Prostaglandin E2 Strongly Inhibits Human Osteoclast Formation

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Endocrinology 146(12):5204 –5214 Copyright © 2005 by The Endocrine Society doi: 10.1210/en.2005-0451

Prostaglandin E2 Strongly Inhibits Human Osteoclast Formation Ikuko Take, Yasuhiro Kobayashi, Yohei Yamamoto, Hideki Tsuboi, Takahiro Ochi, Setsuko Uematsu, Norimasa Okafuji, Saburo Kurihara, Nobuyuki Udagawa, and Naoyuki Takahashi Graduate School of Oral Medicine (I.T.), Institute for Oral Science (Y.K., N.T.), Department of Orthodontics (I.T., S.U., N.O., S.K.), and Department of Biochemistry (N.U.), Matsumoto Dental University, Nagano 399-0781, Japan; Department of Periodontology (Y.Y.), School of Dentistry, Aichi Gakuin University, Nagoya 464-8651, Japan; Department of Orthopedic Surgery, Osaka University Graduate School of Medicine (H.T.), Osaka 565-0871, Japan; and National Hospital Organization Sagamihara National Hospital (T.O.), Kanagawa 228-8522, Japan Prostaglandin E2 (PGE2) enhances osteoclast formation in mouse macrophage cultures treated with receptor activator of nuclear factor-␬B ligand (RANKL). The effects of PGE2 on human osteoclast formation were examined in cultures of CD14ⴙ cells prepared from human peripheral blood mononuclear cells. CD14ⴙ cells differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. CD14ⴙ cells expressed EP2 and EP4, but not EP1 or EP3, whereas CD14ⴙ cell-derived osteoclasts expressed none of the PGE2 receptors. PGE2 and PGE1 alcohol (an EP2/4 agonist) stimulated cAMP production in CD14ⴙ cells. In contrast to mouse macrophage cultures, PGE2 and PGE1 alcohol inhibited RANKL-induced human osteoclast formation in CD14ⴙ cell cultures. H-89 blocked the inhibitory effect of PGE2 on human osteoclast formation. These results suggest that the

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STEOCLASTS, BONE-RESORBING multinucleated cells, are differentiated from the monocyte-macrophage lineage under the tight regulation of osteoblasts (1– 4). Multinucleated cells with osteoclast characteristics, including tartrate-resistant acid phosphatase (TRAP) activity, vitronectin receptors (VNR), and pit-forming activity, are formed in cocultures of mouse osteoblasts and hemopoietic cells in the presence of osteotropic factors such as 1␣,25dihydroxyvitamin D3 [1␣,25(OH)2D3], PTH, and IL-11 (3, 5, 6). Osteoblasts express two cytokines essential for osteoclast differentiation: receptor activator of NF-␬B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) (7, 8). M-CSF is constitutively expressed by osteoblasts, whereas the expression of RANKL is up-regulated by those osteotropic factors. Osteoclast precursors express RANK and cFms, receptors of RANKL and M-CSF, respectively, and difFirst Published Online September 8, 2005 Abbreviations: COX, Cyclooxygenase; GAPDH, glyceraldehyde-3phosphate dehydrogenase; GM-CSF, granulocyte-macrophage colonystimulating factor; G protein, GTP-binding protein; IBMX, 3-isobutyl1-methylxanthine; IFN-␥, interferon-␥; M-CSF, macrophage colonystimulating factor; MNC, multinucleated cell; 1␣,25(OH)2D3, 1␣,25dihydroxyvitamin D3; PGE2, prostaglandin E2; PKA, protein kinase A; RANKL, receptor activator of nuclear factor-␬B ligand; TAK1, TGF␤activated kinase 1; TRAP, tartrate-resistant acid phosphatase; VNR, vitronectin receptor. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

inhibitory effect of PGE2 on human osteoclast formation is mediated by EP2/EP4 signals. SaOS4/3 cells have been shown to support human osteoclast formation in cocultures with human peripheral blood mononuclear cells in response to PTH. PGE2 inhibited PTH-induced osteoclast formation in cocultures of SaOS4/3 cells and CD14ⴙ cells. Conversely, NS398 (a cyclooxygenase 2 inhibitor) enhanced osteoclast formation induced by PTH in the cocultures. The conditioned medium of CD14ⴙ cells pretreated with PGE2 inhibited RANKL-induced osteoclast formation not only in human CD14ⴙ cell cultures, but also in mouse macrophage cultures. These results suggest that PGE2 inhibits human osteoclast formation through the production of an inhibitory factor(s) for osteoclastogenesis of osteoclast precursors. (Endocrinology 146: 5204 –5214, 2005)

ferentiate into osteoclasts in the presence of both cytokines (3, 4, 6). We have established a subclone, SaOS-4/3, of the human osteosarcoma cell line SaOS-2 by transfection with an expression vector of human PTH/PTHrP receptor (9). SaOS4/3 cells supported human osteoclast formation in cocultures with human peripheral blood mononuclear cells in the presence of PTH. Prostaglandin E2 (PGE2) has diverse biological activities in a variety of tissues (10). The actions of PGE2 in the target cells are mediated by four different G protein-coupled receptor subtypes, EP1, EP2, EP3, and EP4 (11, 12). The EP subtypes differ in tissue distribution, ligand binding affinity, and coupling to intracellular signaling pathways. It has been shown that the signal of EP1 predominantly increases intracellular Ca2⫹ and activates protein kinase C (13). EP2- and EP4induced signals activate G␣s, followed by increases in adenylate cyclase activity, cAMP production, and protein kinase A (PKA) activity in the target cells (14, 15). In contrast, signals induced by EP3 act via G␣i to inhibit cAMP generation (16). Like other osteotropic factors, PGE2 stimulates the expression of RANKL in osteoblasts (17, 18). Among PGE2 receptor subtypes, EP4 has been shown to mainly mediate PGE2induced RANKL expression in osteoblasts (17, 19). In addition, PGE2 directly enhances the differentiation of mouse bone marrow macrophages, osteoclast precursors, into osteoclasts induced by RANKL and M-CSF (20, 21). We have shown that the effect of PGE2 on RANKL-induced osteoclast differentiation in mouse bone marrow macrophage cultures

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is mediated through EP2 and EP4 (22). TGF␤-activated kinase 1 (TAK1) acted as an adapter molecule, linking PKAinduced signals and RANKL-induced signals in mouse osteoclast precursors (22). Ono et al. (23) reported that EP2mediated signals, rather than EP4-mediated ones, are mainly involved in the enhancement effect of PGE2 on RANKLinduced mouse osteoclast formation. Thus, PGE2 stimulates osteoclastic bone resorption in mice through two pathways: induction of RANKL expression by osteoblasts and direct enhancement of RANKL-induced osteoclast differentiation of the precursors. PGE2 is believed to be a bone resorption-stimulating factor in humans. However, there have been few reports showing that PGE2 is a potent bone-resorbing factor in humans. Paradoxically, we believe that administration of cyclooxygenase-2 (COX-2) inhibitors to rheumatoid arthritis patients does not always suppress the enhanced bone resorption in the chronic inflammatory lesions. In addition, Itonaga et al. (24) reported that PGE2 inhibited osteoclast formation induced by M-CSF and RANKL in human peripheral blood mononuclear cell cultures. In contrast, Lader and Flanagan (25) reported that PGE2 increased osteoclast formation in human bone marrow cell cultures. In the present study we explored the role of PGE2 in human osteoclast differentiation in more detail. Unlike mouse macrophage cultures, PGE2 strongly inhibited RANKL-induced osteoclast formation in human CD14⫹ cell cultures. Human osteoclast progenitors produced a soluble unidentified factor(s) in response to PGE2 that strongly inhibited RANKL-induced osteoclast formation not only in human CD14⫹ cell cultures, but also in mouse macrophage cultures. These results suggest that COX-2 inhibitors may not be suitable therapeutic agents to suppress osteoclastic bone resorption in inflammatory bone diseases. Materials and Methods Chemicals and antibodies Recombinant human RANKL and M-CSF (Leukoprol) were purchased from PeproTech (London, UK) and Kyowa Hakko (Tokyo, Japan), respectively. PGE2, PGE1, PGF1␣, and PGF2␣ were obtained from Sigma-Aldrich Corp. (St. Louis, MO), suspended in ethanol at 10⫺3 m, and stored at ⫺80 C. Final concentrations of ethanol in cultures were lower than 0.1%. 3-Isobutyl-1-methylxanthine (IBMX) and 17-phenyltrinol-PGE2 were purchased from BIOMOL (Plymouth Meeting, PA). PTH was purchased from Peptide Institute, Inc. (Osaka, Japan). Antihuman vitronectin receptor (CD51/CD61) antibody was obtained from BD Biosciences (San Jose, CA). Ficoll-Paque Plus was purchased from Amersham Biosciences (Uppsala, Sweden). CD14 MicroBeads were obtained from Miltenyi Biotec (Auburn, CA). All other chemicals were of analytical grade.

Cultures of human CD14⫹ cells Human CD14⫹ cells were isolated from peripheral blood obtained from two female volunteers, aged 25 and 28 yr, and eight male volunteers, aged 27–52 yr, who were not receiving drug therapy. No significant difference in osteoclast formation was observed among the volunteers. Peripheral blood mononuclear cells were isolated with FicollPaque (Amersham Biosciences) density gradient centrifugation at 1600 rpm for 35 min. The leukocytes were collected from the interface between the plasma and the Ficoll-Paque, washed with PBS, and resuspended in PBS containing 0.5% BSA at 107 cells/80 ␮l (26). Twenty microliters of the MACS CD14 MicroBeads (Miltenyi Biotec) were added to 80 ␮l of the cell suspension so that the final cell number was 107

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cells/100 ␮l. After incubation for 15 min at 4 C, the cells were collected by centrifugation, resuspended in PBS containing 0.5% BSA, and applied to a positive selection column placed in the magnetic field of the MACS separator. The CD14-negative cells passed through the column. The column was rinsed with 15 ml PBS containing 0.5% BSA, then removed from the MACS separator and placed on a collection tube. CD14⫹ cells were flushed out with the plunger containing 7 ml 0.5% BSA in PBS. CD14⫹ cells were collected by centrifugation and resuspended in ␣MEM (Invitrogen Life Technologies, Inc., Carlsbad, CA) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS) at 5 ⫻ 105 cells/ml. The cells were seeded in 96-well plates (105 cells/well) and cultured with M-CSF (50 ng/ml) and RANKL (100 ng/ml). The culture medium was changed every 3 d in most cultures. In some cultures, the culture medium was changed every 2 d. PGs and cytokines were added to the culture at each medium change. After being cultured for 6 d, cells were fixed and stained with anti-VNR antibody to identify osteoclasts formed in the culture (9). In some experiments, CD14⫹ cells were cultured for 2, 4, 6, and 8 d with M-CSF (50 ng/ml) plus RANKL (100 ng/ml). CD14⫹ cells were also cultured for 8 d with M-CSF (50 ng/ml) plus RANKL (100 ng/ml), and PGE2 (10⫺7 m) was added after various periods of culture. Cells were then fixed and stained with anti-VNR antibody. The number of VNR-positive multinucleated cells (MNCs) containing three or more nuclei per cell was counted as osteoclasts. Informed consent for all procedures was obtained from all volunteers.

Cultures of mouse bone marrow macrophages and peripheral blood mononuclear cells Mouse bone marrow-derived macrophages were prepared as osteoclast precursors as described previously (26). Bone marrow cells were obtained from tibiae of 5- to 8-wk-old male ddY mice (Shizuoka Laboratories Animal Center, Shizuoka, Japan). Bone marrow cells were suspended in ␣MEM supplemented with 10% fetal bovine serum and cultured with M-CSF (100 ng/ml) in a 60-mm diameter dish. After culture for 16 h, nonadherent cells were harvested and also cultured in 96-well plates (104 cells/well) with M-CSF (50 ng/ml) and RANKL (100 ng/ml) in the presence of various concentrations of PGE2. Peripheral blood of 5- to 8-wk-old male ddY mice was collected by heart puncture. Peripheral blood mononuclear cells were isolated with Ficoll-Paque density gradient centrifugation and cultured in 96-well plates (105 cells/ well) with M-CSF (50 ng/ml) plus RANKL (100 ng/ml) in the presence of various concentrations of PGE2. After culture for 7 d, cells were fixed and stained for TRAP (27). The number of TRAP-positive MNCs containing three or more nuclei per cell was counted as osteoclasts. All procedures for animal care were approved by the animal management committees of Matsumoto Dental University.

Cocultures of SaOS4/3 cells and CD14⫹ cells SaOS4/3 cells (104 cells/well) and CD14⫹ cells (2 ⫻ 105 cells/well) were cocultured in 48-well plates in the presence or absence of PTH (100 ng/ml). Some cocultures were treated with PGE2 (10⫺7 m) or NS398 (10⫺7 m). After being cultured for 14 d, cells were fixed and stained with anti-VNR antibody to identify osteoclasts formed in culture. The number of VNR-positive MNCs containing three or more nuclei per cell was counted as osteoclasts.

Spot cocultures of CD14⫹ cells and mouse bone marrow macrophages CD14⫹ cells (104 cells/spot) and mouse bone marrow macrophages (104 cells/spot) were spot-cultured in a dish so as not to come into contact with each other. CD14⫹ cells and mouse bone marrow macrophages were also spot-cultured in the separate culture dishes as controls. Most cells attached firmly to the dish surface within 0.5 h. Then the culture medium containing M-CSF (50 ng/ml) and RANKL (100 ng/ml) was gently added to the dish (3 ml/dish). Some spot cultures were treated with PGE2 (10⫺7 m). After culture for 7 d, cells were stained with TRAP. The number of TRAP-positive MNCs containing three or more nuclei per cell in each spot was counted as osteoclasts.

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Preparation of the conditioned medium of CD14⫹ cells CD14⫹ cells were cultured for 2 d with M-CSF (50 ng/ml) in a 60-mm diameter dish (107 cells/dish). The culture medium was then changed to fresh medium containing M-CSF (50 ng/ml) with or without PGE2 (10⫺7 m). After culture for 2 d, the medium was changed to fresh medium containing M-CSF (50 ng/ml), but not PGE2. The conditioned medium was collected after incubation for 24 h.

RT-PCR for PGE2 receptor mRNAs CD14⫹ cells were cultured for 7 d in a 60-mm diameter dish (107 cells/dish) in the presence of M-CSF (50 ng/ml) together with or without RANKL (100 ng/ml). CD14⫹ cells cultured with M-CSF alone are called CD14⫹ macrophages, and CD14⫹ cells cultured with M-CSF and RANKL are called CD14⫹-derived osteoclasts. For semiquantitative RTPCR analysis, total RNA was extracted from the culture using TRIzol solution (Invitrogen Life Technologies, Inc.). Using a phase contrast microscope, we confirmed that most CD14⫹ cells differentiated into MNCs. First-strand cDNA was synthesized from the total RNA with oligo(deoxythymidine) primers using reverse transcriptase (RevatraAce, Toyobo Biochemicals, Osaka, Japan) and subjected to PCR. Primers used in PCR for EP receptor isoforms were as follows: EP1, 5⬘-CTCGCCGCCCTGGTGTGCAACACGC-3⬘ (forward) and 5⬘-GGCCTCCCAGGCGCTCGGTGTTAGGCC-3⬘ (reverse) for an EP1 fragment of 519 bp; EP2, 5⬘-TTCATCCGGCACGGGCGGACCGC-3⬘ (forward) and 5⬘-GTCAGCCTGTTTACTGGCATCTG-3⬘ (reverse)for an EP2 fragment of 510 bp; EP3, 5⬘-TGTGTCGCGCAGTACCGGCG-3⬘ (forward) and 5⬘-CGGGCCACTGGACGGTGTACT-3⬘ (reverse) for an EP3 fragment of 400 bp; EP4, 5⬘-CCTCCTGAGAAAGACAGTGCT-3⬘ (forward) and 5⬘-AAGACACTCTCTGAGTCCT-3⬘ (reverse) for an EP4 fragment of 366 bp; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5⬘ACCACAGTCCATGCCATCAC-3⬘ (forward) and 5⬘-TCCACCACCCTGTTGCTGTA-3⬘ (reverse) for a fragment of 562 bp, and calcitonin receptor, 5⬘-TACCCAATGGAGAGCTCGTG-3⬘ (forward) and 5⬘TACACGGCCCTGGTAATAGC-3⬘ (reverse) for a fragment of 235 bp. The PCR conditions for EP subtypes were denaturation at 95 C for 60 sec, reannealing at 58 C for 60 sec, and extension at 72 C for 60 sec for 24 cycles. The PCR conditions for GAPDH were denaturation at 94 C for 30 sec, reannealing at 58 C for 45 sec, and extension at 72 C for 60 sec for 28 cycles. The PCR conditions for calcitonin receptor were denaturation at 94 C for 60 sec, reannealing at 64 C for 60 sec, and extension at 72 C for 60 sec for 28 cycles. The PCR products were subjected to electrophoresis in 2% agarose gel, followed by staining with ethidium bromide.

Assay of cAMP production CD14⫹ cells were cultured in six-well plates (105 cells/well) in the presence of M-CSF (50 ng/ml) for 6 d. Cells were preincubated for 5 min at 37 C in ␣MEM containing 1 mm IBMX and incubated for 30 min with 10⫺7 m PGE2. Cells were washed with PBS containing 1 mm IBMX. The content of intracellular cAMP was determined using a cAMP enzyme immunoassay kit (Amersham Biosciences, Piscataway, NJ).

Statistics The results were expressed as the mean ⫾ sd of three or more cultures obtained from an individual volunteer. In experiments using mouse peripheral blood mononuclear cells, blood was collected from three mice and mixed to prepare peripheral blood mononuclear cells for one experiment. Similar results were obtained from at least three independent experiments. Statistical analysis was performed using Student’s t test.

Results Effects of PGE2 on human osteoclast formation

Recent studies have shown that PGE2 enhances RANKLinduced osteoclast formation in cultures of mouse bone marrow macrophages and RAW 264.7 cells (20 –22). We first examined the effect of PGE2 on RANKL-induced osteoclast formation in cultures of human CD14⫹ cells in comparison


with that in mouse osteoclast progenitors (Fig. 1). PGE2 enhanced RANKL-induced TRAP-positive osteoclast formation in cultures of mouse bone marrow macrophages in a dose-dependent manner (Fig. 1A). VNR is used as a reliable marker to identify human osteoclasts formed in vitro (9, 28). VNR-positive osteoclasts were formed in human CD14⫹ cell cultures treated with RANKL and M-CSF. In contrast to mouse osteoclast formation, PGE2 inhibited RANKL-induced VNR-positive osteoclast formation in CD14⫹ cell cultures in a dose-dependent manner (Fig. 1B). Recently, Ono et al. (29) reported that PGE2 has an initial inhibitory effect and a later stimulatory effect on osteoclast formation in mouse spleen cell cultures. CD14⫹ cells were treated with PGE2 (10⫺7 m) at different periods of culture (Fig. 2, A and B). VNR-positive osteoclasts first appeared on d 2, and their number increased up to d 6 – 8 in CD14⫹ cell cultures treated with RANKL plus M-CSF (Fig. 2A). The inhibitory effect of PGE2 on osteoclast formation was strongest after treatment for 2– 4 d (Fig. 2B). The effects of various PGs (PGE1, PGE2, PGF1␣, and PGF2␣) on osteoclast formation were also examined in human CD14⫹ cell cultures treated with RANKL and M-CSF (Fig. 2B). PGE1 and PGF2␣ as well as PGE2 dose-dependently inhibited human osteoclast formation. PGF1␣ had little effect on human osteoclast formation at the doses examined (Fig. 2B). Expression of PGE2 receptors in CD14⫹ cells

We next examined the expression of PGE2 receptor subtypes in freshly isolated CD14⫹ cells, CD14⫹ cells treated with M-CSF, and CD14⫹ cell-derived osteoclasts using RT-PCR (Fig. 3A). CD14⫹ cells cultured with M-CSF alone are hereafter called CD14⫹ macrophages, because they strongly expressed macrophage-specific markers such as CD14 and CD68 (data not shown). Freshly isolated CD14⫹ cells and CD14⫹ macrophages expressed mRNAs of EP2 and EP4, but not EP1 or EP3. In contrast, CD14⫹ cell-derived osteoclasts failed to express detectable levels of EP1, EP2, EP3, or EP4 mRNA (Fig. 3A). These results suggest that the expression of EP2 and EP4 mRNAs is down-regulated during the differentiation of CD14⫹ cells into osteoclasts. We then examined the effects of PGE2 and its agonists on human osteoclast formation induced by RANKL (Fig. 3B). PGE2 and PGE1 alcohol (EP2/4 agonist), but not sulprostone (EP1/EP3 agonist) or 17-phenyl-trinor-PGE2 (EP1 agonist), at 10⫺7 m inhibited RANKL-induced human osteoclast formation (Fig. 3B). PGE2 and PGE1 alcohol, but not sulprostone significantly stimulated cAMP production in CD14⫹ cells treated with M-CSF (Fig. 3C). CD14⫹ cell-derived osteoclasts slightly produced cAMP in response to PGE2 and PGE1 alcohol, but not sulprostone, probably due to CD14⫹ macrophages that remained in the human osteoclast preparation (Fig. 3C). A small number of VNR-negative mononuclear cells were always observed in the osteoclast preparation. H-89 at 10⫺7 and 10⫺6 m blocked the inhibitory effect of PGE2 on human osteoclast formation (Fig. 3D). Inhibitory effects of PGE2 on human osteoclast formation were not affected by H-89 at 10⫺8 m (data not shown). H-89 at 10⫺6 m had no effect on RANKL-induced osteoclast formation in CD14⫹ cell cultures (Fig. 3D). Calcitonin, but not PGE2, enhanced cAMP production in CD14⫹ cell-derived osteoclasts (data not shown). These results suggest that functional



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FIG. 1. Effects of PGE2 on RANKL-induced osteoclast formation in cultures of mouse bone marrow macrophages and human CD14⫹ cells. A, Mouse bone marrow macrophages (104 cells/well) were cultured with increasing concentrations of PGE2 together with RANKL (100 ng/ml) in the presence of M-CSF (50 ng/ml) in 96-well plates. After cells were cultured for 7 d, they were fixed and stained for TRAP. TRAP-positive cells appeared as red cells. B, Human CD14⫹ cells (105 cells/well) were cultured with increasing concentrations of PGE2 together with RANKL (100 ng/ml) in the presence of M-CSF (50 ng/ml) in 96-well plates. After cells were cultured for 6 d, they were fixed and stained with anti-VNR antibody. VNR-positive cells appeared as brown cells. TRAP-positive MNCs and VNR-positive MNCs containing three or more nuclei were counted as mouse and human osteoclasts, respectively. Results were expressed as the mean ⫾ SD of more than three cultures. *, P ⬍ 0.01 (significantly different from cultures treated with RANKL alone).

EP2 and EP4 are expressed in human osteoclast precursors, and that the cAMP-PKA pathway mediated by EP2 and EP4 is involved in the inhibitory effect of PGE2 on human osteoclast formation. Effect of PGE2 on human osteoclast formation in cocultures with SaOS4/3 cells

RANKL locally expressed by osteoblasts appears to be important for osteoclast formation in vivo. PGE2 stimulates osteoclast formation in cocultures of mouse osteoblasts and bone marrow cells through RANKL expression by osteoblasts (17, 18). Therefore, we examined the effects of PGE2 in cocultures of human osteoblasts and CD14⫹ cells. We have established SaOS4/3 cells from the human os-

teosarcoma cell line SaOS-2 by transfection of human PTH/PTHrP receptor cDNA (9). When SaOS4/3 cells were cocultured with human peripheral blood mononuclear cells, human osteoclasts were formed in response to PTH (9). We then examined the effects of PGE2 on PTH-induced human osteoclast formation in cocultures of SaOS-4/3 cells and CD14⫹ cells (Fig. 4). When CD14⫹ cells were cocultured with SaOS4/3 cells, VNR-positive osteoclasts were formed in response to PTH (100 ng/ml). PGE2 (10⫺7 m) added together with PTH to the coculture completely inhibited human osteoclast formation induced by PTH. NS398 (a COX-2 inhibitor; 10⫺7 m) added to the coculture markedly enhanced human osteoclast formation induced by PTH (Fig. 4). PGE2 at 10⫺7 m completely abolished

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osteoclast formation induced by PTH and NS398 in the coculture. This suggested that PGE2 endogenously produced in the coculture is involved in the constitutive suppression of osteoclast differentiation of human progenitor cells. Production of an inhibitory factor(s) for osteoclastogenesis by CD14⫹ cells

To examine the possibility that CD14⫹ cells produce an inhibitory factor(s) in response to PGE2, CD14⫹ cells and mouse bone marrow macrophages were spot-cultured in a dish so as not to come into contact with each other (Fig. 5A). Addition of RANKL together with M-CSF to the spot cultures stimulated TRAP-positive osteoclast formation in both human and mouse macrophage colonies. PGE2 strongly inhibited osteoclast formation not only in the colonies of CD14⫹ cells, but also in the colonies of mouse bone marrow macrophages in a single culture dish (Fig. 5, B and C). CD14⫹ cells and mouse bone marrow macrophages were also spotcultured in the separate culture dishes as controls (Fig. 5A). PGE2 inhibited VNR-positive osteoclast formation in the colonies of CD14⫹ cells and enhanced TRAP-positive osteoclast formation in the colonies of mouse bone marrow macrophages (Fig. 5B). These results suggest that human CD14⫹ cells produced a soluble factor(s) in response to PGE2, which strongly inhibited mouse osteoclast formation. We next examined the effects of the conditioned medium of CD14⫹ cell cultures pretreated with PGE2 on human and mouse osteoclast formation (Fig. 6). The conditioned medium dose-dependently inhibited both human and mouse osteoclast formation induced by RANKL (Fig. 6A). The conditioned medium treated without PGE2 had no effect on human or mouse osteoclast formation (Fig. 6B). RANKL had no effect on the production of an inhibitory factor(s) by CD14⫹ cells (Fig. 6B). In our experiments, human osteoclast precursors were prepared from peripheral blood, whereas mouse osteoclast precursors were from bone marrow cells. Peripheral blood and bone marrow cells may respond differently to PGE2. We finally examined the effects of PGE2 and the conditioned medium of CD14⫹ cell cultures pretreated with PGE2 on osteoclast formation in mouse peripheral blood mononuclear cell cultures (Fig. 6C). PGE2 dose-dependently enhanced RANKL-induced osteoclast formation in mouse peripheral blood mononuclear cell cultures, and the conditioned medium inhibited it in a dose-dependent manner (Fig. 6C). Discussion FIG. 2. Effects of PGs on RANKL-induced osteoclast formation in human CD14⫹ cell cultures. A, Time course of changes in human osteoclast formation in CD14⫹ cell cultures. Human CD14⫹ cells (105 cells/well) were cultured with RANKL (100 ng/ml) plus M-CSF (50 ng/ml) in the presence or absence of PGE2 (10⫺7 M) in 96-well plates. After culture for the indicated periods, cells were fixed and stained with anti-VNR antibody. B, Effects of PGE2 added after different culture periods on human osteoclast formation in CD14⫹ cell cultures. Human CD14⫹ cells (105 cells/well) were cultured with RANKL (100 ng/ml) plus M-CSF (50 ng/ml) in 96-well plates. PGE2 (10⫺7 M) was added at the indicated times of culture. After cells were cultured for 8 d, they were fixed and stained with anti-VNR antibody. C, Effects of various PGs on human osteoclast formation induced by RANKL.

It is well known that PGE2 stimulates osteoclast formation in mouse bone marrow cultures and in cocultures of mouse osteoblasts and hemopoietic cells. PGE2 also stimulates osHuman CD14⫹ cells (105 cells/well) were cultured with increasing concentrations of PGE1, PGE2, PGF1␣, or PGF2␣ together with RANKL (100 ng/ml) plus M-CSF (50 ng/ml) in 96-well plates. After cells were cultured for 6 d, they were fixed and stained with anti-VNR antibody. VNR-positive MNCs containing three or more nuclei were counted as human osteoclasts. Results were expressed as the mean ⫾ SD of more than three cultures. *, P ⬍ 0.01 (significantly different from cultures treated with RANKL alone).



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FIG. 3. Involvement of EP2- and EP4-mediated signals in PGE2-induced inhibition of human osteoclast formation. A, Expression of mRNAs of PGE2 receptor subtypes and calcitonin receptors in human cells. Human CD14⫹ cells were cultured for 6 d with M-CSF (50 ng/ml) in the presence or absence of RANKL (100 ng/ml). Total RNA was extracted from freshly isolated CD14⫹ cells (lane 1), CD14⫹ cells treated with M-CSF (CD14⫹ macrophages; lane 2), and CD14⫹ cells treated with RANKL plus M-CSF (CD14⫹ cell-derived osteoclasts; lane 3), and cDNA was synthesized from the total RNA. The expression of mRNAs of EP1, EP2, EP3, EP4, calcitonin receptor (CTR), and GAPDH was detected by RT-PCR using the respective primers, as described in Materials and Methods. B, Effects of various EP agonists on human osteoclast formation. CD14⫹ cells (105 cells/well) were cultured with M-CSF (50 ng/ml) in the presence or absence of RANKL (100 ng/ml) in 96-well plates. PGE2, sulprostone (EP1/EP3 agonist), 17-phenyl-trinor-PGE2 (EP1 agonist), or PGE1 alcohol (EP2/4 agonist) was added at 10⫺7 M to some cultures of CD14⫹ cells. After cells were cultured for 6 d, they were fixed and stained with anti-VNR antibody. VNR-positive MNCs containing three or more nuclei were counted as human osteoclasts. Results were expressed as the mean ⫾ SD of five cultures. *, P ⬍ 0.01 (significantly different from cultures treated with RANKL alone). C, Effects of EP agonists on cAMP production in CD14⫹ macrophages and CD14⫹ cell-derived osteoclasts. CD14⫹ cells (105 cells/well) were cultured with M-CSF (50 ng/ml) for 6 d in 96-well plates. CD14⫹ cells (105 cells/well) were also cultured with RANKL (100 ng/ml) plus M-CSF (50 ng/ml) to induce human osteoclasts. Cells were then incubated for 30 min with or without PGE2, sulprostone, or PGE1 alcohol at 10⫺7 M, and the amount of cellular cAMP was measured as described in Materials and Methods. Results were expressed as the mean ⫾ SD of three cultures. *, P ⬍ 0.01 (significantly different from control cultures). D, Effects of H-89 on PGE2-induced inhibition of human osteoclast formation. CD14⫹ cells (105 cells/well) were cultured for 6 d with or without RANKL (100 ng/ml) in the presence of M-CSF (50 ng/ml). Some cultures were treated with H-89 at 10⫺7 and 10⫺6 M. After cells were cultured for 6 d, they were fixed and stained with anti-VNR antibody. VNR-positive MNCs containing three or more nuclei were counted as human osteoclasts. Results were expressed as the mean ⫾ SD of five cultures. *, P ⬍ 0.01 (significantly different from cultures treated with RANKL plus PGE2).

teoclastic bone resorption in rodent organ cultures of fetal and neonatal bones (10, 30). These effects of PGE2 on osteoclast formation and bone resorption are believed to be due to the enhancement of RANKL expression by osteoblasts or bone marrow stromal cells in these cultures. Recent studies have also shown that PGE2 directly acts on osteoclast precursors, such as mouse bone marrow macrophages and

mouse macrophage RAW264.7 cells, and enhances RANKLinduced osteoclastic differentiation from these precursor cells (20 –22). In contrast to these effects in mouse culture systems, it was reported that PGE2 inhibited RANKL-induced osteoclast formation in human peripheral blood mononuclear cell cultures (24). In agreement with previous findings (24), PGE2 strongly inhibited human osteoclast for-

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FIG. 4. Effects of PGE2 on human osteoclast formation in cocultures of SaOS4/3 cells and CD14⫹ cells. Human CD14⫹ cells (2 ⫻ 105 cells/well) were cocultured with SaOS4/3 cells (104 cells/well) in the presence or absence of PTH (100 ng/ml) in 48-well plates. Some cocultures were treated with PGE2 (10⫺7 M) or NS398 (10⫺7 M). After cells were cultured for 14 d, they were fixed and stained with anti-VNR antibody. VNR-positive MNCs containing three or more nuclei were counted as human osteoclasts. Results were expressed as the mean ⫾ SD of three cultures. *, P ⬍ 0.01 (significantly different from cultures treated with PTH).

mation in CD14⫹ cell cultures treated with RANKL, and in cocultures of SaOS-4/3cells and CD14⫹ cells treated with PTH. PGE2 occasionally inhibited the growth of CD14⫹ cells. However, PGE2 always inhibited RANKL-induced osteoclast formation. The conditioned medium of CD14⫹ cells pretreated with PGE2 showed no inhibitory effect on the growth of human CD14⫹ cells (see photographs in Fig. 6A). These results suggest that the inhibitory effect of PGE2 on cell growth is not involved in the inhibition of osteoclast differentiation induced by PGE2. Thus, the effect of PGE2 on in vitro osteoclast formation in human culture systems is the opposite of that in mouse culture systems. We have also shown that human CD14⫹ cells produce an inhibitory factor(s) for osteoclast formation not only from human CD14⫹ cells, but also mouse osteoclast precursor cells. Kanatani et al. (31) showed that the conditioned medium of human monocytes inhibited mouse osteoclast formation induced by 1␣,25(OH)2D3 or PTH in the absence of stromal cells. In our experiments, no osteoclasts were formed in the absence of stromal cells even in the presence of 1␣,25(OH)2D3. Different culture conditions or strains of mice may be the cause of the difference. Lader et al. (25) reported that PGE2 is involved in osteoclast formation in human bone marrow cultures treated with IL-1 and TNF␣. This suggests that peripheral blood cells and bone marrow cells may differently respond to PGE2. In our experiments, PGE2 also enhanced RANKL-induced osteoclast formation in cultures of mouse peripheral blood cells, and conditioned medium of CD14⫹ cell cultures pretreated with PGE2 inhibited RANKLinduced osteoclast formation. These results indicate that mouse bone marrow cells and peripheral blood cells respond similarly to PGE2 and CD14⫹ cell-conditioned medium. Additional studies are necessary to elucidate the discrepancy in the effect of PGE2 on human osteoclast formation. Human CD14⫹ cells and CD14⫹ macrophages expressed

EP2 and EP4, but not EP1 or EP3. In contrast, CD14⫹ cellderived osteoclasts failed to express detectable levels of EP1, EP2, EP3, or EP4 mRNA. PGE2 and PGE1 alcohol stimulated cAMP production in the CD14⫹ cell-derived osteoclast preparation. This may be due to CD14⫹ macrophages that remained in the osteoclast preparations. We recently reported that mouse bone marrow macrophage expressed EP1, EP2, EP3␤, and EP4, and the expression of EP2 and EP4 was down-regulated during osteoclastic differentiation induced by RANKL and M-CSF (32). Purified mouse osteoclasts failed to produce cAMP in response to PGE2. These results suggest that human and mouse osteoclast precursors express EP2 and EP4, but down-regulate their own EP2 and EP4 levels during their differentiation into osteoclasts. H-89 is known to have different IC50 values for several protein kinases: 0.048 ␮m for PKA, 0.48 ␮m for cGMP-dependent protein kinase, and 31.7 ␮m for protein kinase C (33). In our experiments, H-89 at 10⫺7 m significantly blocked the inhibitory effect of PGE2 on human osteoclast formation. The potency of PGs to inhibit osteoclast formation was highest for PGE2, followed by PGE1, PGF2␣, and PGF1␣ in that order, and the order was highly correlated with the order of the potency for increasing cAMP production in CD14⫹ cells (data not shown). These results suggest that cAMP-PKA signals are involved in the secretion of inhibitory factor(s) by human osteoclast precursors. We previously reported that PGE2 enhanced osteoclastic differentiation of mouse precursor cells through PKA-dependent phosphorylation of TAK1, a MAPK kinase kinase (22). We found that TAK1 possesses a PKA recognition sequence (Arg409-Arg-Arg-Ser-Ilu-Gln414) in the C-terminal region. PGE2 directly phosphorylated TAK1 in RAW264.7 cells through EP2 or EP4. When RAW264.7 cells were transfected with Ser4123 Ala mutant TAK1, the mutant TAK1 served as a dominant-negative mutant in PGE2-enhanced osteoclastic



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FIG. 5. Effects of PGE2 on human and mouse osteoclast formation in the single dish cultures. A, Schematic representation of spot cultures of human CD14⫹ cells and mouse macrophages. Human CD14⫹ cells (104 cells/spot) and mouse bone marrow macrophages (104 cells/spot) were spotcultured in separate culture dishes or in a single culture dish. Cells attached firmly to the dish surface within 0.5 h. Then the culture medium containing M-CSF (50 ng/ml) and RANKL (100 ng/ml) was added to the dish (3 ml/dish). Some spot cultures were also treated with PGE2 (10⫺7 M). B, Effect of PGE2 on osteoclast formation in each colony of human CD14⫹ cells and mouse macrophages in the separate culture dishes and in the single culture dish. Human CD14⫹ cells and mouse bone marrow macrophages were spot-cultured for 0.5 h, then treated with vehicle, RANKL (100 ng/ml), or RANKL (100 ng/ml) plus PGE2 (10⫺7 M), in the presence of M-CSF (50 ng/ml). After cells were cultured for 7 d, they were stained with TRAP. C, Number of osteoclasts formed in single dish cultures. TRAP-positive MNCs containing three or more nuclei were counted as mouse and human osteoclasts in spot cultures in single culture dishes. Results were expressed as the mean ⫾ SD of three cultures. *, P ⬍ 0.01 (significantly different from cultures treated with RANKL).

differentiation of RAW264.7 cells (22). Interestingly, human TAK1 also possesses the PKA recognition sequence motif, suggesting that EP2 and EP4 signals also enhance RANK signals in human osteoclast progenitors. If the inhibitory factors for osteoclastogenesis produced by human osteoclast progenitors are neutralized by specific antibodies, human osteoclast formation could be enhanced by PGE2 through the crosstalk between TAK1 and RANKL. The conditioned medium of CD14⫹ cells pretreated with PGE2 inhibited not only human, but also mouse, osteoclast formation, but did not affect the cell growth of human and mouse osteoclast progenitors. Particularly, mouse osteoclast formation was strongly inhibited by the conditioned medium, suggesting that mouse cultures would be useful for identification of the inhibitor(s) produced by human CD14⫹ cells. GM-CSF (34), IFN-␥ (35), and IL-4 (36 –38) have been shown to inhibit mouse osteoclast formation. Human IFN-␥ showed no inhibitory effect on mouse osteoclast formation (data not shown). It is also known that human IFN-␥ does not

bind to mouse IFN-␥ receptors. Human recombinant IL-4 at 10 ng/ml did not strongly inhibit mouse osteoclast formation in cultures of mouse bone marrow macrophages (number of TRAP-positive osteoclasts: control, 312 ⫾ 63; after treatment with IL-4, 250 ⫾ 62; mean ⫾ sd of four cultures). Neutralizing antibodies against GM-CSF failed to rescue human and mouse osteoclast formation inhibited by the CD14⫹ cellconditioned medium (data not shown). These results suggest that CD14⫹ cells produce an inhibitor(s) for osteoclastogenesis that does not correspond to known inhibitory factors. Human osteoclasts expressed calcitonin receptor mRNA, but not EP mRNAs. This suggests that the expression of EP2 and EP4 was down-regulated during osteoclastic differentiation of CD14⫹ cells. Similarly, mouse bone marrow macrophages expressed EP2 and EP4, but mouse osteoclasts did not (32). When EP4 was expressed in mouse osteoclasts cultured on dentine slices using an adenovirus carrying EP4 cDNA, the pit-forming activity of osteoclasts was inhibited in an infectious unit-dependent manner (32). Treatment of

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EP4-expressing osteoclasts with PGE2 also inhibited their pit-forming activities. Such inhibitory effects of EP4-mediated signals on osteoclast function are quite similar to those of calcitonin receptor-mediated ones. These results suggest that osteoclast precursors of both mice and humans downregulate EP2 and EP4 levels during their differentiation into osteoclasts and thereby escape the inhibitory effects of PGE2 on bone resorption. PGE2 is believed to be a bone resorption-stimulating factor in humans. However, there have been few reports showing that PGE2 is a potent bone-resorbing factor in humans. In our experiments, NS-398 stimulated human osteoclast formation in cocultures containing SaOS4/3 cells in the presence of PTH. Therefore, it is suggested that COX-2 inhibitors suppress inflammation in rheumatoid arthritis, but stimulate osteoclastic bone resorption due to suppression of endogenous production of PGE2. Salvi and Lang (39) summarized the effects of COX-1 and COX-2 inhibitors on the treatment of periodontal diseases and reported that COX inhibitors are mainly responsible for the stabilization of periodontal conditions by reducing the rate of alveolar bone resorption. In contrast, Head et al. (40) reported that short-term treatment of normal men with ibuprofen and acetaminophen had differential effects on urinary excretion of peptide-bound and free deoxypyridinoline cross-links of type I collagen. They reported that short-term ibuprofen use may alter the renal handling of collagen cross-links and increase bone resorption to a greater extent than acetaminophen. Therefore, the effect of COX inhibitors on osteoclastic bone resorption in humans is still a matter of controversy. The usefulness of COX inhibitors as antiinflammatory drugs in rheumatoid arthritis and periodontitis should be carefully studied in the future. The mechanism by which the conditioned medium of CD14⫹ cells inhibits osteoclast differentiation, and what kind of cytokine(s) is involved in the inhibition of osteoclast differentiation are of particular interest. These subjects are currently under investigation in our laboratories. Acknowledgments We thank Dr. Teruhito Yamashita (Matsumoto Dental University) for critical reading of the manuscript and helpful discussion.


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Received April 18, 2005. Accepted August 30, 2005. Address all correspondence and requests for reprints to: Dr. Naoyuki Takahashi, Institute for Oral Science, Matsumoto Dental University, 1780 Hiro-oka Gobara, Shiojiri, Nagano 399-0781, Japan. E-mail: [email protected]. This work was supported in part by Health and Labor Sciences Research Grants.

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FIG. 6. Effects of conditioned medium of CD14⫹ cell cultures on human and mouse osteoclast formation. A, Effects of the conditioned medium of PGE2-pretreated CD14⫹ cells on human and mouse osteoclast formation. CD14⫹ cells were cultured in ␣MEM supplemented with 10% fetal calf serum in the presence of PGE2 (10⫺7 M) and M-CSF (50 ng/ml) for 48 h in a 60-mm diameter dish (107 cells/dish). After cells were cultured for 48 h, they were washed with PBS and cultured in ␣MEM supplemented with 10% fetal calf serum in the presence of M-CSF (50 ng/ml), but not PGE2, for another 24 h. The conditioned medium of CD14⫹ cell cultures was then collected. Human CD14⫹ cells (105 cells/well) or mouse bone marrow macrophages (104 cells/well) were cultured in 96-well plates in the presence of M-CSF (50 ng/ml). Some cultures were also treated with RANKL (100 ng/ml) in the presence of increasing amounts of the conditioned medium of CD14⫹ cells. After they were cultured for 7 d, human CD14⫹ cell cultures were stained with anti-VNR antibody. Mouse macrophage cultures were stained for TRAP. VNR-positive MNCs and TRAP-positive MNCs containing three or more nuclei were counted as human and mouse osteoclasts, respectively. Results were expressed as the mean ⫾ SD of three cultures. B, Effects of conditioned medium of CD14⫹ cells treated with M-CSF alone and those pretreated with PGE2 plus RANKL. CD14⫹ cells were cultured with M-CSF (50 ng/ml) alone for 48 h in a 60-mm diameter dish (107 cells/dish), washed with PBS, and cultured with M-CSF (50 ng/ml) for another 24 h. The conditioned medium of CD14⫹ cell cultures was then collected. CD14⫹ cells were cultured in the presence of PGE2 (10⫺7 M) together with RANKL (100 ng/ml) plus M-CSF (50 ng/ml) for 48 h in a 60-mm diameter dish (107 cells/dish), washed with PBS, and cultured in the presence of M-CSF (50 ng/ml), but not PGE2 or RANKL, for another 24 h. The conditioned medium of CD14⫹ cell cultures was then collected. The effects of those conditioned media on human and mouse osteoclast formation were examined as described in A. C, Effects of PGE2 and the conditioned medium of CD14⫹ cell cultures pretreated with PGE2 on RANKL-induced osteoclast formation in mouse peripheral blood mononuclear cell cultures. Mouse peripheral blood mononuclear cells (105 cells/well) were cultured with increasing concentrations of PGE2 together with RANKL (100 ng/ml) plus M-CSF (50 ng/ml) in 96-well plates. Some cultures were also treated with increasing amounts of the conditioned medium of CD14⫹ cells pretreated with PGE2. After cells were cultured for 7 d, they were fixed and stained for TRAP. TRAP-positive MNCs containing three or more nuclei were counted as mouse osteoclasts. Results were expressed as the mean ⫾ SD of more than three cultures. *, P ⬍ 0.01 (significantly different from cultures treated with RANKL).

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