclasts. These results indicated that the majority of LPS-responsive OCPs as well as RANKL-responsive OCPs in freshly prepared mev/mev BM cell populations might be present in the Kit+ cell fraction (29).

It is noted that a majority of c-Kit+ cells in freshly prepared BM cells are immature before expressing RANK (41). Lam et al. (20) proposed that TNF{alpha} could induce osteoclast differentiation only in precursors simultaneously or previously exposed to RANKL. Their conclusion was based on the results that BM cells cultured for 3 d with M-CSF and OPG lost the potential of TNF{alpha}-induced osteoclastogenesis, although they also mentioned that overnight preincubation with M-CSF and OPG did not affect their potential of responsiveness to TNF{alpha} (20). A majority of OCPs that respond to LPS and differentiate into osteoclasts are Kit+ cells, which might not express RANK. Even if previous exposure to RANKL is needed to maintain the responsiveness to LPS or TNF{alpha} in OCPs, OPG addition from 3 d before or from the initiation of culture must result in the same effect. We confirmed that overnight incubation with M-CSF and OPG did not affect the potential of LPS-induced osteoclastogenesis in mev/mev BM cells (data not shown). As Lam et al. (20) demonstrated, the presence of RANKL might be optimal for the maintenance of this potential; however, regardless of presence or absence of OPG, 3-d preculture with M-CSF reduced the potential to differentiate into osteoclasts induced by TNF{alpha} (29) or LPS. Moreover, several recent studies using RANK-KO mice showed that osteoclastogenesis is induced without RANK/RANKL signaling (18, 19). Therefore, LPS-induced osteoclastogenesis from mev/mev BM cells may be independent of RANK/RANKL signaling.

To assess whether mev/mev BM contains more Kit+ cells than +/? BM, flow cytometric analyses were performed repeatedly (data not shown). Some mev/mev BM contained a slightly higher ratio of Kit+ cells than +/? BM, but others were comparable to the wild-type BM. As mev/mev Kit+ cell-enriched populations still generated higher numbers of osteoclasts than Kit+ cell-enriched +/? populations, the presence of more Kit+ cells in mev/mev BM might not account for the accelerated osteoclastogenesis. Using a limiting dilution assay, we assessed the frequency of OCPs in BM (40). The mev/mev mice and +/? littermates contained, on the average, one OCP per 45.3 BM cells and one OCP per 34.8 BM cells, respectively. Single OCPs of mev/mev and +/? BM gave rise to 7.1 ± 9.6 and 11.8 ± 21.7 TRAP+ cells, respectively. The frequency of OCPs in BM cells and the growth of OCPs in culture are comparable to those in +/? littermates.

LPS, but not PGN or CpG, induced osteoclastogenesis of mev/mev BM cells in the presence of M-CSF. These three TLRs (TLR2, -4, and -9) share the downstream signaling, MyD88, TRAF6, NF-{kappa}B, and MAPK, but only TLR4, a receptor for LPS, is known to be another signaling pathway independent of independent of MyD88 (23, 43, 44). LPS might mimic the function of RANKL/RANK signaling, but not that of M-CSF/Fms signaling, in mev/mev BM osteoclastogenesis. In the absence of M-CSF, mev/mev BM osteoclastogenesis was not observed even if RANKL and LPS were added to the culture (data not shown). SHP-1 is reported to negatively regulate signaling via receptor protein tyrosine kinases, but the ligands, such as stem cell factor, vascular endothelial growth factor 164, platelet-derived growth factor, or insulin could not replace M-CSF function (45) (Yamada, T., unpublished observation). In addition to M-CSF, at least 1 ng/ml RANKL or TNF{alpha} is necessary to induce osteoclastogenesis in both mev/mev and their littermate (+/?) BM cells. M-CSF plus LPS-induced osteoclastogenesis was not inhibited by either OPG or anti-TNF{alpha} Ab. A recent report demonstrated that enriched BM macrophages cultured with M-CSF and thioglycolate-activated peritoneal macrophages produced less than 400 pg/ml TNF{alpha} ((28). As we used whole BM cells in the steady state, less than 1/20th of the cell populations and approximately 1/50th of the LPS concentration were comparable to this report, and it is unlikely that our cultures contained more than 1 ng/ml TNF{alpha}. Therefore, production of RANKL or TNF{alpha} might not be involved in LPS-induced osteoclastogenesis of mev/mev BM cells.

Recently, it was shown that RANKL/RANK signaling activates SHP-1 recruitment to the complex containing TRAF6, and SHP-1 blocked the interaction of TRAF6 with the RANK signaling pathway (25). This suggests that SHP-1 might function in the TLR and TRAF6 signaling pathway. Mice lacking either triggering receptor expressed on myeloid cells 2 (TREM2) (46) or DAP12 are reported to develop osteopetrosis (47). These mice have fewer osteoclasts and lack the ability for bone resorption. DAP12, containing a cytoplasmic immunoreceptor tyrosine-based activation motif, is a TREM-related receptor, which recruits SHP-1. The mev/mev BM cells accelerate multinucleation (Fig. 1Go) and bone resorption (12, 13). DAP12 dephosphorylation may be delayed in mev/mev BM cells, resulting in an increase in multinucleated osteoclasts.

After addition of PD098059, a MAPK kinase inhibitor, to the culture for 6 d, the total number of TRAP+ cells was relatively comparable to that in the absence of this reagent (data not shown). However, the number of TRAP+ MNCs was significantly reduced. MEK/ERK signaling may be involved in multinucleation of osteoclasts, suggesting that SHP-1 regulates the MEK/ERK signaling pathway. Moreover, as it is known that downstream signaling of TREM and DAP12 activate ERK, SHP-1 deficiency may accelerate ERK activation, resulting in an increase in multinucleated osteoclasts.

Recently, we demonstrated that peritoneal OCPs lose the potential to differentiate into mature osteoclasts if they were exposed to TLR ligands, TNF{alpha}, or even RANKL before an encounter with M-CSF and RANKL as a differentiation signal (29). Because SHP-1 deficiency accelerates this signaling pathway, OCPs in the mev/mev peritoneal cavity may lose the potential by the exposure of their ligands or unknown natural ligands. Finally, experiments using mev/mev mice lacking RANK or RANKL will provide conclusive evidence as to whether LPS may induce osteoclastogenesis in vivo. If so, LPS injection should cure the osteopetrosis in the double-mutant mice.


   Acknowledgments
 
We acknowledge Drs. Kensuke Miyake (Tokyo University, Tokyo, Japan), Masato Ogata (Mie University, Mie, Japan), and Nobuyuki Udagawa (Matsumoto Dental University, Nagano, Japan) for helpful suggestions, and Masayuki Takahashi and Takao Taki (Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan) for M-CSF. We also thank Drs. Tomohiro Kurosaki (RIKEN Yokohama Institute, Yokohama, Japan) for his warm encouragement, and Ms. Toshie Shinohara for her secretarial assistance.


   Footnotes
 
This work was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science, and Technology; the Japanese government (to S.I.H. and H.Y.); the Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd.; and NIH Grant CA20408 (to L.D.S.).

Current address for T.Y.: Laboratory for Lymphocyte Differentiation, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama 230-0045, Japan.

Abbreviations: Ab, Antibody; BM, bone marrow; FBS, fetal bovine serum; Hprt, hypoxanthine phosphoribosyl transferase; IRAK, IL-1 receptor-associated kinase; KO, knockout; LPS, lipopolysaccharide; M-CSF, macrophage-colony-stimulating factor; MNC, multinucleated cell; NF-{kappa}B, nuclear factor-{kappa}B; OCP, osteoclast precursor; ODN, oligonucleotide; OPG, osteoprotegerin; PE, phycoerythrin; PEC, peritoneal cavity cell; PGN, peptidoglycan; RANK, receptor activator of nuclear factor-{kappa}B; RANKL, receptor activator of nuclear factor-{kappa}B ligand; SHP, Src homology 2-domain phosphatase; TLR, Toll-like receptor; TRAF, TNF receptor-associated factor; TRAP, tartrate-resistant acid phosphatase; TREM, triggering receptor expressed on myeloid cell.

Received February 10, 2004.

Accepted for publication February 17, 2004.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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