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Endocrinology Vol. 140, No. 2 925-932
Copyright © 1999 by The Endocrine Society

ARTICLES

Human Osteoclast-Like Cells Are Formed from Peripheral Blood Mononuclear Cells in a Coculture with SaOS-2 Cells Transfected with the Parathyroid Hormone (PTH)/PTH-Related Protein Receptor Gene

Kenichiro Matsuzaki, Kazuhiko Katayama, Yasuyuki Takahashi, Ichiro Nakamura, Nobuyuki Udagawa, Taro Tsurukai, Ryuichi Nishinakamura, Yoshiaki Toyama, Yutaka Yabe, Masayuki Hori, Naoyuki Takahashi and Tatsuo Suda

Department of Biochemistry, School of Dentistry, Showa University (K.M., I.N., N.U., T.T., N.T., T.S.), 1–5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555; Asahi Chemical Industry Company, Ltd., Laboratory for Bone Metabolism (K.K., Y.T., M.H.), 632–1 Mifuku, Ohito-cho, Tagata-gun, Shizuoka 410-2321; Institute of Medical Science University of Tokyo (R.N.), 4–6-1 Shiroganedai, Minato-ku, Tokyo 108-0071; and Department of Orthopaedic Surgery, School of Medicine, Keio University (K.M., Y.T., Y.Y.), 35 Shinanomachi, Shinjuku-ku, Tokyo 160-0016, Japan

Address all correspondence and requests for reprints to: Yasuyuki Takahashi, Asahi Chemical Industry Company, Ltd., Laboratory for Bone Metabolism, 632–1 Mifuku, Ohito-cho, Tagata-gun, Shizuoka 410-2321, Japan. E-mail: a7911076{at}ut.asahi-kasei.co.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Subclones of the human osteosarcoma cell line SaOS-2 were established by transfecting with an expression vector containing the human PTH/PTH-related protein (PTHrP) receptor, and their abilities to support osteoclast-like multinucleated cell (OCL) formation were examined in coculture with mouse or human hemopoietic cells. Of four subclones examined, SaOS-2/4 and SaOS-4/3 bound high levels of [125I]-PTH and produced a significant amount of cAMP in response to PTH. OCLs were formed in response to PTH in the cocultures of mouse bone marrow cells with either SaOS-2/4 cells or SaOS-4/3 cells. Human OCLs were also formed in response to PTH in the coculture of SaOS-4/3 cells and human peripheral blood mononuclear cells. Adding dexamethasone together with PTH greatly enhanced PTH-induced human OCL formation. Like mouse OCLs, human OCLs formed in response to PTH were tartrate-resistant acid phosphatase positive, expressed abundant calcitonin receptors and vitronectin receptors, and formed resorption pits on dentine slices. Other osteotropic factors such as 1{alpha},25-dihydroxyvitamin D3, prostaglandin E2, and interleukin 6 plus soluble interleukin 6 receptors failed to induce mouse and human OCLs in cocultures with SaOS-4/3 cells. Both mouse and human OCL formation supported by SaOS-4/3 cells were inhibited by either adding an antibody against macrophage-colony stimulating factor or adding granulocyte/macrophage-colony stimulating factor. Thus, it is likely that human and mouse OCL formation supported by SaOS-4/3 cells are similarly regulated. These results indicate that the target cells of PTH for inducing osteoclast formation are osteoblast/stromal cells but not osteoclast progenitor cells in the coculture. This coculture model will be useful for investigating the abnormalities of osteoclast differentiation and function in human metabolic bone diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOCLASTS ARE MULTINUCLEATED cells responsible for bone resorption (1, 2, 3, 4). Evidence indicates that osteoclasts are derived from hematopoietic cells, probably from monocyte-macrophage lineage cells (2, 5). We previously reported that osteoclast-like multinucleated cells (OCLs) were formed in response to several bone-resorbing factors in mouse bone marrow cultures and in cocultures of mouse hemopoietic cells and osteoblastic stromal cells (6, 7, 8, 9). Using these culture systems, we have demonstrated that either osteoblasts or bone marrow-derived stromal cells play a critical role in OCL formation through cell-cell interaction with OCL progenitors (1, 2).

Reports have suggested that human osteoclast development differs from mouse osteoclast development in several ways. Reports have shown that human hemopoietic progenitors differentiate into mature osteoclasts in the absence of osteoblasts/stromal cells in culture (3, 10, 11, 12, 13). Hemopoietic growth factors required for osteoclast development also differ between mouse and human culture systems: macrophage-colony stimulating factor (M-CSF) is an essential factor for mouse osteoclast formation (14, 15, 16, 17), whereas granulocyte/macrophage-colony stimulating factor (GM-CSF) is needed as the corresponding growth factor in human osteoclast formation (3, 13, 18). Mouse OCLs are formed within a week in culture, whereas a relatively long culture period (2–3 weeks) is required for human OCL formation (3, 13, 18). The physiological importance of these differences are unknown.

PTH and PTH-related protein (PTHrP) trigger many process in target cells by binding to PTH/PTHrP receptors (PTH/PTHrPR) (19, 20, 21). To compare the regulatory mechanisms of human and mouse osteoclast formation, we established subclones of the human osteosarcoma cell line SaOS-2 (22, 23) by transfection with an expression vector containing the human PTH/PTHrPR gene. One of these subclones, designated SaOS-4/3, expressed a high level of functional PTH/PTHrPR and supported OCL formation in response to PTH in cocultures with mouse or human hemopoietic cells. Both mouse and human OCL formation required cell-to-cell contact with SaOS-4/3 cells. M-CSF was an essential factor for both mouse and human OCL formation, whereas GM-CSF inhibited OCL formation in both cocultures. The present study indicates that the target cells of PTH for inducing osteoclast formation are osteoblasts/stromal cells, but not osteoclast progenitors in the coculture. It is likely that human osteoclast formation is regulated in a similar fashion to mouse osteoclast formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and chemicals
Five-week-old male ddY mice and newborn ddY mice were obtained from Sankyo Laboratories Animal Center (Tokyo, Japan). Mice deficient for the IL-3/GM-CSF/IL-5 ßc Receptor were generated on a hybrid background (129 x C57BL/6 F2) and also on a 129/Sv background as described (24). Human PTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) [hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)] and Elcatonin, a synthetic analog of eel calcitonin, were synthesized by Asahi Chemical Industry (Tokyo, Japan). [Nle8,18,Tyr34]hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) was chemically synthesized and purified on HPLC by Asahi Chemical Industry. The peptide was commercially labeled with Na125I by Amersham Co. (Buckinghamshire, UK) using the chloramine-T method. 1{alpha},25-dihydroxyvitamin D3 [1{alpha},25(OH)2D3] were purchased from Wako Pure Chemicals Co. (Osaka, Japan). Prostaglandin E2 (PGE2) and HISTOPAQUE 1077 were obtained from Sigma Chemical Co. (St. Louis, MO). Monoclonal antibody 23C6 was kindly provided by Dr. Michael A. Horton (The Middlesex Hospital, UK). 125I-labeled salmon calcitonin (specific activity, 74 TBq/mmol) was purchased from Amersham Co.. Human interleukin 6 (IL-6), human soluble IL-6 receptor (sIL-6R), recombinant human GM-CSF [hGM-CSF], recombinant mouse GM-CSF [mGM-CSF], and monoclonal antihuman M-CSF neutralizing antibody were obtained from R & D Systems Co. (Minneapolis, MN).

Transfection of the human PTH/PTHrP receptor (PTH/PTHrPR) gene into SaOS-2 cells
Human and rat kidney complementary DNA (cDNA) libraries were constructed from respective poly (A)+ RNAs (CLONTECH Laboratories, Inc., Palo Alto, CA) using a Time Saver cDNA synthesis kit (Pharmacia, Uppsala, Sweden). The rat kidney cDNA library was screened with the [32P]-Iabeled oligonucleotide (5'-TGGTGTCCCAGCCCTTGTCTGACACCATTATGT-3') encoding a complementary sequence to rat PTH/PTHrPR cDNA from 285 to 254. One clone designated as RR2 had a 1.8 kb insert of rat PTH/PTHrPR cDNA. The human cDNA containing an entire open reading frame was obtained by screening a human kidney cDNA library using a [32P]-labeled 1.4 kb BstXI fragment of clone RR2. The human cDNA clone HR3 obtained had 2119 bp insert of human PTH/PTHrPR cDNA containing 216 bp of the 5'-noncoding region and 120 bp of the 3'-noncoding region.

A mammalian expression vector pSR{alpha}-dhfr-neo was constructed from pcDL-SR{alpha}296 with the dhfr gene of pSV2-dhfr (Gibco BRL) and with the neo gene of pSV2-neo (Gibco BRL) (K. Katayama, unpublished results). The vector pcDL-SR{alpha}296 was kindly provided by Y. Takebe (National Institute of Health, Tokyo). A 2.1-kb fragment from the human cDNA clone HR3 was inserted into the downstream of the SR{alpha} promoter in pSR{alpha}-dhfr-neo. The human osteosarcoma cell line SaOS-2 was obtained from American Type Culture Collection and maintained in RPMI1640 supplemented with 10% FBS. SaOS-2 cells were transfected with the expression vector using a calcium phosphate-DNA precipitation method (25) and cultured in RPMI1640 supplemented with 10% FBS and 2 mg/ml of Geneticin. Individual Geneticin-resistant colonies were selected and expanded in multiwell dishes. Four clones designated as SaOS-125, SaOS-77, SaOS-2/4, and SaOS-4/3 were chosen for a more detailed examination.

Measurement of PTH binding and PTH-dependent cAMP production
The parent SaOS-2 and its subclonal cells (SaOS-125, SaOS-77, SaOS-2/4, and SaOS-4/3) (5 x 104 cells/well) were cultured for 48 h in 48-well plates and incubated with [125I]-hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (5 x 104 cpm/well) in the presence of increasing concentrations of hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) at 15 C. After incubation for 4 h, cells were washed three times with PBS, and solubilized in 0.4 ml of 0.1 M NaOH. The radioactivity of the cell lysate was measured using a {gamma} counter. Binding of [125I]-hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) was expressed as cpm/well. For cAMP determination, SaOS-2-derived subclones (1.5 x 105 cells/well) cultured in 24-well plates were incubated with increasing concentrations of hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) in the presence of 1 mM isobutylmethylxanthine for 20 min. Intracellular cAMP was extracted with 65% ethanol, and the amount of cAMP in the extracts was determined using a cAMP ELISA kit.

Coculture system
Human peripheral blood was collected in syringes containing 1,000 U/ml preservative-free heparin from healthy normal donors. Informed consent was obtained in all cases before blood aspiration. Peripheral blood mononuclear cells were isolated by centrifugation over HISTOPAQUE 1077 density gradients, washed, and resuspended in {alpha}-MEM. Human studies were approved by the Showa University Institutional Review Board. Mouse bone marrow cells were obtained from tibiae of 6- to 9-week-old ddY male mice or 5-week-old GM-CSF receptor-deficient mice as described previously (26, 27). Mouse calvarial cells were obtained from calvariae of newborn ddY mice (6). The procedures for experiments using mice were approved by the Showa University Animal Management Committee.

Mouse bone marrow cells (1 x 105 cells/well) or human peripheral blood mononuclear cells (2 x 105 cells/well) were cocultured for 6–12 days with either the parent SaOS-2 cells or its subclonal cells (1 x 104 cells/well) in 0.3 ml of {alpha}-MEM containing 10% FBS in 48-well plates (Corning, Corning, NY). Cocultures were treated with or without bone-resorbing factors such as hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)(100 ng/ml), 1{alpha},25(OH)2D3 (10-8 M), PGE2 (10-6 M), and human IL-6 (50 ng/ml) plus human sIL-6R (200 ng/ml) in the presence or absence of dexamethasone (10-7 M). Culture medium was replaced every 3 days with fresh medium supplemented with cytokines and hormones.

Determination of osteoclast characteristics
After culture for indicated periods, cells were fixed and stained for tartrate resistant acid phosphatase (TRAP) as described previously (9). TRAP-positive cells containing more than three nuclei were counted as OCLs. For autoradiography using [125I]-labeled salmon calcitonin, cocultures were performed on coverslips placed in 24-well plates. Cultures were then incubated with 2 x 10-10 M [125I]-calcitonin, stained for TRAP, and processed for autoradiography as previously described (27). Nonspecific binding was assessed in the presence of an excess amount (20 nM) of unlabeled Elcatonin (eel calcitonin). For immunohistochemical staining, cells were fixed with cold methanol-acetone (50:50, vol/vol) for 10 min and incubated with a monoclonal antibody against vitronectin receptors (23C6) as described (28, 29). The bound antibodies were visualized with biotinylated second antibodies, avidin-biotin conjugated peroxidase, and an AEC substrate kit (Histofine, Nichirei Co, Tokyo, Japan). Pit-forming activity of OCLs formed in cocultures with SaOS-4/3 cells was assayed according to the procedure described previously (6, 30). Mouse bone marrow cells (1 x 105 cells/well) or human peripheral blood mononuclear cells (2 x 105 cells/ml) and SaOS-4/3 cells (1 x 104 cells/well) were cocultured on dentine slices (diameter, 4 mm) in the presence of hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (100 ng/ml) in 48-well plates. After culture for 7 days, adherent cells were removed from the slices, and the resorption pits on the slices were stained with Mayer’s hematoxylin (30).

Statistical analysis
The results were expressed as the means ± SEM (SEM) of quadruplicate cultures. Significance of the differences was determined using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SaOS-2 cells were transfected with a pSR{alpha}-dhfr-neo expression vector containing the human PTH/PTHrPR gene by a calcium phosphate-DNA precipitation method. A total of 44 distinct subclones displaying different levels of PTH-binding activity and PTH-induced cAMP production were obtained as neomycin-resistant SaOS-2-derived subclones. Four subclonal cell lines designated as SaOS-125, SaOS-77, SaOS-2/4, and SaOS-4/3 were chosen for further examination. Figure 1AGo shows [125I]-hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)-binding activity of the parent SaOS-2 and four subclonal cell lines in the presence of increasing concentrations of unlabeled hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). These cell lines bound different levels of [125I]-hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), which were dose dependently displaced by simultaneously adding unlabeled hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). SaOS-2/4 and SaOS-4/3 had the highest hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)-binding activity, followed by SaOS-77, SaOS-125 and the parent SaOS-2. A marked increase in cAMP production in response to hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) was observed in SaOS-2/4 cells and SaOS-4/3 cells (Fig. 1BGo). To examine the ability of the parent SaOS-2 and its subclonal cells to support OCL formation, cells were cocultured with mouse bone marrow cells in the presence or absence of hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (10-8 M) (Fig. 1CGo). OCLs were formed in response to hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) only in coculture with SaOS-2/4 cells or SaOS-4/3 cells. SaOS-4/3 was then selected for further studies.



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  		Figure 1. Characterization of SaOS-2 and its subclones transfected with the human PTH/PTHrPR gene. A, The parent SaOS-2 and its subclones (SaOS-125, SaOS-77, SaOS-2/4, and SaOS-4/3) (5 x 104 cells/well) cultured in 48-well plates were incubated with [125I]-hPTH (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (5 x 104 cpm/well) together with increasing concentrations of unlabeled hPTH (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ). After incubation for 4 h, cells were washed with PBS and dissolved in 0.4 ml of 0.1 M NaOH. The radioactivity of the cell lysate were measured. (B) The parent SaOS-2 cells and its subclonal cells (1.5 x 105 cells/well) were cultured in 24-well plates and then incubated for 20 min with increasing concentrations of hPTH (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ). cAMP in the cell layer was then measured. C, The parent SaOS-2 and its subclones (2 x 104 cells/well) were cocultured with mouse bone marrow cells (2 x 105 cells/well) in {alpha}-MEM containing 10% FBS in 24-well plates with or without hPTH (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (10-8 M). After 7 days, cells were fixed and stained for TRAP. TRAP-positive multinucleated cells were counted. Results are expressed as the mean ± SEM of triplicate cultures. Similar results were obtained in an additional set of experiments.

 
Fujikawa et al. (31) first demonstrated that human peripheral blood mononuclear cells differentiated into osteoclasts when cocultured with the mouse stromal cell line, ST2, or the rat osteoblastic cell line, UMR-106. Therefore, human peripheral blood mononuclear cells were cocultured with SaOS-4/3 cells in the presence or absence of hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (Fig. 2Go). Addition of hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) alone to coculture of SaOS-4/3 cells and human peripheral blood mononuclear cells slightly stimulated OCL formation, but adding 10-7 M dexamethasone together with hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) greatly stimulated PTH-induced OCL formation (Fig. 2BGo). On the contrary, dexamethasone was not required for mouse OCL formation induced by hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (Fig. 2AGo). Therefore, dexamethasone (10-7 M) was added to cocultures with human peripheral blood mononuclear cells in subsequent experiments ( Figs. 3–8GoGoGoGoGoGo).



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  		Figure 2. The requirement of dexamethasone for OCL formation in human coculture system. Mouse bone marrow cells (1 x 105 cells/well) (A) or human peripheral blood mononuclear cells (2 x 105 cells/well) (B) were cocultured with SaOS-4/3 cells (1 x 104 cells/well) in 0.3 ml of {alpha}-MEM containing 10% FBS in 48-well plates. Cocultures were treated with or without 100 ng/ml of hPTH (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) in the presence or absence of dexamethasone (Dex) (10-7 M). After 7 days, cells were fixed and stained for TRAP. TRAP-positive multinucleated cells were counted. Results are expressed as the mean ± SEM of quadruplicate cultures. Significantly different from the culture treatment with vehicle: (*) P < 0.01. Similar results were obtained in three additional sets of experiments.

 


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  		Figure 3. Effects of various osteotropic factors on TRAP-positive multinucleated cell formation in cocultures of SaOS-4/3 cells with either mouse bone marrow cells (A) or human peripheral blood mononuclear cells (B). Mouse bone marrow cells (1 x 105 cells/well) or human peripheral blood mononuclear cells (2 x 105 cells/well) were cocultured with SaOS-4/3 cells (1 x 104 cells/well) in 0.3 ml of {alpha}-MEM containing 10% FBS in 48-well plates. Cocultures were treated with or without bone-resorbing factors such as hPTH (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (100 ng/ml), 1{alpha},25(OH)2D3 (1,25D3) (10-8 M), PGE2 (10-6 M), and human IL-6 (50 ng/ml) plus human sIL-6R (200 ng/ml). Dexamethasone (10-7 M) was added to all the cocultures with human peripheral blood mononuclear cells. After 7 days, cells were fixed and stained for TRAP. TRAP-positive multinucleated cells were counted. Results are expressed as the mean ± SEM of quadruplicate cultures. Similar results were obtained in three additional sets of experiments.

 


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  		Figure 4. Time course of change in the TRAP-positive multinucleated cell formation in cocultures of SaOS-4/3 cells with either mouse bone marrow cells (A) or human peripheral blood mononuclear cells (B). Mouse bone marrow cells (1 x 105 cells/well) or human peripheral blood mononuclear cells (2 x 105 cells/well) were cocultured with SaOS-4/3 cells (1 x 104 cells/well) in 0.3 ml of {alpha}-MEM containing 10% FBS in 48-well plates. Cocultures were treated with (•) or without ({circ}) hPTH (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (100 ng/ml). Dexamethasone (10-7 M) was added to all the cocultures with human peripheral blood mononuclear cells. After coculture for indicated periods, cells were fixed and stained for TRAP. TRAP-positive multinucleated cells were counted. Results are expressed as the mean ± SEM of quadruplicate cultures. Similar results were obtained in three additional sets of experiments.

 


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  		Figure 5. Characteristics of osteoclasts expressed by multinucleated cells formed in the cocultures of SaOS-4/3 cells with either mouse bone marrow cells (A–C) or human peripheral blood mononuclear cells (D–F). Mouse bone marrow cells (1 x 105 cells/well) (A) or human peripheral blood mononuclear cells (2 x 105 cells/well) (B) were cocultured with SaOS-4/3 cells (1 x 104 cells/well) in 0.3 ml of {alpha}-MEM containing 10% FBS in 48-well plates. Cocultures were treated with 100 ng/ml of hPTH (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ). Dexamethasone (10-7 M) was added to all the cocultures with human peripheral blood mononuclear cells. After 7 days, cultures were fixed and stained for TRAP (A, D). Bar, 200 µm. For immunohistochemical staining specific for human osteoclasts, cultures were incubated with monoclonal antibodies against vitronectin receptors (23C6). Cells expressing antigens were stained brown (E). Bar, 200 µm. To examine pit-forming activity of MNCs, cocultures were performed on dentine slices (diameter, 4 mm) placed in 48-well plates. After culture for 7 days, adherent cells were removed from the slices, and the resorption pits on the slices were stained with Mayer’s hematoxylin (C, F). Bar, 200 µm.

 


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  		Figure 6. Autoradiography of [125I]-calcitonin binding to TRAP-positive multinucleated cells formed in cocultures of SaOS-4/3 cells with either mouse bone marrow cells (A, B) or human peripheral blood mononuclear cells (C, D). Mouse bone marrow cells (2 x 105 cells/well) (A and B) or human peripheral blood mononuclear cells (4 x 105 cells/well) (C and D) were cocultured with SaOS-4/3 cells (2 x 104 cells/well) in 0.5 ml of {alpha}-MEM containing 10% FBS on coverslips placed in 24-well plates. Cocultures were treated with 100 ng/ml of hPTH (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ). Dexamethasone (10-7 M) was added to all the cocultures with human peripheral blood mononuclear cells. After 7 days, cultures were incubated with 2 x 10-10 M [125I]-calcitonin in the presence or absence of an excess amount (20 nM) of unlabeled Elcatonin (eel calcitonin). After incubation for 1 h, cells were fixed and stained for TRAP, and processed for autoradiography as described (27 ). Numerous grains due to [125I]-calcitonin binding were observed on TRAP-positive MNCs (A, C). Simultaneously adding an excess amount (20 nM) of unlabeled Elcatonin caused the dense grains to disappear completely (Fig. 6Go, B and D). Bar, 200 µm.

 


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  		Figure 7. Requirement of human M-CSF for OCL formation in the human coculture system. SaOS-4/3 cells (1 x 104 cells/well) were cocultured with either mouse bone marrow cells (1 x 105 cells/well) (A) or human peripheral blood mononuclear cells (2 x 105 cells/well) (B) in the presence of hPTH (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (100 ng/ml) in 48-well plates. Dexamethasone (10-7 M) was added to all the cocultures with human peripheral blood mononuclear cells. Mouse bone marrow cells and calvarial cells were cocultured in the presence of hPTH (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (100 ng/ml) in 48-well plates (C). Various concentrations of antihuman M-CSF antibodies were added to the cocultures. After 7 days, cells were fixed and stained for TRAP. TRAP-positive multinucleated cells were counted. Results are expressed as the mean ± SEM of quadruplicate cultures. Significantly different from the culture treated with PTH: **, P < 0.05; *, P < 0.01. Similar results were obtained in three additional sets of experiments.

 


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  		Figure 8. Effects of GM-CSF on OCL formation. Mouse bone marrow cells (1 x 105 cells/well) (A) obtained from wild-type mice or human peripheral blood mononuclear cells (2 x 105 cells/well) (B) or bone marrow cells (1 x 105 cells/well) (C) obtained from GM-CSF receptor deficient mice were cocultured with SaOS-4/3 cells (1 x 104 cells/well) in the presence or absence of hPTH (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) (100 ng/ml). Some of the cocultures were treated with hGM-CSF (10 ng/ml) or mGM-CSF (10 ng/ml). After 7 days, cells were fixed and stained for TRAP. TRAP-positive multinucleated cells were counted. Results are expressed as the mean ± SEM of quadruplicate cultures. Significantly different from the culture treated with PTH: *, P < 0.01. Similar results were obtained in three additional sets of experiments.

 
Effects of various osteotropic factors, such as hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), 1{alpha},25(OH)2D3, PGE2, and human IL-6 plus human sIL-6R on mouse and human OCL formation were examined (Fig. 3Go). Mouse and human OCLs were formed from respective hemopoietic cells in response to hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) in coculture with SaOS-4/3 cells. However, none of the other bone-resorbing factors induced mouse or human OCL formation (Fig. 3Go). The concentrations of 1{alpha},25(OH)2D3, PGE2, and human IL-6 plus human sIL-6R used in this experiment stimulated OCL formation in cocultures of mouse calvarial cells and bone marrow cells (data not shown).

Figure 4Go shows the time course of changes in OCL formation in cocultures of SaOS-4/3 cells with either mouse bone marrow cells or human peripheral blood mononuclear cells. Mouse OCL formation peaked around day 6 and slightly decreased on day 9 in coculture treated with hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (Fig. 4AGo). Similarly, maximal formation of human OCLs was observed on day 6 (Fig. 4BGo). No TRAP-positive cells appeared in the control cocultures treated without hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) throughout the experimental period (Fig. 4Go).

Figure 5Go shows characteristics of multinucleated cells formed from mouse and human hemopoietic cells in response to hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) in cocultures with SaOS-4/3 cells. Multinucleated cells and some mononuclear cells that appeared in the cocultures with mouse or human hemopoietic cells stained for TRAP (Fig. 5Go, A and D). Multinucleated cells and some mononuclear cells formed from human peripheral blood mononuclear cells stained with the antivitronectin receptor antibody, 23C6 (Fig. 5EGo). Consistent with previous findings that 23C6 does not recognize mouse vitronectin receptors, multinucleated cells formed from mouse bone marrow cells failed to react with 23C6 antibody (Fig. 5BGo). When mouse bone marrow cells or human peripheral blood mononuclear cells were cocultured on dentine slices, resorption pits were similarly formed on the slices recovered from the cocultures treated with hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (100 ng/ml) (Fig. 5Go, C and F).

Autoradiography using [125I]-calcitonin showed that numerous grains due to the binding of [125I]-calcitonin accumulated on TRAP-positive mononuclear and multinucleated cells of both mouse and human origin (Fig. 6Go, A and C). About 90% of TRAP-positive multinucleated cells expressed calcitonin receptors. Simultaneously adding an excess amount (20 nM) of unlabeled Elcatonin (eel calcitonin) caused the dense grains to disappear completely (Fig. 6Go, B and D).

M-CSF is considered to be an essential factor that regulates proliferation and differentiation of mouse osteoclast precursors (14, 15, 16, 17). Mouse (Fig. 7AGo) and human (Fig. 7BGo) OCL formation in cocultures with SaOS-4/3 cells were dose dependently inhibited by adding a monoclonal antibody against human M-CSF. Human OCL formation in coculture with SaOS-4/3 cells treated with hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) in the absence of dexamethasone (28.8 ± 2.2/well, the mean ± SEM of quadruplicate cultures) was completely blocked by adding monoclonal antibody at 5 µg/ml. In agreement with the view that this antibody does not recognize mouse M-CSF, OCL formation in the coculture of mouse calvarial cells and bone marrow cells was unaffected by its addition (Fig. 7CGo). Addition of exogenous human M-CSF (10–100 ng/ml) had no stimulatory effect on PTH-induced OCL formation in coculture of SaOS-4/3 cells with mouse bone marrow cells or human peripheral blood mononuclear cells (data not shown).

GM-CSF is strong inhibitor of osteoclastogenesis in mouse culture systems (32, 33, 34). In contrary, GM-CSF has been shown to stimulate osteoclast formation in human hemopoietic cell cultures (3, 13, 18). However, in this study human GM-CSF strongly inhibited PTH-induced OCL formation in coculture with human peripheral blood mononuclear cells but not in cocultures with mouse bone marrow cells (Fig. 8Go, A and B). In contrast, mouse GM-CSF inhibited mouse OCL formation in cocultures with mouse bone marrow cells but not in cocultures with human peripheral blood mononuclear cells (Fig. 8Go, A and B). Considering the fact that mouse GM-CSF fails to act on human cells and that human GM-CSF fails to act on mouse cells, it is likely that GM-CSF acts on the hemopoietic cell population.

Finally, cocultures of SaOS-4/3 cells with bone marrow cells prepared from GM-CSF receptor-deficient mice were treated with or without mouse GM-CSF. OCL formation induced by hPTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) in this coculture was not inhibited by adding mouse GM-CSF (Fig. 8CGo). This indicates that GM-CSF acts specifically on hemopoietic cells and not on osteoblasts/stromal cells to inhibit human and mouse OCL formation.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Of the four subclones of human osteosarcoma SaOS-2 cells stably transfected with the PTH/PTHrPR gene, two cell lines, SaOS-2/4 and SaOS-4/3, exhibited a high capacity for [125I]-labeled PTH binding and produced a large amount of cAMP in response to PTH. Both SaOS-4/3 and SaOS-2/4 cells supported OCL formation in cocultures with mouse bone marrow cells in the presence of PTH. In contrast, parent SaOS-2 and the other two subclones (SaOS-125 and SaOS-77), which were less sensitive to PTH, failed to support OCL formation. These findings indicate that SaOS-4/3 cells with high levels of functionally active PTH/PTHrPR are critical for OCL formation induced by PTH in coculture with mouse hemopoietic cells. Human OCLs were also formed in response to PTH when human peripheral blood mononuclear cells were cocultured with SaOS-4/3 cells. Treatment of the coculture with dexamethasone markedly enhanced human OCL formation. The stimulatory effect of dexamethasone was not observed in the coculture with mouse bone marrow cells. This indicates that cells responding to dexamethasone are human peripheral blood mononuclear cells, because SaOS-4/3 cells were used as the common supporting cells in the two cocultures. Further studies are necessary to elucidate the precise mechanism of the enhancement of human OCL formation by glucocorticoids.

Multinucleated cells formed from human and mouse hemopoietic cells in cocultures with SaOS-4/3 cells satisfied the major criteria for authentic osteoclasts. They were positive for TRAP, a marker enzyme of osteoclasts. Further, [125I]-calcitonin specifically bound to TRAP-positive mononuclear and multinucleated cells formed from mouse and human hemopoietic cells. Resorption pits were formed on dentine slices on which mouse and human hemopoietic cells had been cocultured with SaOS-4/3 cells in the presence of PTH. Furthermore, multinucleated cells formed from human peripheral blood mononuclear cells but not from mouse bone marrow cells stained with the 23C6 antibody, which recognizes human but not mouse vitronectin receptors. Thus, we conclude that SaOS-4/3 cells support mouse and human OCL formation in cocultures with the respective hemopoietic cells in the presence of PTH. These results also indicate that osteoblasts/stromal cell lines stably transfected with the PTH/PTHrP receptor gene are innovative tools for studying the molecular aspects of PTH-induced osteoclastogenesis.

The regulatory mechanism of human OCL formation in the coculture of human peripheral blood mononuclear cells and SaOS-4/3 cells was quite similar to that of mouse OCL formation in the coculture of mouse bone marrow cells and SaOS-4/3 cells. Of several osteotropic hormones and cytokines examined, only PTH stimulated OCL formation in both cocultures. Maximal human and mouse OCL formation was observed around day 6 in both cocultures. When direct contact between SaOS-4/3 cells and human peripheral blood mononuclear cells was inhibited by a membrane filer, no OCLs were formed even in the presence of PTH (data not shown). This indicates that direct contact between human osteoclast progenitors and osteoblasts/stromal cells is required for their differentiation into osteoclasts in the coculture, as in the case of mouse osteoclast progenitors.

Our results are consistent with the findings of Fujikawa et al. (31), who demonstrated that UMR-106 cells and ST2 cells supported human OCL formation in coculture with human peripheral blood mononuclear cells in the presence of 1{alpha},25(OH)2D3 and dexamethasone. In their experiments, addition of human M-CSF to cocultures was essential to induce human OCLs, because UMR-106 and ST2 cells produce rat and mouse M-CSF, respectively, which do not act on human M-CSF receptors (c-Fms) (31). On the other hand, human OCLs were formed in our cocultures with SaOS-4/3 cells of human origin without adding further human M-CSF. This is probably due to the presence of sufficient amounts of human M-CSF produced by SaOS-4/3 cells. Antihuman M-CSF antibody inhibited both mouse and human OCL formation in our coculture system with SaOS-4/3 cells. This antibody had no inhibitory effect on mouse OCL formation in cocultures with mouse calvarial cells because the antibody failed to neutralize mouse M-CSF produced by mouse calvarial cells. These results are also consistent with the findings of Sarma et al. (35, 36), who demonstrated a critical role for M-CSF in the formation of human osteoclasts.

GM-CSF is a potent inhibitor of human and mouse OCL formation supported by SaOS-4/3 cells. However, adding human GM-CSF inhibited human OCL formation but not mouse OCL formation. Similarly, mouse GM-CSF inhibited mouse but not human OCL formation supported by SaOS-4/3 cells. It has been shown that human GM-CSF acts on human cells but not mouse cells, whereas mouse GM-CSF acts on mouse cells but not human cells (37). Mouse GM-CSF exhibited no inhibitory effect on OCL formation induced by PTH in coculture with bone marrow cells prepared from GM-CSF receptor knockout mice. These results strongly suggest that the target cells of GM-CSF in inhibiting OCL formation are hemopoietic cells and not SaOS-4/3 cells.

Several research groups have reported that human and mouse osteoclasts are formed in cultures of hemopoietic progenitor cells in the absence of osteoblasts/stromal cells (3, 10, 11, 12, 13). It has also been reported that mouse hemopoietic blast cells express specific binding sites for PTH and differentiate into osteoclast-like cells in response to PTH in the absence of osteoblasts/stromal cells (38). Using transgenic mice constitutively expressing human IL-6R, we previously showed that the expression of membrane-bound IL-6R in calvarial cells is indispensable for OCL formation induced by IL-6 in coculture (39). Recently, it has been shown that spleen cells obtained from vitamin D receptor (VDR) knockout mice differentiate into osteoclasts in response to 1{alpha},25(OH)2D3 in cocultures with normal calvarial cells (40). Liu et al. (41) also reported that osteoclasts are formed in response to PTH in coculture of hemopoietic cells obtained from PTH/PTHrPR knockout mice and normal calvarial cells. These results together with the present study indicate that the signals induced by all bone-resorbing factors are transduced into osteoblasts/stromal cells to recruit osteoclasts in the coculture system. We propose that osteoblasts/stromal cells express osteoclast differentiation factor (ODF) on their plasma membrane, which is induced by osteotropic factors (1, 2). Osteoclast progenitors recognize ODF through a mechanism involving cell-cell interaction to differentiate into osteoclasts in the present model. The present study, however, does not exclude other possibilities, in which osteoclast development occurs without the help of osteoblasts/stromal cells.

In summary, SaOS-2-derived subclonal cell lines stably expressing recombinant human PTH/PTHrPR support OCL formation in coculture with mouse or human hemopoietic cells in response to PTH. The availability of SaOS-4/3 cells should facilitate further examination of the regulatory mechanism of human osteoclast formation and function. The coculture system using SaOS-4/3 cells may provide a new tool to investigate osteoclast generation and function in metabolic bone diseases such as osteoporosis and rheumatoid arthritis.


   Acknowledgments
 
We thank Dr. Richard Murray (DNAX Research Institute of Molecular and Cellular Biology) for preparation of IL-3/GM-CSF/IL-5 ßc receptor-deficient mice.

Received April 27, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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