ABSTRACT. osteoclastogenesis, macrophage colony stimulating factor, receptor activator nuclear factor- B ligand, parathyroid hormone

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1 JOURNAL OF BONE AND MINERAL RESEARCH Volume 15, Number 9, American Society for Bone and Mineral Research Importance of Membrane- or Matrix-Assoicated Forms of M-CSF and RANKL/ODF in Osteoclastogenesis Supported by SaOS-4/3 Cells Expressing Recombinant PTH/PTHrP Receptors KANAMI ITOH, 1 NOBUYUKI UDAGAWA, 1 KENICHIRO MATSUZAKI, 1 MASAMICHI TAKAMI, 1 HITOSHI AMANO, 2 TOSHIMASA SHINKI, 1 YUTAKA UENO, 1 NAOYUKI TAKAHASHI, 1 and TATSUO SUDA 1 ABSTRACT SaOS-4/3, a subclone of the human osteosarcoma cell line SaOS-2, established by transfecting the human parathyroid hormone/parathyroid hormone related protein (PTH/PTHrP) receptor complementary DNA (cdna), supported osteoclast formation in response to PTH in coculture with mouse bone marrow cells. Osteoclast formation supported by SaOS-4/3 cells was completely inhibited by adding either osteoprotegerin (OPG) or antibodies against human macrophage colony stimulating factor (M-CSF). Expression of messenger RNAs (mrnas) for receptor activator of NF- B ligand/osteoclast differentiation factor (RANKL/ODF) and both membrane-associated and secreted forms of M-CSF by SaOS-4/3 cells was up-regulated in response to PTH. SaOS-4/3 cells constitutively expressed OPG mrna, expression of which was down-regulated by PTH. To elucidate the mechanism of PTH-induced osteoclastogenesis, SaOS-4/3 cells were spot-cultured for 2 h in the center of a culture well and then mouse bone marrow cells were uniformly plated over the well. When the spot coculture was treated for 6 days with both PTH and M-CSF, osteoclasts were induced exclusively inside the colony of SaOS-4/3 cells. Osteoclasts were formed both inside and outside the colony of SaOS-4/3 cells in coculture treated with a soluble form of RANKL/ODF (srankl/sodf) in the presence of M-CSF. When the spot coculture was treated with srankl/sodf, osteoclasts were formed only inside the colony of SaOS-4/3 cells. Adding M-CSF alone failed to support osteoclast formation in the spot coculture. PTH-induced osteoclast formation occurring inside the colony of SaOS-4/3 cells was not affected by the concentration of M-CSF in the culture medium. Mouse primary osteoblasts supported osteoclast formation in a similar fashion to SaOS-4/3 cells. These findings suggest that the up-regulation of RANKL/ODF expression is an essential step for PTH-induced osteoclastogenesis, and membrane- or matrix-associated forms of both M-CSF and RANKL/ ODF are essentially involved in osteoclast formation supported by osteoblasts/stromal cells. (J Bone Miner Res 2000;15: ) Key words: osteoclastogenesis, macrophage colony stimulating factor, receptor activator nuclear factor- B ligand, parathyroid hormone 1 Department of Biochemistry, School of Dentistry, Showa University, Tokyo, Japan. 2 Department of Pharmacology, School of Dentistry, Showa University, Tokyo, Japan. 1766

2 THE MECHANISM OF OSTEOCLASTOGENESIS INTRODUCTION OSTEOCLASTS, MULTINUCLEATED cells responsible for bone resorption, are derived from monocyte-macrophage lineage cells. We have established a coculture system of mouse hemopoietic cells and osteoblasts/stromal cells, in which osteoclasts were formed in response to boneresorbing factors such as 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ], parathyroid hormone (PTH), and prostaglandin E 2 (PGE 2 ). (1 3) When direct contact between osteoblasts/stromal cells and hemopoietic cells was inhibited by a membrane filter, no osteoclasts were formed, even in the presence of 1,25(OH) 2 D 3, PTH, or PGE 2.Wehypothesized that a membrane bound factor designated osteoclast differentiation factor (ODF) is expressed on osteoblasts/stromal cells in response to bone-resorbing factors, and osteoclast progenitors recognize ODF through cell-to-cell interaction with osteoblasts/stromal cells. (3) Recently, this hypothesis was supported by the discovery of a new tumor necrosis factor (TNF) ligand family member, ODF (4) (also called osteoprotegerin ligand [OPGL] (5) / TNF-related activation-induced cytokine (TRANCE) (6) / receptor activator of NF- B ligand (RANKL) (7) ], expressed by osteoblasts/stromal cells. Osteoclast precursors express RANK, a TNF receptor family member. (7,8) When a soluble form of RANKL/ODF (srankl/sodf) was added to pure spleen cell cultures, osteoclasts were formed in the presence of macrophage colony stimulating factor (M-CSF; also called colony-stimulating factor 1), another factor essential for osteoclast formation, (9) even in the absence of osteoblasts/ stromal cells. (4,5,8) OPG (10) (also called osteoclastogenesis inhibitory factor (11) ) is a soluble decoy receptor for RANKL/ODF, which inhibits osteoclastogenesis induced by RANKL/ODF. Because RANKL/ODF is a type II transmembrane protein, this cytokine seems to support osteoclast formation as a membrane-associated factor. (4,5) However, recent reports indicate another possibility that RANKL/ ODF acts as a soluble form to induce osteoclast formation. Lum et al. (12) reported that RANKL/ODF was released from cells by shedding with a TNF- converting enzyme (TACE). Kong et al. (13) also showed that activated murine T cells secreted an active form of RANKL/ODF into the culture medium. These findings indicate a possibility that a factor(s) other than RANKL/ODF may be required for cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors for inducing osteoclast differentiation. M-CSF also produced by osteoblasts/stromal cells has been believed to act as a soluble factor on osteoclast progenitors. (14 16) However, the synthesis of a membraneassociated form as well as a secreted form of M-CSF has been shown in human cells (17,18) and murine cells. (19,20) Human cells express at least four transcripts of M-CSF messenger RNA (mrna) by alternative splicing. (21) The secreted form of M-CSF is encoded by two transcripts with 2.5 kilobases (kb) and 4.0 kb, which differ in their 3 ends, alternatively using exon 9 and exon 10 of the M-CSF gene, respectively. Both transcripts have exon 6, which contains the sequence for a proteolytic cleavage site. The other two mrnas of 1.6 kb and 3.1 kb also differ in the alternative use of exon 9 and exon 10, respectively, and lack the sequence for the proteolytic cleavage site in exon 6. Consequently, M-CSF proteins are also expressed as membraneassociated forms. In this respect, Yao et al. (22) clearly showed that the membrane-associated M-CSF expressed by NIH 3T3 cells, which had been transfected with 1.6 kb complementary DNA (cdna) encoding a membraneassociated form of M-CSF, was functionally active in inducing osteoclast formation in coculture with mouse bone marrow cells. It also was shown that M-CSF produced by L929 cells (23) and by Chinese hamster ovary cells (24) transfected with 4.0 kb human M-CSF cdna, which encodes a secreted form, is post-translationally modified by the attachment of a glycosaminoglycan side chain. Because of this modification, the secreted form of M-CSF binds to the matrix. (25) Indeed, the proteoglycan form of this cytokine was isolated from bone. (26) These findings suggest that M-CSF also acts as a membrane-associated or a matrixassociated form to support osteoclastogenesis. PTH and PTH-related protein (PTHrP) induce a biological response in the target cells through their binding to the PTH/PTHrP receptor. (27) We have established subclones of the human osteosarcoma cell line SaOS-2 by transfection with an expression vector containing the human PTH/ PTHrP receptor cdna to examine the regulatory mechanism of osteoclast formation induced by PTH. (28) One subclone, designated SaOS-4/3, which expressed a high level of PTH/PTHrP receptors, supported osteoclast formation in cocultures with mouse or human hemopoietic cells in the presence of PTH, but not other osteotropic factors such as 1,25(OH) 2 D 3 and PGE 2. (26) In the present study, we explored the mechanism of osteoclastogenesis supported by SaOS-4/3 cells in more detail. RANKL/ODF and M-CSF were the two major factors essential for osteoclast formation in the PTH-treated coculture with SaOS-4/3 cells. Although PTH stimulated expression of mrnas for both RANKL/ODF and M-CSF in SaOS-4/3 cells, up-regulation of RANKL/ODF expression appeared to be an essential step for the PTH-induced osteoclastogenesis. It also was suggested that a membraneor a matrix-associated form of M-CSF as well as that of RANKL/ODF are essential for osteoclastogenesis supported by SaOS-4/3 cells. Primary osteoblasts induced osteoclast formation in a similar manner as SaOS-4/3 cells. Such a mechanism of action of RANKL/ODF and M-CSF on osteoclast progenitors may be important for the selective formation of osteoclasts in bone. MATERIALS AND METHODS Animals and chemicals 1767 Five-week-old male and newborn ddy mice were obtained from Sankyo Laboratories Animal Center (Tokyo, Japan). Recombinant murine srankl/sodf and human OPG were kindly provided by Dr. K. Higashio of Snow Brand Milk Products (Tochigi, Japan). Recombinant human M-CSF (Leukoprol) was obtained from Yoshitomi Pharmaceuticals (Osaka, Japan). Anti-human M-CSF rabbit polyclonal antibodies were purchased from New England Bio Labs (Lake Placid, NY, U.S.A.). [Nle 8,18, Tyr 34 ]-human PTH(1 34) (PTH) was kindly provided by Dr. M. Hori of

3 1768 ITOH ET AL. Asahi Chemical Industry (Shizuoka, Japan). PGE 2 and 1,25(OH) 2 D 3 were purchased from Wako Pure Chemicals (Osaka, Japan). An ELISA kit for human M-CSF was obtained from Genzyme TECHNE (Minneapolis, MN, U.S.A.). Cells and coculture systems SaOS-4/3, a subclone of the human osteosarcoma cell line SaOS-2 established by transfecting the human PTH/ PTHrP receptor cdna, was kindly provided by Mr. Y. Takahashi of Asahi Chemical Industry. (28) Mouse bone marrow cells were obtained from tibias of 6- to 9-week-old ddy male mice, and primary osteoblasts were prepared from calvariae of newborn ddy mice as described previously. (29) All procedures for experiments using mice were approved by the Showa University Animal Management Committee. SaOS-4/3 cells ( cells/well) were cocultured with bone marrow cells ( cells/well) in 48-well plates in -minimal essential medium ( -MEM; 0.3 ml/well) containing 10% fetal bovine serum (FBS) in the presence or absence of PTH (10 8 M). Some cultures also were treated with OPG (100 ng/ml) or antibodies against human M-CSF (0.5 g/ml). The culture medium was replaced with fresh medium on day 3. After culture for 6 days, cells were fixed and double-stained for tartrate-resistant acid phosphatase (TRAP; a marker enzyme of osteoclasts) and alkaline phosphatase (ALP; a marker enzyme of osteoblasts). (29) TRAPpositive cells appeared as dark red cells and ALP-positive cells appeared as blue cells. The number of TRAP-positive cells including mononuclear and multinucleated cells was scored using a microscope. In spot cocultures, SaOS-4/3 cells or primary osteoblasts ( cells/0.05 ml/well) were spot-cultured for 2hin -MEM containing 10% FBS in the center of a single culture well of 12-well plates. Nonadherent cells were then removed by washing two times with -MEM, and mouse bone marrow cells ( cells/well) were uniformly plated over the culture well. The spot coculture was further treated for 6 days with or without PTH (10-8 M) in 0.5 ml of -MEM containing 10% FBS. Some spot cocultures also were treated with srankl/sodf (100 ng/ml), M-CSF (100 ng/ml), or srankl/sodf (100 ng/ml) plus M-CSF (100 ng/ml). Cells were then fixed and stained for TRAP, and the numbers of TRAP-positive cells that formed inside and outside the colony of SaOS-4/3 cells or primary osteoblasts were scored separately. The experiments were repeated at least three times. The results obtained from a typical experiment were expressed as the means SEM of three cultures. Measurement of M-CSF in the conditioned medium SaOS-4/3 cells ( cells/well) were cultured for 3 days in a 12-well plate in the presence or absence of PTH (10 8 M). The culture medium was then recovered and filtrated through a 0.45-mm filter. The concentration of M-CSF in the filtrate was determined using an ELISA kit for human M-CSF. SaOS-4/3 cells ( cells) and bone marrow cells ( cells) suspended in 0.1 ml of -MEM containing 10% FBS were spot-cultured for 2 h in thecenter of a single well of 12-well plates (small volume culture) or in the center of 60-mm culture dishes (large volume culture). The cocultures were then treated for 6 days with or without PTH (10 8 M) in 0.5 ml (small volume culture) or 5.0 ml (large volume culture) of -MEM containing 10% FBS. The culture medium was replaced with fresh medium on day 3. After culture for 6 days, the culture medium was recovered and the concentration of M-CSF was determined as described above. The cells on the well or dish were fixed and stained for TRAP. The number of TRAP-positive cells that formed inside the colony of SaOS-4/3 cells and bone marrow cells were scored. The results were expressed as the means SEM of three cultures. Northern blot and reverse-transcriptase polymerase chain reaction analyses SaOS-4/3 cells and the parent SaOS-2 cells precultured in a 10-cm culture dish were treated with and without PTH (10 8 M), 1,25(OH) 2 D 3 (10 8 M), or PGE 2 (10 6 M) for indicated periods. Total cellular RNA was extracted using Trizol solution (Gibco Laboratories, Gland Island, NY, U.S.A.), and poly(a) RNA was prepared from total RNA using a MACS mrna Isolation kit (Miltenyi Biotec GmbH, Köln, Germany). Poly(A) RNA (2.5 mg) was electrophoresed in 1.4% agarose-formaldehyde gels and transferred onto nylon membrane filters (Hybond-N; Amersham International, Little Chalfont, U.K.). The membranes were hybridized for 15 h at 42 C with radioactive cdna probes for human RANKL/ODF, OPG, and M-CSF prepared using a multirandom primer oligonucleotide labeling kit (Takara Biomedical, Shiga, Japan). Probes for human RANKL/ODF and OPG cdna were kindly provided by Dr. K. Higashio of Snow Brand Milk Products. Human M-CSF cdna was cloned by reverse-transcriptase polymerase chain reaction (RT-PCR). As an internal control, the membrane was rehybridized with a radioactive cdna probe for human -tubulin. Each membrane was exposed to an X-ray film. Expression of mrnas for soluble and membraneassociated forms of M-CSF was determined using semiquantitative RT-PCR analysis. First-strand cdna was synthesized from total RNA (5 g) extracted from SaOS-4/3 cells treated with or without PTH for 48 h with random primers. Exon 6 of the human M-CSF gene (nucleotides ) contains a sequence for a proteolytic cleavage site. The first-strand cdna was subjected to PCR amplification with EX Taq polymerase (Takara Biomedical) using specific PCR primers, which allowed us to detect both membrane-associated and secreted forms of human M-CSF, 5 -GCTTTGCTGAATGCTCCAGC-3 (forward, nucleotides ) and 5 -CAGAGGGACATTGGACAAACG-3 (reverse, nucleotides ). Expression of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was determined as an internal control, 5 -ACCACAGTC- CATGCCATCAC-3 (forward, nucleotides ) and 5 - TCCACCACCCTGTTGCTGCTGTA-3 (reverse, nucleotides ). PCR products were separated by electrophoresis on a 2% agarose gel. Transcripts for the membrane-associated and secreted forms of M-CSF were

4 THE MECHANISM OF OSTEOCLASTOGENESIS 1769 expected to be signals with 308 base pairs (bp) and 1201 bp, respectively. RESULTS TRAP-positive cells were formed within 6 days in response to PTH in the coculture of SaOS-4/3 cells and mouse bone marrow cells (Figs. 1A and 1B). Other osteotropic factors such as 1,25(OH) 2 D 3 and PGE 2 failed to induce TRAP-positive cells in the coculture with SaOS-4/3 cells (Fig. 1A). TRAP-positive cell formation induced by PTH was completely blocked by adding either OPG or antihuman M-CSF antibodies (Figs. 1A and 1B). Because TRAP-positive cells were formed in mouse bone marrow cultures treated with srankl/sodf together with M-CSF, (30) this suggests that RANKL/ODF and M-CSF expressed by SaOS-4/3 cells are two critical factors essential for osteoclast formation in the coculture treated with PTH. Expression of RANKL/ODF and OPG mrnas by SaOS- 4/3 cells was examined by Northern blot analysis. A time course study showed that the expression of RANKL/ODF mrna by SaOS-4/3 cells was up-regulated as early as 6 h after addition of PTH and the level reached a maximum between 48 and 72 h (Fig. 2A). SaOS-4/3 cells constitutively expressed OPG mrna, and its expression was downregulated by adding PTH. Treatment of SaOS-4/3 cells with PTH also up-regulated the expression of M-CSF mrna of the 4.0-kb transcript in a time-dependent manner. Neither 1,25(OH) 2 D 3 nor PGE 2 affected the expression of mrnas for RANKL/ODF, OPG, and M-CSF by SaOS-4/3 cells (Fig. 2B). M-CSF mrna of the shorter transcripts (2.5 kb and 1.6 kb) was not detected under the experimental condition used. It was difficult to distinguish the 3.1-kb transcript of the membrane-associated form of M-CSF from the 4.0-kb transcript of the secreted form of M-CSF in the Northern blot analysis. Therefore, the expression of mrna encoding membrane-associated and secreted forms of M-CSF was examined by RT-PCR (Fig. 2C). Treatment of SaOS-4/3 cells with PTH up-regulated the expression of both a membrane-associated (308 bp) and a secreted form (1201 bp) of M-CSF mrna (Fig. 2C). The concentration of M-CSF in the conditioned medium of SaOS-4/3 cells cultured in 12-well plates for 3 days also was increased in the presence of PTH (control culture, 120 pg/ml; PTH-treated culture, 1110 pg/ml). A spot coculture system was employed to examine the mechanism of osteoclastogenesis supported by SaOS-4/3 cells. When the spot coculture was treated for 6 days with PTH, TRAP-positive cells were formed in the colony of SaOS-4/3 cells (Fig. 3). No TRAP-positive cells were formed outside the colony of SaOS-4/3 cells. TRAPpositive cells were formed exclusively inside but not outside the colony of SaOS-4/3 cells in the coculture treated with PTH plus M-CSF (Fig. 3). These findings suggest that the membrane-associated RANKL/ODF is functionally active in inducing osteoclastogenesis in the coculture with SaOS- 4/3 cells. To determine the distribution of M-CSF, the spot coculture with SaOS-4/3 cells was treated for 6 days with FIG. 1. Effects of OPG and anti M-CSF antibodies on PTH-induced osteoclast formation in cocultures of SaOS- 4/3 cells with bone marrow cells. (A) Mouse bone marrow cells ( cells/well) were cocultured with SaOS-4/3 cells ( cells/well) in 0.3 ml of -MEM containing 10% FBS in 48-well plates. Cocultures were treated with and without 1,25(OH) 2 D 3 (1,25D 3,10 8 M), PGE 2 (10 6 M), or PTH (10 8 M). OPG (100 ng/ml) or anti-human M-CSF antibodies (0.5 g/ml) was added to some cocultures treated with PTH. After culture for 6 days, cells were fixed and stained for TRAP. Total TRAP-positive cells including mononuclear and multinucleated cells were counted. Results were expressed as the means SEM of three cultures. (B) Enzyme histochemistry for TRAP activity in cocultures with SaOS-4/3 cells. TRAP-positive cells appeared as dark red cells. Because osteoclasts formed in the coculture tend to push away osteoblasts/stromal cells from their neighborhood, some TRAP-positive cells do not appear to contact directly with SaOS-4/3 cells. Bar 100 m. M-CSF, srankl/sodf, or srankl/sodf plus M-CSF (Fig. 4). Adding of M-CSF alone to the coculture failed to induce osteoclasts both inside and outside the colony of

5 1770 ITOH ET AL. FIG. 2. Expression of mrna for RANKL/ODF, OPG, and M-CSF by SaOS-4/3 cells treated with several osteotropic factors. (A) SaOS-4/3 cells ( cells/well) were cultured for 2 days in 10-cm dishes. Then the cells were treated with or without PTH (10 8 M) for the indicated periods. (B) After culture for 2 days, SaOS-4/3 cells were treated with or without PTH (10 8 M), 1,25(OH) 2 D 3 (1,25D 3,10 8 M), or PGE 2 (10 6 M) for an additional 48 h. Poly(A) RNA (2.5 g) was electrophoresed in an agaroseformaldehyde gel and hybridized with radioactive cdna probes for human RANKL/ODF, OPG, M-CSF, and -tubulin. (C) First-strand cdna synthesized from total RNA was subjected to PCR amplification using specific PCR primers for the membrane-associated and secreted forms of human M-CSF. The PCR products were separated by electrophoresis on an agarose gel. PCR products for mrnas encoding a membrane-associated and a secreted form of M-CSF are expected to be 308 bp and 1201 bp transcripts, respectively. The expression of GAPDH mrna (453 bp) was determined as the internal control. SaOS-4/3 cells. TRAP-positive cells were formed only inside the colony of SaOS-4/3 cells, when the coculture was treated with srankl/sodf. No TRAP-positive cells were observed outside the colony of SaOS-4/3 cells. When the coculture was treated with srankl/sodf plus M-CSF, TRAP-positive cells were formed not only inside but also outside the colony of SaOS-4/3 cells (Fig. 4). The number of TRAP-positive cells that formed inside the colony of SaOS-4/3 cells treated with both srankl/sodf and M-CSF was higher than that with srankl/sodf alone. When the spot coculture was treated with PTH plus srankl/sodf, TRAP-positive cells were formed only inside the colony of SaOS-4/3 cells (data not shown). It is therefore likely that a membrane- or a matrix-associated form of M-CSF as well as that of ODF/RANKL is critical for inducing osteoclastogenesis in the coculture. The osteoclast-inducing activity of SaOS-4/3 cells was compared between cocultures with a small and a large volume of the culture medium at the same cell number (Fig. 5). PTH markedly increased the concentration of M-CSF in the conditioned medium obtained from the small and large volume cultures (Fig. 5A). High concentrations of M-CSF ( 40 ng/ml) have been considered to rather inhibit osteoclast formation in the hemopoietic cell cultures. (4,31) The concentration of M-CSF in the small volume culture treated with PTH ( 5 ng/ml) was much lower than that required to inhibit osteoclast formation in the cocultures. The concentration of M-CSF in the large volume culture was only one-ninth of that in the small volume culture. Nevertheless, the number of TRAP-positive cells formed inside the colony was quite similar between the large volume culture and the small volume culture (Fig. 5B). These findings suggest that the secreted forms of M-CSF and RANKL/ODF in the conditioned medium are not important for TRAP-positive cell formation in the coculture. The mechanism of osteoclastogenesis was further examined using the spot coculture system with primary osteoblasts (Fig. 6). When the spot coculture was treated with PTH, TRAP-positive cells were formed only inside the colony of osteoblasts. The TRAP-positive cell formation occurred only inside the colony of osteoblasts in response to PTH, even when M-CSF or srankl/odf was added simultaneously. TRAP-positive cells also were formed only inside the colony of primary osteoblasts in the coculture treated with srankl/sodf. No TRAP-positive cells were formed in the spot coculture treated with M-CSF alone. DISCUSSION TRAP-positive osteoclast formation supported by SaOS- 4/3 cells was completely inhibited by adding either OPG or antibodies against human M-CSF. Expression of both RANKL/ODF and M-CSF mrnas was up-regulated by the treatment of SaOS-4/3 cells with PTH. Other osteotropic factors such as 1,25(OH) 2 D 3 and PGE 2 did not induce osteoclast formation in cocultures with SaOS-4/3 cells either. They did not stimulate expression of RANKL/ODF and M-CSF mrnas by SaOS-4/3 cells. Osteoclasts were formed in mouse bone marrow cultures treated with srankl/sodf and M-CSF in the absence of osteoblasts/ stromal cells. (32) These findings suggest that the two factors, RANKL/ODF and M-CSF, satisfy necessary and sufficient conditions for osteoclastogenesis supported by SaOS-4/3 cells. The spot coculture of SaOS-4/3 cells and bone marrow cells clearly showed that the RANKL/ODF and M-CSF expressed as a membrane- or a matrix-associated form were involved in osteoclast differentiation from their progenitors

6 THE MECHANISM OF OSTEOCLASTOGENESIS 1771 FIG. 3. Localization of RANKL/ODF activity in the spot coculture of SaOS-4/3 cells and mouse bone marrow cells. (A) SaOS-4/3 cells ( cells/0.05 ml) were spot-cultured for 2hin -MEM containing 10% FBS in the center of a single culture well of 12-well plates. Mouse bone marrow cells ( cells) were then uniformly plated over the culture well and further cultured for 6 days in the presence of PTH (10 8 M) together with or without M-CSF (100 ng/ml). Cells were then fixed and stained for TRAP and ALP. TRAP-positive cells (dark red cells) that formed inside and outside the colony of SaOS-4/3 cells (blue cells) were counted separately. Results were expressed as the means SEM of three cultures. (B) Enzyme histochemistry for TRAP and ALP activity in the spot coculture treated with PTH (upper panels) or PTH plus M-CSF (lower panels). Left panels show a low power view of the spot coculture. Right panels show a high power view of the portions in squares indicated in the left panels. Bar 100 m. in the coculture. Osteoclasts were never formed outside the colony of SaOS-4/3 cells or primary osteoblasts, even when M-CSF was simultaneously added to the coculture treated with PTH. These findings indicate that RANKL/ODF expressed by osteoblasts/stromal cells functions as the membrane-associated form in inducing osteoclast formation in the coculture. In contrast, activated murine T cells have been shown to secrete a soluble form of RANKL/ODF into the conditioned medium, which in turn enhances osteoclast formation in mouse bone marrow cultures in the presence of M-CSF. (13) Such a difference in the RANKL/ODF activity in the conditioned medium may be caused by the difference in cell types. Osteoblasts/stromal cells may predominantly produce the membrane-associated form of RANKL/ODF, whereas T cells preferentially produce its secreted form. Further studies are necessary to elucidate the difference in the precise mechanism between osteoclast formation supported by osteoblasts/stromal cells and activated T cells. It has been shown that the membrane-associated form of M-CSF expressed by NIH 3T3 cells support osteoclast formation. (12) Consistent with the previous finding, M-CSF acted as a membrane- or a matrix-associated form in osteoclast formation in our coculture system. When the spot coculture was treated with either PTH or srankl/sodf, osteoclast formation occurred only inside the colony of SaOS-4/3 cells or osteoblasts. Although the concentration of M-CSF in the conditioned medium of the large volume culture was much lower than that in the small volume culture of SaOS-4/3 cells, a similar number of osteoclasts was formed inside the colony in both culture conditions. These findings suggest that the SaOS-4/3 cell induced osteoclastogenesis is little affected by the concentration of M-CSF secreted into the culture medium. The exact form of M-CSF in the cell layers of SaOS-4/3 cells and osteoblasts is not known at present. As has been proposed, (22) a membrane-associated form of M-CSF appears to contribute to osteoclast formation supported by SaOS-4/3 cells or osteoblasts. In fact, SaOS-4/3 cells constitutively expressed mrna for a membrane-associated form of M-CSF, expression of which was up-regulated by PTH. A matrixassociated form of M-CSF is produced from a secreted form of M-CSF through the attachment of a glycosaminoglycan side chain. (24-26) Therefore, it is likely that a secreted form of M-CSF also is involved as a matrix-associated form in osteoclast formation supported by osteoblasts/stromal cells.

7 1772 ITOH ET AL. FIG. 4. Localization of M-CSF activity in the spot coculture of SaOS-4/3 cells and mouse bone marrow cells. (A) SaOS-4/3 cells (1 103 cells/0.05 ml) were spot-cultured for 2 h in -MEM containing 10% FBS in the center of a single culture well (12-well plate). Mouse bone marrow cells (5 104 cells) were then uniformly plated over the culture well and further cultured for 6 days in the presence of M-CSF (100 ng/ml), srankl/sodf (100 ng/ml), or srankl/sodf (100 ng/ml) plus M-CSF (100 ng/ml). Cells were then fixed and stained for TRAP and ALP. TRAP-positive cells (dark red cells) that formed inside and outside the colony of SaOS-4/3 cells (blue cells) were counted separately. Results were expressed as the means SEM of three cultures. (B) Enzyme histochemistry for TRAP and ALP activity in the spotcoculture treated with M-CSF (upper panels), srankl/sodf (middle panels), or M-CSF plus srankl/sodf (lower panels). Left panels show a low power view of the spot coculture. Right panels show a high power view of the portions in squares indicated in the left panels. Bar 100 m.

8 THE MECHANISM OF OSTEOCLASTOGENESIS 1773 FIG. 5. Effects of increasing volumes of the culture medium on the concentration of M-CSF secreted and TRAPpositive cell formation supported by SaOS-4/3 cells. A mixture of SaOS-4/3 cells ( cells/0.1 ml) and bone marrow cells ( cells/0.1 ml) were spot-cultured for 2 h in the center of a single well (22 mm) of 12-well plates (small volume culture) or in the center of 60-mm culture dishes (large volume culture). The cocultures were then treated for 6 days with or without PTH (10 8 M) in 0.5 ml (small volume culture) or 5.0 ml (large volume culture) of -MEM containing 10% FBS. (A) The conditioned medium was then collected and the concentration of M-CSF was measured using the ELISA kit. Cells also were fixed and stained for TRAP and ALP. (B) TRAP-positive cells formed inside the colony were counted. Results were expressed as the means SEM of three cultures. PTH and PTHrP have been shown to increase expression of M-CSF in human and rat osteoblastic cells. (22,32) Production of M-CSF as well as expression of its mrna by SaOS-4/3 cells also was up-regulated by adding PTH. However, up-regulation of M-CSF production by PTH was not essential for osteoclast formation supported by SaOS-4/3 cells, because adding M-CSF alone to the spot coculture with SaOS-4/3 cells failed to induce osteoclast formation. In contrast, srankl/sodf added to the spot coculture induced osteoclasts inside the colony of SaOS-4/3 cells or osteoblasts. PTH up-regulated RANKL/ODF mrna expression, whereas it down-regulated OPG mrna expression by SaOS-4/3 cells. These findings suggest that an essential step in PTH-induced osteoclast formation in the coculture is the stimulation of RANKL/ODF-RANK interaction. The number of osteoclasts formed inside the colony of SaOS-4/3 cells treated with srankl/sodf alone was lower than that with srankl/sodf plus M-CSF. Sarma et al. (33) also reported that estradiol down-regulated mrna expression and protein synthesis of the membraneassociated form of M-CSF in human osteoblasts. Therefore, FIG. 6. Localization of RANKL/ODF and M-CSF activities in the spot coculture with primary osteoblasts. Primary osteoblasts ( cells/0.05 ml) were spot-cultured for 2hin -MEM containing 10% FBS in the center of a single culture well of 12-well plates. Mouse bone marrow cells ( cells) were then uniformly plated over the culture well. The spot coculture was treated for 6 days with PTH (10 8 M), PTH (10 8 M) plus M-CSF (100 ng/ml), PTH (10 8 M) plus sodf/srankl (100 ng/ml), sodf/ srankl (100 ng/ml), or M-CSF (100 ng/ml). Cells were then fixed and stained for TRAP and ALP. (A) TRAPpositive cells (dark red cells) that formed inside and outside the colony of primary osteoblasts (blue cells) were counted separately. Results were expressed as the means SEM of three cultures. (B) Enzyme histochemistry for TRAP and ALP activity in the spot coculture with primary osteoblasts. Left and right panels show portions inside and outside the colony of primary osteoblasts, respectively. Bar 100 m.

9 1774 ITOH ET AL. alteration of M-CSF production by osteoblasts/stromal cells is an important event in the regulation of osteoclastogenesis supported by osteoblasts/stromal cells. In spite of the relatively wide distribution of RANKL/ ODF (4 7) and M-CSF (34) expression, osteoclasts are formed only in bone. It is not known how osteoclast progenitors recognize the precise site where they differentiate into osteoclasts. Both RANKL/ODF and M-CSF are capable of acting on osteoclast progenitors as a soluble form. Injection of M-CSF into M-CSF deficient op/op mice induces osteoclasts in the precise site of bone where bone resorption takes place. (14,15) Disruption of the gene encoding OPG developed severe osteoporosis in mice as a result of enhanced osteoclastic bone resorption. (35,36) Histochemical analysis showed that the increased number of TRAPpositive osteoclasts in OPG-deficient mice was found only in calcified bone but not in soft tissues. There must be other specific mechanisms that induce osteoclast formation at an appropriate site of bone. Hofstetter et al. (37) reported that M-CSF is strongly expressed in mineralized cartilage, where osteoclasts appear in mouse embryonic bone rudiments. A proteoglycan form of M-CSF was immunohistochemically detected on the surface of human bone. (38) These findings suggest that the local production and distribution of M-CSF are important for osteoclastogenesis. Our present findings showed that both RANKL/ODF and M-CSF act as a membrane- or a matrix-associated form on osteoclast progenitors in inducing osteoclast formation. Such a mechanism of action of RANKL/ODF and M-CSF on osteoclast progenitors may be important for the selective formation of osteoclasts in bone. Recently, Arai et al. (39) described that differentiation of osteoclasts from bipotential precursors of macrophages and osteoclasts in methylcellulose culture was poorer than that observed in the liquid culture system. 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