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• W2012496504 abstract "Development of bone depends on a continuous supply of bone-degrading osteoclasts. Although several factors such as the matrix metalloproteinases and the integrins have been shown to be important for osteoclast recruitment, the mechanism of action remains poorly understood. In this study we investigated the molecular mechanisms homing osteoclasts to their future site of resorption during bone development. We show that RANKL and VEGF, two cytokines known to be present in bone, possess chemotactic properties toward osteoclasts cultured in modified Boyden chambers. Furthermore, in ex vivo cultures of embryonic murine metatarsals, a well established model of osteoclast recruitment, antagonists of RANKL and VEGF reduced calcium release, showing that both cytokines play roles during bone development. In cultures of purified osteoclasts both RANKL and VEGF induced phosphorylation of ERK1/2 MAP kinase. M-CSF, a well-known chemoattractant of osteoclast, also induced activation of ERK1/2, although this activation followed a kinetic pattern differing from that of RANKL and VEGF. RANKL and VEGF-induced, but not M-CSF-induced, osteoclast invasion was completely blocked by the specific inhibitor of ERK1/2 phosphorylation, PD98059. In addition, PD98059 was able to inhibit calcium release in cultures of embryonic metatarsals. In contrast, PD98059 was unable to abrogate the RANKL-induced calcium release in the tibia model, demonstrating that only some of the RANKL functions on osteoclast physiology are regulated through the ERK1/2 pathway. Taken together, these results show that RANKL and VEGF, in addition to their role in osteoclast differentiation and activation of resorption, are important components of the processes regulating osteoclast chemotaxis. Development of bone depends on a continuous supply of bone-degrading osteoclasts. Although several factors such as the matrix metalloproteinases and the integrins have been shown to be important for osteoclast recruitment, the mechanism of action remains poorly understood. In this study we investigated the molecular mechanisms homing osteoclasts to their future site of resorption during bone development. We show that RANKL and VEGF, two cytokines known to be present in bone, possess chemotactic properties toward osteoclasts cultured in modified Boyden chambers. Furthermore, in ex vivo cultures of embryonic murine metatarsals, a well established model of osteoclast recruitment, antagonists of RANKL and VEGF reduced calcium release, showing that both cytokines play roles during bone development. In cultures of purified osteoclasts both RANKL and VEGF induced phosphorylation of ERK1/2 MAP kinase. M-CSF, a well-known chemoattractant of osteoclast, also induced activation of ERK1/2, although this activation followed a kinetic pattern differing from that of RANKL and VEGF. RANKL and VEGF-induced, but not M-CSF-induced, osteoclast invasion was completely blocked by the specific inhibitor of ERK1/2 phosphorylation, PD98059. In addition, PD98059 was able to inhibit calcium release in cultures of embryonic metatarsals. In contrast, PD98059 was unable to abrogate the RANKL-induced calcium release in the tibia model, demonstrating that only some of the RANKL functions on osteoclast physiology are regulated through the ERK1/2 pathway. Taken together, these results show that RANKL and VEGF, in addition to their role in osteoclast differentiation and activation of resorption, are important components of the processes regulating osteoclast chemotaxis. The development and continuous remodeling of the skeleton demand a tightly regulated balance between the bone-forming and bone-resorbing processes. Although many aspects of the resorptive processes remain elusive, there is general consensus that the hematopoietically derived osteoclast is the pivotal cell in the degradation of the bone matrix (reviewed in Ref. 1.Karsenty G. Genes Dev. 1999; 13: 3037-3051Crossref PubMed Scopus (273) Google Scholar). Inevitably, the focus centers on the factors that direct the resorptive activity of the osteoclasts and in particular the recruitment of the immature osteoclasts to the future site of resorption. Understanding the control of osteoclast recruitment to the future resorption site is of great importance, as incorrect regulation of recruitment it likely to constitute at least part of the underlying cause of the majority of bone metabolic disorders. During embryonic development of long bones, osteoclast precursors appear in the mesenchymal tissue surrounding the primitive bone collar. Along with endothelial cells they are recruited into the calcified tissue where they mature and begin formation of the marrow cavity (2.Blavier L. Delaisse J.M. J. Cell Sci. 1995; 108: 3649-3659PubMed Google Scholar). Cytokines are likely to constitute the molecular mediator through which the osteoclasts are directed to their future site of resorption. We, as well as others (3.Engsig M.T. Chen Q.J. Vu T.H. Pedersen A.C. Therkidsen B. Lund L.R. Henriksen K. Lenhard T. Foged N.T. Werb Z. Delaisse J.M. J. Cell Biol. 2000; 151: 879-890Crossref PubMed Scopus (491) Google Scholar, 4.Felix R. Cecchini M.G. Hofstetter W. Elford P.R. Stutzer A. Fleisch H. J. Bone Miner. Res. 1990; 5: 781-789Crossref PubMed Scopus (251) Google Scholar, 5.Weir E.C. Horowitz M.C. Baron R. Centrella M. Kacinski B.M. Insogna K.L. J. Bone Miner. Res. 1993; 8: 1507-1518Crossref PubMed Scopus (100) Google Scholar, 6.Carlevaro M.F. Cermelli S. Cancedda R. Descalzi C.F. J. Cell Sci. 2000; 113: 59-69PubMed Google Scholar, 7.Schlaeppi J.M. Gutzwiller S. Finkenzeller G. Fournier B. Endocrinol. Res. 1997; 23: 213-229Crossref PubMed Scopus (80) Google Scholar), have previously shown that cytokines expressed in the cellular environment of the bone surface are able to act at various stages of the osteoclast lifecycle. Macrophage colony-stimulating factor (M-CSF) 1The abbreviations used are: M-CSFmacrophage colony-stimulating factorRANKLreceptor activator of nuclear factor κB ligandVEGFvascular endothelial growth factorPlGF-2placenta growth factor-2hOPG-Fchuman osteoprotegerin fused to an Fc domainsFlt-Fcsoluble Flt-1 fused to an Fc domainERK1/2extracellular-regulated signal kinase 1/2MAPKmitogen-activated protein kinasePIphosphatidylinositolVitD31,25α-dihydroxy-vitamin D3. has long been known to play an important role in osteoclast physiology. At least part of that role may be attributable to its well-documented stimulatory action on osteoclast migration (8.Fuller K. Owens J.M. Jagger C.J. Wilson A. Moss R. Chambers T.J. J. Exp. Med. 1993; 178: 1733-1744Crossref PubMed Scopus (313) Google Scholar, 9.Owens J. Chambers T.J. Biochem. Biophys. Res. Commun. 1993; 195: 1401-1407Crossref PubMed Scopus (26) Google Scholar). In in vitro cultures, osteoclasts have been shown to migrate toward sources of M-CSF, marking the cytokine as one of the potential mediators of osteoclast recruitment. Other cytokines that are expressed in the immediate vicinity of the bone surface and whose receptors are present on the osteoclast include vascular endothelial growth factor (VEGF) and receptor activator of nuclear factor κB ligand (RANKL). VEGF has been shown to partially rescue M-CSF deficiency in the op/op mice (10.Niida S. Kaku M. Amano H. Yoshida H. Kataoka H. Nishikawa S. Tanne K. Maeda N. Nishikawa S. Kodama H. J. Exp. Med. 1999; 190: 293-298Crossref PubMed Scopus (361) Google Scholar). Furthermore, systemic injection of a soluble chimeric VEGF receptor (mFlt-1-IgG) induced morphological changes in the growth plate suggesting impaired chondroclast (a subset of osteoclasts present during bone development) function (11.Gerber H.P. Vu T.H. Ryan A.M. Kowalski J. Werb Z. Ferrara N. Nat. Med. 1999; 5: 623-628Crossref PubMed Scopus (1723) Google Scholar). Finally, we have previously mentioned an effect of VEGF on osteoclast migration (3.Engsig M.T. Chen Q.J. Vu T.H. Pedersen A.C. Therkidsen B. Lund L.R. Henriksen K. Lenhard T. Foged N.T. Werb Z. Delaisse J.M. J. Cell Biol. 2000; 151: 879-890Crossref PubMed Scopus (491) Google Scholar). Similarly, RANKL has been shown to have numerous effects on osteoclast physiology, including induction of differentiation, activation of resorption and stimulation of survival (12.Lacey D.L. Timms E. Tan H.L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4656) Google Scholar, 13.Burgess T.L. Qian Y. Kaufman S. Ring B.D. Van G. Capparelli C. Kelley M. Hsu H. Boyle W.J. Dunstan C.R. Hu S. Lacey D.L. J. Cell Biol. 1999; 145: 527-538Crossref PubMed Scopus (613) Google Scholar, 14.Lacey D.L. Tan H.L. Lu J. Kaufman S. Van G. Qiu W. Rattan A. Scully S. Fletcher F. Juan T. Kelley M. Burgess T.L. Boyle W.J. Polverino A.J. Am. J. Pathol. 2000; 157: 435-448Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). macrophage colony-stimulating factor receptor activator of nuclear factor κB ligand vascular endothelial growth factor placenta growth factor-2 human osteoprotegerin fused to an Fc domain soluble Flt-1 fused to an Fc domain extracellular-regulated signal kinase 1/2 mitogen-activated protein kinase phosphatidylinositol 1,25α-dihydroxy-vitamin D3. RANKL, VEGF, and M-CSF all exert their functions through multiple signal transduction pathways, which have not yet been completely characterized. Especially, the pathways involving the mitogen-activated protein kinases (MAPK) have attracted considerable interest in recent years due to their central role in a range of osteoclastic activities. The p38 MAPK was shown to be essential for osteoclast differentiation in vitro (15.Matsumoto M. Sudo T. Saito T. Osada H. Tsujimoto M. J. Biol. Chem. 2000; 275: 31155-31161Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 16.Matsumoto M. Sudo T. Maruyama M. Osada H. Tsujimoto M. FEBS Lett. 2000; 486: 23-28Crossref PubMed Scopus (76) Google Scholar). The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are activated by M-CSF, the interleukins IL-1α and IL-1β and are likely involved in osteoclast survival (17.Miyazaki T. Katagiri H. Kanegae Y. Takayanagi H. Sawada Y. Yamamoto A. Pando M.P. Asano T. Verma I.M. Oda H. Nakamura K. Tanaka S. J. Cell Biol. 2000; 148: 333-342Crossref PubMed Scopus (343) Google Scholar, 18.Lee Z.H. Lee S.E. Kim C.W. Lee S.H. Kim S.W. Kwack K. Walsh K. Kim H.H. J. Biochem. (Tokyo). 2002; 131: 161-166Crossref PubMed Scopus (78) Google Scholar). In addition, the ERK1/2 kinases have also been implicated in fibroblast growth factor-2 (FGF-2) and Gas6-induced resorption (19.Katagiri M. Hakeda Y. Chikazu D. Ogasawara T. Takato T. Kumegawa M. Nakamura K. Kawaguchi H. J. Biol. Chem. 2001; 276: 7376-7382Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 20.Chikazu D. Hakeda Y. Ogata N. Nemoto K. Itabashi A. Takato T. Kumegawa M. Nakamura K. Kawaguchi H. J. Biol. Chem. 2000; 275: 31444-31450Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Although, RANKL activation of ERK1/2 in mature osteoclasts has been demonstrated, the relevance of this activation has yet to be clarified (21.Wong B.R. Besser D. Kim N. Arron J.R. Vologodskaia M. Hanafusa H. Choi Y. Mol. Cell. 1999; 4: 1041-1049Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Here, we used a combination of ex vivo bone cultures and in vitro cell migration assays to study the role of RANKL and VEGF in osteoclast recruitment during bone development. We demonstrate that addition of antagonists of RANKL and VEGF, osteoprotegerin (OPG) and endostatin, respectively, led to reduced recruitment of osteoclasts in ex vivo cultures of embryonic bones. Moreover, both RANKL and VEGF dose-dependently induced osteoclast recruitment in the Boyden chamber invasion assay. We explored cytokine activation of the MAPKs in cultures of purified osteoclasts and found that RANKL and VEGF, as well as M-CSF, activated the ERK1/2, but failed to activate p38 MAPK. Further studies of the ERK1/2 kinases demonstrated that ERK1/2 is indeed involved in osteoclast recruitment in embryonic bones. Interestingly, we discovered that RANKL and VEGF utilize an ERK1/2-dependent pathway for inducing osteoclast migration; whereas M-CSF-mediated migration is independent of the ERK1/2 cascade. Finally, we demonstrate that RANKL-induced bone resorption is independent of ERK1/2 activity, further underlining the important differences in the signaling cascades employed by the various cytokines. Reagents and Mice—All animal experiments were performed according to approved protocols following guidelines at Nordic Bioscience A/S, Denmark. Recombinant murine RANKL, human VEGF, human M-CSF, and recombinant human Flt-Fc chimeric protein were from R&D Systems. Recombinant human OPG-Fc chimeric protein was from Alexis Corporation. Endostatin was purchased from Calbiochem. Antibodies toward ERK1/2, p38, phospho-ERK1/2, and phospho-p38 were provided by Cell Signaling Technology. The MAP kinase inhibitors PD98059 and SB203580 were from Calbiochem. Preparation of Osteoclast-like Cells and Spleen Cell-derived Osteoclasts—Osteoclast-like cells were generated in the co-culture system (22.Akatsu T. Tamura T. Takahashi N. Udagawa N. Tanaka S. Sasaki T. Yamaguchi A. Nagata N. Suda T. J. Bone Miner. Res. 1992; 7: 1297-1306Crossref PubMed Scopus (160) Google Scholar). Briefly, primary calvarial osteoblast were isolated by mincing calvariae from 1 day old BalbC CF1 mice and then culturing them in αMEM containing 10% fetal bovine serum. Hematopoietic cells were isolated by dissecting the femur and tibiae of 6-week-old BalbC CF1 mice, and subsequent coculturing (1.8 × 107) with the calvarial osteoblasts (2.5 × 105) and grown for 6–7 days in αMEM containing 10% fetal bovine serum and 10 nm 1,25α-dihydroxy-vitamin D3 (VitD3). Spleen cell-derived osteoclasts were generated as described (12.Lacey D.L. Timms E. Tan H.L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W.J. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4656) Google Scholar). Briefly, spleens were isolated from BalbC CF1 mice and mashed through a 70-μm filter (Invitrogen A/S). The cells were placed on a Ficoll-Hypaque gradient, centrifuged for 20 min. The cells at the interface were collected, washed in media, and grown for 6–7 days in αMEM +10% heat-inactivated fetal bovine serum in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml). Chemotaxis Assays—Chemotaxis assays were modified from previous procedures (23.Sato T. Foged N.T. Delaisse J.M. J. Bone. Miner. Res. 1998; 13: 59-66Crossref PubMed Scopus (93) Google Scholar). The assays were performed in modified Boyden chambers with polycarbonate filters containing 12-μm pore membranes (Corning Costar). The filters were coated with 10 μl of reconstituted type I collagen (Nitta Collagen) at a concentration of 2.4 mg/ml. Osteoclast-like cells (OCLs) were prepared from osteoclast/osteoblast co-cultures by sequential treatment with collagenase 0.1% (w/v) and dispase 0.1% (w/v) to remove osteoblastic cells. The remaining cells were loosened by trypsin treatment and then gently lifted off the plates with a rubber policeman. The OCLs were seeded in the culture inserts in αMEM containing 0,1% (w/v) albumax. The inserts were placed in 12-well plates, and the cells were cultured for 22 h with or without additions as described under “Results.” The cells were fixed in 3.7% formaldehyde, and OCLs were visualized TRAP staining using a leukocyte acid phosphatase kit (Sigma-Aldrich). In all assays where inhibitors were used, the OCLs were allowed to attach for 1 h before the addition of the inhibitor and cytokines to reduce any activity of the inhibitor on attachment of the cells. Invasion was determined as the ratio of OCLs that has invaded through the collagen gel to reach the lower side of the membrane compared with the total number of OCLs in the insert. Bone Resorption Models—Bone resorption was evaluated with well established models (2.Blavier L. Delaisse J.M. J. Cell Sci. 1995; 108: 3649-3659PubMed Google Scholar), on littermates. Timed pregnant mothers were injected subcutaneously with 100 μCi of 45CaCl2 (45Ca) at day 16 after the confirmation of the vaginal plug. The middle three metatarsals (preserved as a triad) and the tibiae were isolated from each hindlimb of day 17 embryos. One metatarsal triad or tibia from each embryo was used for treatments and the other as a control. The bone explants were cultured floating on Millipore filter membrane in 400 μl of BGJb medium supplemented with NaHCO3 (2.2 g/liter), NaCl (0.9 g/liter), albumax (1 g/liter), glutamax (0.5 g/liter), ascorbate (50 mg/liter), and (except for the experiment described in Fig. 7) VitD3 (50 nm) and the factors/inhibitors specified in the figure legends. The bones were cultured for 4 days, with measurement of 45Ca release every day. On day 4 the 45Ca remaining in the tissue was released by formic acid treatment and measured by scintillation counting. The demineralization of the bone explants was then expressed cumulatively as the percentage of total 45Ca amount. Immunoblotting—Spleen cell-derived osteoclasts were starved for 2 h in αMEM containing 0,1% (w/v) albumax to minimize the activation of kinases induced by the presence of serum and cytokines in the cultures. This was followed by incubation with the cytokines for various times as specified in the figure legends. After incubation with or without stimuli the spleen cell-derived osteoclasts were washed twice in ice-cold phosphate-buffered saline, followed by lysis in modified radioimmune precipitation assay buffer (30 mm NaCl, 50 mm Tris, 5 mm EDTA, 1% Nonidet P-40, 1% deoxycholic acid, 0,1% SDS, 5 mm NaF, 1 mm NaVO3, and protease inhibitor mixture from Calbiochem). Cell lysates were centrifuged for 30 min at 4 °C at 15,000 × g.10 μg of total protein were loaded and electrophoresed on 10% SDS-PAGE gels. The proteins were transferred to a nitrocellulose membrane (BA85; pore size: 0.45 μm from Schleicher & Schuell). The quality of the protein loading was confirmed by staining with 0.2% Ponceau S in trichloroacetic acid. Nonspecific binding was blocked by incubation of the membrane in TBS-T buffer (50 mm Tris, 150 mm NaCl, 0.1% Tween 20) containing 5% skim milk powder for 1 h, followed by incubation with the appropriate primary antibody overnight at 4 °C, and then a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. All blots were visualized using the ECL kit from Amersham Biosciences. Statistics—The statistical analysis of the migration experiments was performed using the two-tailed Student's t test. The statistical analysis on the 45Ca release experiments was performed using a paired two-tailed Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. RANKL and VEGF Induce Osteoclast Invasion—Through their function in osteoclastogenesis and angiogenesis, respectively, RANKL and VEGF have already been ascribed important but distinct roles in the development of bone. We hypothesized an additional and overlapping role for the two growth factors as integral parts of the osteoclast recruitment machinery. To study the effect of RANKL and VEGF on osteoclast motility we used the modified Boyden chambers coated with a type I collagen gel. When RANKL was exclusively added to the lower chamber we observed a dose-dependent increase in OCL invasion from the upper chamber through the type I collagen matrix into the lower chamber (Fig. 1A). Maximal activation was obtained at RANKL concentrations of 100 ng/ml or higher, resulting in an increase of 70–90% of the invasion. Addition of RANKL to the upper chamber did not significantly activate OCL invasion, showing that RANKL induces invasion in a chemotactic manner. Addition of 300 ng/ml of hOPG-Fc completely abrogated the RANKL-induced invasion, without affecting the basal level of migration. Importantly, we did not observe any differences in osteoclast number between the treated and non-treated conditions (data not shown). We have previously mentioned that VEGF stimulates osteoclast invasion (3.Engsig M.T. Chen Q.J. Vu T.H. Pedersen A.C. Therkidsen B. Lund L.R. Henriksen K. Lenhard T. Foged N.T. Werb Z. Delaisse J.M. J. Cell Biol. 2000; 151: 879-890Crossref PubMed Scopus (491) Google Scholar). Here we observed a biphasic dose-dependent induction of invasion against the VEGF concentration gradient (Fig. 1B), with optimal stimulation at 1 ng/ml where the invasion is increased by 40–50% when compared with the non-stimulated controls. Similar to RANKL, addition of VEGF to the upper chamber did not result in a significant activation, showing that VEGF also stimulates OCL invasion in a chemotactic manner. Addition of soluble VEGF-receptor sFlt-Fc completely inhibited VEGF-induced invasion, whereas basal invasion was unaffected. Several VEGF receptors exist, of which both Flt-1 and Flk-1 have been observed in osteoclasts (10.Niida S. Kaku M. Amano H. Yoshida H. Kataoka H. Nishikawa S. Tanne K. Maeda N. Nishikawa S. Kodama H. J. Exp. Med. 1999; 190: 293-298Crossref PubMed Scopus (361) Google Scholar, 11.Gerber H.P. Vu T.H. Ryan A.M. Kowalski J. Werb Z. Ferrara N. Nat. Med. 1999; 5: 623-628Crossref PubMed Scopus (1723) Google Scholar, 24.Nakagawa M. Kaneda T. Arakawa T. Morita S. Sato T. Yomada T. Hanada K. Kumegawa M. Hakeda Y. FEBS Lett. 2000; 473: 161-164Crossref PubMed Scopus (314) Google Scholar). Addition of the Flt-1-specific ligand placenta growth factor-2 (PlGF-2) to the modified Boyden chambers resulted in increased invasion similar to addition of VEGF, indicating at least a partial role of Flt-1 in the process. M-CSF has previously been described as a powerful chemotactic factor for osteoclasts (8.Fuller K. Owens J.M. Jagger C.J. Wilson A. Moss R. Chambers T.J. J. Exp. Med. 1993; 178: 1733-1744Crossref PubMed Scopus (313) Google Scholar, 25.Nakamura I. Lipfert L. Rodan G.A. Le T.D. J. Cell Biol. 2001; 152: 361-373Crossref PubMed Scopus (91) Google Scholar); however, studies with M-CSF in the modified Boyden Chamber have never been reported. In our system M-CSF acted as a chemoattractant, activating OCL invasion to levels comparable with RANKL and VEGF stimulation, with maximal activation (50–60% increase when compared with baseline levels) of motility being reached at 5 ng/ml of M-CSF and higher. Endostatin Inhibits VEGF-stimulated Osteoclast Migration— Endostatin is a known antagonist of VEGF-mediated endothelial cell migration and proliferation (26.Yamaguchi N. Anand-Apte B. Lee M. Sasaki T. Fukai N. Shapiro R. Que I. Lowik C. Timpl R. Olsen B.R. EMBO J. 1999; 18: 4414-4423Crossref PubMed Scopus (426) Google Scholar, 27.Taddei L. Chiarugi P. Brogelli L. Cirri P. Magnelli L. Raugei G. Ziche M. Granger H.J. Chiarugi V. Ramponi G. Biochem. Biophys. Res. Commun. 1999; 263: 340-345Crossref PubMed Scopus (90) Google Scholar). Its effects on other cell types and cytokine-mediated signals, however, still remain to be studied. We tested the effect of endostatin on VEGF-, RANKL-, M-CSF-, and PlGF-2-stimulated osteoclast invasion through type I collagen in the modified Boyden chamber. Endostatin completely inhibited VEGF (Fig. 2A) and PlGF-2 (Fig. 2D)-mediated invasion, whereas no effect was observed on RANKL- (Fig. 2B) or M-CSF- (Fig. 2C) mediated invasion, suggesting it is specific for VEGF receptor-mediated signaling. We did not observe any differences in osteoclast number between the treated and non-treated conditions (data not shown). Preincubation of cells with endostatin for 30 min was necessary for endostatin to exert its inhibitory effect (data not shown), corresponding with previous findings in endothelial cell migration studies (26.Yamaguchi N. Anand-Apte B. Lee M. Sasaki T. Fukai N. Shapiro R. Que I. Lowik C. Timpl R. Olsen B.R. EMBO J. 1999; 18: 4414-4423Crossref PubMed Scopus (426) Google Scholar). Antagonists of RANKL and VEGF Inhibit Calcium Release from Cultured Primitive Long Bones—The murine metatarsal system provides an excellent model for studying osteoclast recruitment during bone development. When metatarsals are isolated from 17-day old embryos, osteoclast precursors are present in the periosteum surrounding the calcified cartilage of the primitive long bone. During subsequent culture the osteoclast precursors mature, fuse, and begin invasion through the type I collagen-rich bone collar into the soon-to-be marrow cavity. In contrast, in tibiae isolated from 17-day-old mouse embryos, mature actively resorbing osteoclasts are already present on the bone surface of the marrow cavity, and no further recruitment is necessary for resorption (2.Blavier L. Delaisse J.M. J. Cell Sci. 1995; 108: 3649-3659PubMed Google Scholar, 28.Dieudonne S.C. Foo P. van Zoelen E.J. Burger E.H. J. Bone Miner. Res. 1991; 6: 479-487Crossref PubMed Scopus (85) Google Scholar). In order to investigate whether RANKL plays a physiologically relevant role in osteoclast recruitment during bone development, we studied the effect of recombinant hOPG-Fc in the two bone models. Addition of 500 ng/ml of hOPG-Fc led to complete inhibition of the 45Ca release from the metatarsals showing that RANKL indeed plays a role in bone development (Fig. 3A). Although it can not be ruled out that part of this inhibition is due to the effect of hOPG-Fc on osteoclastic differentiation, we observed the presence of osteoclasts inside, as well as outside the periosteum of the hOPG-Fc treated metatarsals by histological analysis (data not shown), thus confirming that recruitment and not only osteoclastogenesis is impaired. We also found that hOPG-Fc inhibited the demineralization of the tibia to ∼50% of the control levels, thus confirming previous findings (29.Tsukii K. Shima N. Mochizuki S. Yamaguchi K. Kinosaki M. Yano K. Shibata O. Udagawa N. Yasuda H. Suda T. Higashio K. Biochem. Biophys. Res. Commun. 1998; 246: 337-341Crossref PubMed Scopus (221) Google Scholar). We have previously reported that addition of a soluble VEGF receptor led to a decrease in osteoclast recruitment in the metatarsals (3.Engsig M.T. Chen Q.J. Vu T.H. Pedersen A.C. Therkidsen B. Lund L.R. Henriksen K. Lenhard T. Foged N.T. Werb Z. Delaisse J.M. J. Cell Biol. 2000; 151: 879-890Crossref PubMed Scopus (491) Google Scholar). Here we find that, although not as efficient as hOPG-Fc, addition of 60 ng/ml endostatin inhibits osteoclast recruitment in this system (Fig. 3B), thus further supporting an important role of VEGF in bone development. Interestingly we found that endostatin did not inhibit Ca45 release in the tibia model, suggesting that inhibition of VEGF does not affect resorption, but only recruitment. RANKL, VEGF, and M-CSF induce MAPK (ERK1/2) Activation—The MAP kinases ERK1/2 and p38 are involved in several cellular processes, such as proliferation, cell survival, differentiation, and cytokine-activated migration. We hypothesized that RANKL- and VEGF-mediated osteoclast recruitment might involve MAPK phosphorylation. In order to have cell populations devoid of contaminating cell types such as stromal cells we studied MAPK activation in spleen cell-derived osteoclasts (SOCs) instead of OCLs. Addition of RANKL led to activation of ERK1/2 within 30 min after stimulation (upper panels, Fig. 4A). This activation was sustained throughout the 2-hour period tested. Total level of ERK1/2 protein was unchanged for the entire period. No activation of p38 was observed (lower two panels, Fig. 4A). VEGF induces ERK1/2 activation with kinetics highly similar to those of RANKL (Fig. 4B). Correspondingly, VEGF stimulation did not lead to activation of p38 (Fig. 4B). As expected from Refs. 17.Miyazaki T. Katagiri H. Kanegae Y. Takayanagi H. Sawada Y. Yamamoto A. Pando M.P. Asano T. Verma I.M. Oda H. Nakamura K. Tanaka S. J. Cell Biol. 2000; 148: 333-342Crossref PubMed Scopus (343) Google Scholar and 25.Nakamura I. Lipfert L. Rodan G.A. Le T.D. J. Cell Biol. 2001; 152: 361-373Crossref PubMed Scopus (91) Google Scholar, M-CSF activated ERK1/2. However, with a maximal activation already after 10 min, followed by a reduction in the activation level, as previously described (17.Miyazaki T. Katagiri H. Kanegae Y. Takayanagi H. Sawada Y. Yamamoto A. Pando M.P. Asano T. Verma I.M. Oda H. Nakamura K. Tanaka S. J. Cell Biol. 2000; 148: 333-342Crossref PubMed Scopus (343) Google Scholar). Thus, the biphasic kinetic profile of M-CSF differed from those of RANKL and VEGF. Again, no effect on p38 activation was observed (Fig. 4C). Finally, we found that PlGF-2 activated ERK1/2 with a kinetic pattern similar to VEGF (Fig. 4D), indicating that Flt-1 is responsible for the ERK1/2 activation by VEGF. Inhibition of ERK1/2 Activation by PD98059 in Cytokine-activated Osteoclast Migration—To study whether the cytokine-induced ERK1/2 activation is involved in osteoclast migration, we used PD98059, which specifically blocks the ERK1/2, but not the p38 MAP kinase pathways (30.Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2595) Google Scholar, 31.Alessi 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 (3259) Google Scholar), in the modified Boyden chamber assay. Addition of PD98059 completely abrogated both RANKL- and VEGF-stimulated OCLs invasion in the modified Boyden chamber (Fig. 5, A and B), without affecting the osteoclast number (data not shown), suggesting that ERK1/2 play an important role in osteoclast" @default.
• W2012496504 created "2016-06-24" @default.
• W2012496504 creator A5001350750 @default.
• W2012496504 creator A5049717703 @default.
• W2012496504 creator A5062465642 @default.
• W2012496504 creator A5082542112 @default.
• W2012496504 date "2003-12-01" @default.
• W2012496504 modified "2023-10-07" @default.
• W2012496504 title "RANKL and Vascular Endothelial Growth Factor (VEGF) Induce Osteoclast Chemotaxis through an ERK1/2-dependent Mechanism" @default.
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