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• W1977681806 abstract "This study investigated the actions of fibroblast growth factor (FGF)-18, a novel member of the FGF family, on osteoblasts, chondrocytes, and osteoclasts and compared them with those of FGF-2 and FGF-10. FGF-18 stimulated the proliferation of cultured mouse primary osteoblasts, osteoblastic MC3T3-E1 cells, primary chondrocytes, and prechondrocytic ATDC5 cells, although it inhibited the differentiation and matrix synthesis of these cells. FGF-18 up-regulated the phosphorylation of extracellular signal-regulated kinase in both osteoblasts and chondrocytes and up-regulated the phosphorylation of p38 mitogen-activated protein kinase only in chondrocytes. FGF-18 mitogenic actions were blocked by a specific inhibitor of extracellular signal-regulated kinase in both osteoblasts and chondrocytes and by a specific inhibitor of p38 mitogen-activated protein kinase in chondrocytes. With regard to the action of FGF-18 on bone resorption, FGF-18 not only induced osteoclast formation through receptor activator of nuclear factor-κB ligand and cyclooxygenase-2 but also stimulated osteoclast function to form resorbed pits on a dentine slice in the mouse coculture system. All these effects of FGF-18 bore a close resemblance to those of FGF-2, whereas FGF-10 affects none of these cells. FGF-18 may therefore compensate for the action of FGF-2 on bone and cartilage. This study investigated the actions of fibroblast growth factor (FGF)-18, a novel member of the FGF family, on osteoblasts, chondrocytes, and osteoclasts and compared them with those of FGF-2 and FGF-10. FGF-18 stimulated the proliferation of cultured mouse primary osteoblasts, osteoblastic MC3T3-E1 cells, primary chondrocytes, and prechondrocytic ATDC5 cells, although it inhibited the differentiation and matrix synthesis of these cells. FGF-18 up-regulated the phosphorylation of extracellular signal-regulated kinase in both osteoblasts and chondrocytes and up-regulated the phosphorylation of p38 mitogen-activated protein kinase only in chondrocytes. FGF-18 mitogenic actions were blocked by a specific inhibitor of extracellular signal-regulated kinase in both osteoblasts and chondrocytes and by a specific inhibitor of p38 mitogen-activated protein kinase in chondrocytes. With regard to the action of FGF-18 on bone resorption, FGF-18 not only induced osteoclast formation through receptor activator of nuclear factor-κB ligand and cyclooxygenase-2 but also stimulated osteoclast function to form resorbed pits on a dentine slice in the mouse coculture system. All these effects of FGF-18 bore a close resemblance to those of FGF-2, whereas FGF-10 affects none of these cells. FGF-18 may therefore compensate for the action of FGF-2 on bone and cartilage. Fibroblast growth factors (FGFs) 1FGFfibroblast growth factorFGFRfibroblast growth factor receptorRANKLreceptor activator of nuclear factor-κB ligandBMPbone morphogenetic proteinαMEMα modified minimum essential mediumFBSfetal bovine serumERKextracellular signal-regulated kinaseMAPKmitogen-activated protein kinaseJNKc-Jun N-terminal kinasePBSphosphate-buffered saline 1FGFfibroblast growth factorFGFRfibroblast growth factor receptorRANKLreceptor activator of nuclear factor-κB ligandBMPbone morphogenetic proteinαMEMα modified minimum essential mediumFBSfetal bovine serumERKextracellular signal-regulated kinaseMAPKmitogen-activated protein kinaseJNKc-Jun N-terminal kinasePBSphosphate-buffered saline are potent mitogens for a wide variety of cells of mesenchymal and neuroectodermal origin (1Ornitz D.M. Itoh N. Genome Biol. 2001; 2: 3005.1-3005.12Crossref Google Scholar). FGFs also play a role in the differentiation of a variety of cells and are involved in morphogenesis, angiogenesis, and development. The FGF family now consists of 23 members, FGF-1 to FGF-23, and there are 4 structurally related high-affinity receptors (FGFR1 to FGFR4) belonging to receptor tyrosine kinases that have an intrinsic protein tyrosine kinase activity and elicit tyrosine autophosphorylation of the receptor (1Ornitz D.M. Itoh N. Genome Biol. 2001; 2: 3005.1-3005.12Crossref Google Scholar, 2Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Crossref PubMed Scopus (1175) Google Scholar). Recent reports showing that mutations of FGFRs cause several genetic diseases with severe impairment of bone and cartilage formation, such as achondroplasia (3Rousseau F. Bonaventure J. Legeal-Mallet L. Pelet A. Rozet J.-M. Maroteaux P., Le Merrer M. Munnich A. Nature. 1994; 371: 252-254Crossref PubMed Scopus (744) Google Scholar, 4Shiang R. Thompson L.M. Zhu Y.-Z. Church D.M. Fielder T.J. Bocian M. Winokur S.T. Wasmuth J.J. Cell. 1994; 78: 335-342Abstract Full Text PDF PubMed Scopus (1092) Google Scholar) and thanatophoric dysplasia type II (5Webster M.K. D'Avis R.Y. Robertson S.C. Donoghue D.J. Mol. Cell. Biol. 1996; 8: 4081-4087Crossref Scopus (160) Google Scholar), indicate the essential role of FGF signalings on bone and cartilage metabolism. fibroblast growth factor fibroblast growth factor receptor receptor activator of nuclear factor-κB ligand bone morphogenetic protein α modified minimum essential medium fetal bovine serum extracellular signal-regulated kinase mitogen-activated protein kinase c-Jun N-terminal kinase phosphate-buffered saline fibroblast growth factor fibroblast growth factor receptor receptor activator of nuclear factor-κB ligand bone morphogenetic protein α modified minimum essential medium fetal bovine serum extracellular signal-regulated kinase mitogen-activated protein kinase c-Jun N-terminal kinase phosphate-buffered saline Among FGFs, FGF-2 is well known as a potent regulator of functions of bone and cartilage cells. It is produced by cells of osteoblastic lineage, accumulated in bone matrix, and acts as an autocrine/paracrine factor for bone cells (6Canalis E. Centrella M. McCarthy T.L. J. Clin. Invest. 1988; 81: 1572-1577Crossref PubMed Scopus (328) Google Scholar, 7Rodan S.B. Wesolowski G. Kyonggeun Y. Rodan G.A. J. Biol. Chem. 1989; 264: 19934-19941Abstract Full Text PDF PubMed Google Scholar, 8Hurley M.M. Abreu C. Gronowicz G.A. Kawaguchi H. Lorenzo J.A. J. Biol. Chem. 1994; 269: 9392-9396Abstract Full Text PDF PubMed Google Scholar). We and others have reported that the exogenous application of FGF-2 has stimulatory effects on bone formation in several in vivo models as a pharmacological action (9Aspenberg P. Lohmander L.S. Acta Orthop. Scand. 1989; 60: 473-476Crossref PubMed Scopus (89) Google Scholar, 10Kawaguchi H. Kurokawa T. Hanada K. Hiyama Y. Tamura M. Ogata E. Matsumoto T. Endocrinology. 1994; 135: 774-781Crossref PubMed Scopus (273) Google Scholar, 11Kawaguchi H. Nakamura K. Tabata Y. Ikada Y. Aoyama I. Anzai J. Nakamura T. Hiyama Y. Tamura M. J. Clin. Endocrinol. Metab. 2001; 86: 875-880Crossref PubMed Scopus (140) Google Scholar). In addition, theFgf-2-deficient mouse exhibits decreased bone mass and bone formation, although these changes were rather moderate (12Montero A. Okada Y. Tomira M. Ito M. Tsurukami H. Nakamura T. Doetschman T. Coffin J.D. Hurley M.M. J. Clin. Invest. 2000; 105: 1085-1093Crossref PubMed Scopus (402) Google Scholar). Paradoxically, FGF-2 is also known as a potent stimulator of bone resorption (13Kawaguchi H. Pilbeam C.C. Gronowicz G. Abreu C. Fletcher B.S. Herschman H.R. Raisz L.G. Hurley M.M. J. Clin. Invest. 1995; 96: 923-930Crossref PubMed Scopus (130) Google Scholar, 14Hurley M.M. Lee S.K. Raisz L.G. Bernecker P. Lorenzo J. Bone (NY). 1998; 22: 309-316Crossref PubMed Scopus (120) Google Scholar, 15Kawaguchi H. Chikazu D. Nakamura K. Kumegawa M. Hakeda Y. J. Bone Miner. Res. 2000; 15: 466-473Crossref PubMed Scopus (72) Google Scholar, 16Chikazu D. Hakeda Y. Ogata N. Nemoto K. Itabashi A. Takao T. Kumegawa M. Nakamura K. Kawaguchi H. J. Biol. Chem. 2000; 275: 31444-31450Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 17Chikazu D. Katagiri M. Ogasawara T. Ogata N. Shimoaka T. Takato T. Nakamura K. Kawaguchi H. J. Bone Miner. Res. 2001; 16: 2074-2081Crossref PubMed Scopus (67) Google Scholar) and is involved in joint destruction of rheumatoid arthritis patients (18Manabe N. Oda H. Nakamura K. Kuga Y. Uchida S. Kawaguchi H. Rheumatology. 1999; 38: 714-720Crossref PubMed Scopus (79) Google Scholar). The stimulatory effect of FGF-2 on osteoclast formation is mediated by the induction of cyclooxygenase-2, a main regulatory enzyme for prostaglandin production in bone, and receptor activator of nuclear factor-κB ligand (RANKL), a key membrane-associated molecule regulating osteoclast differentiation, in osteoblasts (13Kawaguchi H. Pilbeam C.C. Gronowicz G. Abreu C. Fletcher B.S. Herschman H.R. Raisz L.G. Hurley M.M. J. Clin. Invest. 1995; 96: 923-930Crossref PubMed Scopus (130) Google Scholar, 14Hurley M.M. Lee S.K. Raisz L.G. Bernecker P. Lorenzo J. Bone (NY). 1998; 22: 309-316Crossref PubMed Scopus (120) Google Scholar, 15Kawaguchi H. Chikazu D. Nakamura K. Kumegawa M. Hakeda Y. J. Bone Miner. Res. 2000; 15: 466-473Crossref PubMed Scopus (72) Google Scholar, 17Chikazu D. Katagiri M. Ogasawara T. Ogata N. Shimoaka T. Takato T. Nakamura K. Kawaguchi H. J. Bone Miner. Res. 2001; 16: 2074-2081Crossref PubMed Scopus (67) Google Scholar). Other than this indirect action through the mediation of osteoblasts, we recently reported that FGF-2 acts directly on mature osteoclasts to stimulate bone resorption (15Kawaguchi H. Chikazu D. Nakamura K. Kumegawa M. Hakeda Y. J. Bone Miner. Res. 2000; 15: 466-473Crossref PubMed Scopus (72) Google Scholar, 16Chikazu D. Hakeda Y. Ogata N. Nemoto K. Itabashi A. Takao T. Kumegawa M. Nakamura K. Kawaguchi H. J. Biol. Chem. 2000; 275: 31444-31450Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Another FGF, FGF-10, was recently cloned and shown to be expressed predominantly in the embryo and adult lung (19Yamasaki M. Miyake A. Tafashira S. Itoh N. J. Biol. Chem. 1996; 271: 15918-15921Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Further investigation revealed that it emanated from the prospective limb mesoderm to serve as an endogenous initiator for limb bud formation and that ectopic application of FGF-10 induced FGF-8 expression in the adjacent ectoderm (20Ohuchi H. Nakagawa T. Yamamoto A. Araga A. Ohata T. Ishimaru Y. Yoshioka H. Nohno T. Yamasaki M. Itoh N. Noji S. Development. 1997; 124: 2235-2244Crossref PubMed Google Scholar). FGF-10 was recently shown to be essential for limb bud formation because the Fgf-10-deficient mouse exhibited complete truncation of limbs (21Sekine K. Ohuchi H. Fujiwara M. Yamasaki M. Yoshizawa T. Sato T. Yagishita N. Matsui D. Koga Y. Itoh N. Kato S. Nat. Genet. 1999; 21: 138-141Crossref PubMed Scopus (982) Google Scholar). However, the effect of FGF-10 on bone and cartilage cells remains unknown. Two different groups have recently cloned a novel member of the FGF family, designated FGF-18 (22Ohbayashi N. Hoshikawa M. Kimura S. Yamasaki M. Fukui S. Itoh N. J. Biol. Chem. 1998; 273: 18161-18164Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 23Hu M.C. Qiu W.R. Wang Y.P. Hill D. Ring B.D. Scully S. Bolon B. DeRose M. Luethy R. Simonet W.S. Arakawa T. Danilenko D.M. Mol. Cell. Biol. 1998; 18: 6063-6074Crossref PubMed Scopus (108) Google Scholar). Sequence comparison indicates that FGF-18 is highly conserved between humans and mice and is most homologous to FGF-8 and FGF-17 among the FGF family members. FGF-18 is expressed primarily in the adult lungs and kidneys as well as in several discrete regions at embryonic days 14.5 and 19.5. The temporal and spatial patterns of FGF-18 expression in embryos are quite different from those of FGF-8 and FGF-17, suggesting that FGF-18 is a unique secreted signaling molecule in several adult and developing tissues. It is a pleiotropic growth factor that stimulates proliferation in some tissues such as the liver and small intestine (23Hu M.C. Qiu W.R. Wang Y.P. Hill D. Ring B.D. Scully S. Bolon B. DeRose M. Luethy R. Simonet W.S. Arakawa T. Danilenko D.M. Mol. Cell. Biol. 1998; 18: 6063-6074Crossref PubMed Scopus (108) Google Scholar, 24Hu M.C. Wang Y.P. Qui W.R. Oncogene. 1999; 18: 2635-2642Crossref PubMed Scopus (16) Google Scholar); however, its role in bone and cartilage remains undetermined. This study therefore investigated the actions of FGF-18 on cultured osteoblasts, chondrocytes, and osteoclasts and compared them with those of FGF-2 and FGF-10. Neonatal and 8-week-old ddY mice were purchased from Shizuoka Laboratories Animal Center (Shizuoka, Japan). Rat recombinant FGF-18 was generously provided by Amgen Inc. (Thousand Oaks, CA), human recombinant FGF-2 was provided by Kaken Pharmaceutical Co., Ltd. (Chiba, Japan), rat recombinant FGF-10 was provided by Sumitomo Pharmaceutical Co., Ltd. (Osaka, Japan), bone morphogenetic protein (BMP)-2 was provided by Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan), NS-398 was provided by Taisho Pharmaceutical Co., Ltd. (Tokyo), and osteoprotegerin was provided by Snow Brand Milk Products Co., Ltd. (Tochigi, Japan). α Modified minimum essential medium (αMEM) was purchased from Invitrogen, and fetal bovine serum (FBS) was from the Cell Culture Laboratory (Cleveland, OH). Bacterial collagenase and 1,25(OH)2 vitamin D3 were purchased from Wako Pure Chemicals Co. (Osaka, Japan), and dispase was purchased from Nitta Gelatin Co. (Osaka, Japan). Polyclonal rabbit antibody against phosphotyrosine was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Polyclonal rabbit antibodies against phospho-ERK, phospho-p38 MAPK, phospho-JNK, and PD98059 (2′- amino-3′-methoxyflanone) were obtained from New England Biolabs, Inc. (Beverly, MA). SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole) and Pronase were purchased from Calbiochem-Novabiochem. Other chemicals were obtained from Sigma. All animal experiments were performed according to the guidelines of the International Association for the Study of Pain (25Zimmermann M. Pain. 1983; 16: 109-110Abstract Full Text PDF PubMed Scopus (6856) Google Scholar). In addition, the experimental work was reviewed by the committee of Tokyo University charged with confirming ethics. Calvariae dissected from 1–4-day-old mice were washed in phosphate-buffered saline (PBS) and digested with 1 ml of trypsin/EDTA (Invitrogen) containing 10 mg of collagenase (Sigma; type 7) for 10 min × 5 times, and cells from fractions 3–5 were pooled. Cells were plated in 6-multiwell dishes at a density of 5,000 cells/cm2 and grown to confluence in αMEM containing 10% FBS. Chondrocytes were isolated from the ventral parts of the rib cages of 2-day-old mice digested with collagenase D as described previously (26Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar). Cells were plated in 6-multiwell dishes at a density of 5,000 cells/cm2 and grown to confluence in Dulbecco's modified Eagle's medium containing 10% FBS. Mouse osteoblastic MC3T3-E1 cells were maintained and subcultured for 3 or 4 days at 37 °C in a humidified atmosphere of 95% room air:5% CO2 in αMEM containing 10% FBS, 50 units/ml penicillin, 50 mg/ml streptomycin, and 0.2 mml-ascorbic acid phosphate ester sodium salt in a humidified CO2incubator. Mouse prechondrocytic ATDC5 cells were obtained from Riken Cell Bank (Saitama, Japan). The cells were grown in a medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium containing 5% FBS, 10 μg/ml bovine insulin, 10 μg/ml transferin, 3 × 10−8m sodium selenite (Roche Molecular Biochemicals), and antibiotics in a humidified CO2 incubator. Mouse primary osteoblastic cells, MC3T3-E1 cells, mouse primary chondrocytes, or ATDC5 cells were inoculated at a density of 5 × 104cells/well in 24-multiwell plates and cultured to confluence in αMEM or Dulbecco's modified Eagle's medium with 10% FBS for 2 days. The medium was deprived of serum for 12 h before the addition of experimental medium with or without FGFs (FGF-18 at 10−12 to 10−8m, FGF-2 at 10−8m, and FGF-10 at 10−8m), heparin (2 μg/ml), PD98059 (1, 3, 10, and 30 μm), and SB203580 (1, 3, and 10 μm). [3H]Thymidine (1 μCi/ml in the medium) incorporated for the final 2 h was measured at 18 h. For alkaline phosphatase staining, primary osteoblasts or MC3T3-E1 cells were inoculated at a density of 105 cells/well in 12-multiwell plates and cultured in αMEM containing 10% FBS and 50 μg/ml ascorbic acid with or without FGFs or BMP-2 (300 ng/ml). After 14 days of culture, the cells were fixed with 3.7% (v/v) formaldehyde in PBS and stained for alkaline phosphatase using naphthol AS-MX phosphate (Sigma) in N,N-dimethyl formamide as the substrate and Fast BB salt (Sigma) as coupler. For matrix nodule formation, these cells were inoculated at a density of 105 cells/well in 12-multiwell plates and cultured in αMEM containing 10% FBS, 50 μg/ml ascorbic acid, 10 nm dexamethasone, and 10 mm β-glycerophosphate with or without FGFs or BMP-2 for 21 days. The cells were fixed with 3.7% formaldehyde in PBS and stained for Alizarin red at pH 4.0. Primary chondrocytes or ATDC5 cells were inoculated at a density of 105 cells/well in 12-multiwell plates and cultured in the medium described above with or without FGFs or BMP-2 (300 ng/ml). After 21 days of culture, the cells were fixed with 3.7% formaldehyde in PBS, stained for 0.1% Alcian blue (Wako Pure Chemicals Co.) in 0.1 n HCl overnight, and rinsed with distilled water. For quantitative analyses, the amount of extractable dye was measured. Alcian blue-stained cultures were extracted with 1 ml of 6 m guanidine-HCl for 6 h at room temperature. The optical density of the extracted dye was measured at a wavelength of 630 nm with a microplate reader (MTP-300; Corona Electric Co.). Mouse primary osteoblasts and chondrocytes and MC3T3-E1 and ATDC5 cells were incubated in αMEM/0.5% FBS for 24 h before treatment with FGF-18 (5 × 10−9m) for various periods (0–30 min) and lysed with TNE buffer (10 mm Tris-HCl, 150 mm NaCl, 1% Nonidet P-40, 1 mm EDTA, 10 mm NaF, 2 mmNa3VO4, 1 mmaminoethyl-benzenesulfonyl fluoride, and 10 μg/ml aprotinin). The protein concentration in the cell lysate was measured using a Protein Assay Kit II (Bio-Rad). Equivalent amounts (30 μg) of cell lysates were electrophoresed by 8% SDS-PAGE and transferred to nitrocellulose membrane. After blocking with 5% bovine serum albumin, the membrane was incubated with monoclonal mouse antibody against phosphotyrosine and with peroxidase-conjugated anti-mouse IgG antibody. Phosphotyrosine-containing proteins were visualized using the ECL chemiluminescence reaction (Amersham Biosciences, Inc.) following the manufacturer's instructions. After the antibody was stripped from the membrane, to block nonspecific binding, membranes were incubated with 5% bovine serum albumin and then incubated with monoclonal mouse antibodies against phospho-ERK and phospho-JNK and polyclonal rabbit antibodies against phospho-p38 MAPK, and the immunoreactive bands were visualized as described above. Mouse calvarial primary osteoblasts described above (2 × 104 cells/well) and bone marrow cells prepared from 8-week-old mice (1 × 106 cells/well) were cocultured in 24-multiwell dishes containing αMEM/10% FBS with or without FGFs for 6 days with a medium change at 3 days. After 6 days of culture, the cells were fixed with 3.7% formaldehyde in PBS and ethanol-acetone (50:50, v/v) and stained at pH 5.0 in the presence ofl(+)-tartaric acid using naphthol AS-MX phosphate inN,N-dimethyl formamide as the substrate. Tartrate-resistant acid phosphatase-positive-multinucleated cells containing more than three nuclei were counted as osteoclasts. Mouse osteoblasts (1 × 106 cells/dish) and bone marrow cells (2 × 107 cells/dish) prepared as described above were cocultured on 10-cm culture dishes coated with 0.24% collagen gel matrix (Nitta Gelatin, Tokyo, Japan) containing αMEM with 10% FBS, 1,25(OH)2 vitamin D3 (10−8m), and prostaglandin E2 (10−6m) for 6 days, with a medium change every 3 days, and for 1 additional day in αMEM/10% FBS without bone-resorbing factors. After 7 days of culture, nonadherent cells were washed with PBS, and adherent cells were stripped by 0.2% bacterial collagenase. An aliquot of crude osteoclast preparation (0.1 ml) was further cultured on a dentine slice placed in each well of 96-well dishes containing αMEM/10% FBS in the presence or absence of FGFs. After 24 h of culture, cells were removed with 1 n NH4OH solution, and stained with 0.5% toluidine blue. Total area was estimated under a light microscope with a micrometer to assess osteoclastic bone resorption using an image analyzer (System Supply Co., Nagano, Japan). At the same time, cells on a dentine slice in the independent culture were fixed with 3.7% (v/v) formaldehyde in PBS and ethanol-acetone (50:50, v/v), and stained at pH 5.0 in the presence of l(+)-tartaric acid using naphthol AS-MX phosphate (Sigma) inN,N-dimethyl formamide as substrate. Tartrate-resistant acid phosphatase-positive multinucleated cells containing more than three nuclei were counted as osteoclasts. Means of groups were compared by analysis of variance, and the significance of differences was determined by post hoc testing using Bonferroni's method. Effects of FGF-18 (10−12 to 10−8m), FGF-2 (10−8m), and FGF-10 (10−8m) on the proliferation of primary osteoblasts were determined by [3H]thymidine incorporation into DNA (Fig.1). FGF-18 (≥10−9m) dose-dependently stimulated cell proliferation up to approximately10-fold of control cultures. Although heparan sulfate or heparin is reported to modulate the mitogenic activity of several FGFs (27Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Abstract Full Text PDF PubMed Scopus (450) Google Scholar, 34Turnbull J.E. Fernig D.G., Ke, Y. Wilkinson M.C. Gallagher J.T. J. Biol. Chem. 1992; 267: 10337-10341Abstract Full Text PDF PubMed Google Scholar), the mitogenic effect of FGF-18 was not altered by adding heparin to the culture (data not shown). This mitogenic action was similar to those of FGF-2, whereas FGF-10 did not affect cell proliferation. All of these results were reproducible in the culture of mouse osteoblastic MC3T3-E1 cells (data not shown). To further study the effects of FGFs on differentiation and matrix synthesis of osteoblasts, cultured primary osteoblasts were stained with alkaline phosphatase, a marker for osteoblast differentiation, at 14 days and with Alizarin red, a marker for matrix synthesis, at 21 days, respectively (Fig. 2). FGF-18 dose-dependently decreased both stainings in both cell culture systems. These inhibitory actions on differentiation and matrix synthesis were similarly seen in FGF-2-treated cultures, whereas FGF-10 affected none of these stainings. BMP-2, a positive control, potently stimulated both stainings. The effects of these growth factors were similarly seen in the MC3T3-E1 cell culture (data not shown). Fig.3 A shows the time course of effects of FGF-18 on tyrosine phosphorylation of cellular proteins in the primary osteoblast culture. Several proteins were selectively phosphorylated by FGF-18 (10−9m) at a time as early as 2 min. Western blot analyses using antibodies against specific proteins related to MAPKs revealed that phosphorylation of ERK was induced by FGF-18 at 2 min and was maintained for 30 min, although neither p38 nor JNK MAPK phosphorylation was affected for 30 min (Fig.3 A). To examine the functional relevance of the activation of MAPKs by FGF-18 in osteoblasts, PD98059, a specific inhibitor of the upstream kinase of ERK (28Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3255) Google Scholar), and SB203580, a specific inhibitor of p38 MAPK (29Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3133) Google Scholar), were added to the primary osteoblast culture. PD98059 and SB203580 were confirmed to show specific inhibition of the phosphorylation of ERK and p38 MAPK, respectively, in both the presence and absence of FGF-18 by Western blot analyses (data not shown). PD98059 dose-dependently inhibited the stimulation of FGF-18 on [3H]thymidine incorporation to the levels of the control culture (Fig. 3 B), whereas SB203580 (up to the maximum concentration of 10 μm) did not affect FGF-18 stimulation (data not shown). Similar results were seen when MC3T3-E1 cells were used instead of primary osteoblasts (data not shown). These results suggest that the ERK pathway functionally contributes to the FGF-18 mitogenic action on osteoblasts. Similar to the investigation on osteoblasts, the effects of FGF-18, FGF-2, and FGF-10 on chondrocyte proliferation were examined by [3H]thymidine incorporation into cultured primary chondrocytes (Table I). FGF-18 (≥10−10m) stimulated cell proliferation in the cultures. These effects also did not differ in the presence or absence of heparin (data not shown). These mitogenic effects were similar to those of FGF-2, whereas FGF-10 did not affect these cells. To study the effects of FGFs on differentiation/matrix synthesis of chondrocytes, accumulation of sulfated glycosaminoglycan in cultured primary chondrocytes was determined by Alcian blue staining (Table I). FGF-18 (≥10−9m) decreased the intensity of Alcian blue staining, whereas BMP-2 significantly stimulated it. This inhibitory action on chondrocyte differentiation was also seen in FGF-2-treated cultures, but not in FGF-10-treated cultures. These results were reproducible when mouse prechondrocytic ATDC5 cells were used instead of primary chondrocytes (data not shown).Table IEffects of FGFs on proliferation and differentiation of cultured primary chondrocytesThymidine incorporationAlcian blue×10 4 dpm×10 −1 ODControl3.53 ± 0.274.09 ± 0.11FGF-18 10−12m3.74 ± 0.473.80 ± 0.04 10−11m5.94 ± 1.073.85 ± 0.13 10−10m23.00 ± 3.171-ap < 0.01, significantly different from control.3.74 ± 0.11 10−9m25.06 ± 2.671-ap < 0.01, significantly different from control.2.72 ± 0.111-ap < 0.01, significantly different from control. 10−8m28.05 ± 4.711-ap < 0.01, significantly different from control.1.97 ± 0.121-ap < 0.01, significantly different from control.FGF-2 (10−8m)32.08 ± 3.081-ap < 0.01, significantly different from control.1.96 ± 0.081-ap < 0.01, significantly different from control.FGF-10 (10−8m)3.67 ± 0.344.00 ± 0.13BMP-2 (300 ng/ml)5.54 ± 0.191-ap < 0.01, significantly different from control.Data are expressed as means ± S.D. for 8 wells/group.1-a p < 0.01, significantly different from control. Open table in a new tab Data are expressed as means ± S.D. for 8 wells/group. Immunoblot analysis using an anti-phosphotyrosine antibody revealed that several intracellular proteins were phosphorylated by FGF-18 (10−9m) at a time as early as 2 min in cultured primary chondrocytes (Fig. 4 A). Among MAPKs, FGF-18 induced not only phosphorylation of ERK but also that of p38 MAPK at 2–5 min, although JNK MAPK was hardly phosphorylated for 30 min. Both PD98059 and SB203580, whose specific inhibitions of phosphorylation were also confirmed by Western blottings in this culture system, dose-dependently inhibited the stimulation of FGF-18 on [3H]thymidine incorporation to the levels of the control culture (Fig. 4 B). This result indicates the involvement of the activation of ERK and p38 MAPK in chondrocyte proliferation by FGF-18. Similar results were seen when the ATDC5 cell culture system was used (data not shown). To study the effects of FGF-18 on bone resorption, tartrate-resistant acid phosphatase-positive multinucleated osteoclast formation in the coculture system of mouse primary osteoblasts and bone marrow cells was examined. FGF-18 (≥10−10m) dose-dependently stimulated osteoclast formation (TableII). This induction was similar to that of FGF-2, whereas FGF-10 did not affect it. Because we reported previously that FGF-2 stimulated osteoclast formation through induction of cyclooxygenase-2 and RANKL (13Kawaguchi H. Pilbeam C.C. Gronowicz G. Abreu C. Fletcher B.S. Herschman H.R. Raisz L.G. Hurley M.M. J. Clin. Invest. 1995; 96: 923-930Crossref PubMed Scopus (130) Google Scholar, 14Hurley M.M. Lee S.K. Raisz L.G. Bernecker P. Lorenzo J. Bone (NY). 1998; 22: 309-316Crossref PubMed Scopus (120) Google Scholar, 15Kawaguchi H. Chikazu D. Nakamura K. Kumegawa M. Hakeda Y. J. Bone Miner. Res. 2000; 15: 466-473Crossref PubMed Scopus (72) Google Scholar, 17Chikazu D. Katagiri M. Ogasawara T. Ogata N. Shimoaka T. Takato T. Nakamura K. Kawaguchi H. J. Bone Miner. Res. 2001; 16: 2074-2081Crossref PubMed Scopus (67) Google Scholar), we added NS-398 (10−6m), a specific inhibitor of cyclooxygenase-2, and osteoprotegerin (10 ng/ml), a soluble decoy receptor of RANKL. The stimulation of osteoclastogenesis by FGF-18 was decreased ∼80% by NS-398 and almost completely abolished by osteoprotegerin. Hence, like FGF-2, FGF-18 stimulation of osteoclastogenesis was shown to be mediated by both cyclooxygenase-2 and RANKL.Table IIEffects of FGF-18 on osteoclast formation in the mouse coculture system in the presence and absence of specific inhibitors of cyclooxygenase 2 and RANKLData are expressed as means ± S.D. for 8 cultures/group. Representative TRAP staining pictures are shown on the right.2-150 p < 0.01, significant stimulation by FGFs.2-159 p < 0.01, significant inhibition by NS-398 or OPG (versus 10−8m FGF-18 alone). Open table in a new tab Data are expressed as means ± S.D. for 8 cultures/group. Representative TRA" @default.
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