RANKL and Vascular Endothelial Growth Factor (VEGF) Induce Osteoclast Chemotaxis through an ERK1/2-dependent Mechanism

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: PubMed Scopus Google Scholar). Furthermore, of a VEGF receptor induced in the growth of osteoclasts present during bone Vu T.H. J. Werb Z. N. Med. 1999; 5: PubMed Scopus Google Scholar). we have previously an 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). RANKL has been shown to have on osteoclast of activation of resorption and of T. R. A. G. S. H. J. N. A. S. G. J. J. PubMed Scopus Google Scholar, S. G. M. H. S. J. Cell Biol. 1999; PubMed Scopus Google Scholar, J. S. G. W. A. S. T. M. J. 2000; PubMed Scopus Google Scholar). colony-stimulating factor receptor activator of nuclear factor κB ligand vascular endothelial growth factor growth osteoprotegerin fused to an Fc Flt-1 fused to an Fc signal kinase protein kinase D3. VEGF, and M-CSF their functions through signal which have not been completely the the protein have in to their role in a of The was shown to be for osteoclast differentiation in vitro M. T. T. H. M. J. Biol. 2000; PubMed Scopus Google Scholar, M. T. M. H. M. 2000; PubMed Scopus Google Scholar). The and are by M-CSF, the and and are likely in osteoclast T. H. H. A. T. H. K. S. J. Cell Biol. 2000; PubMed Scopus Google Scholar, K. K. J. Biochem. PubMed Scopus Google Scholar). In addition, the ERK1/2 have also been in growth and resorption M. T. T. M. K. H. J. Biol. PubMed Scopus Google Scholar, N. K. A. T. M. K. H. J. Biol. 2000; PubMed Scopus Google Scholar). RANKL activation of ERK1/2 in mature osteoclasts has been the of this activation has to be N. M. H. 1999; PubMed Scopus Google Scholar). we used a of ex vivo bone cultures and in vitro cell migration to study the role of RANKL and VEGF in osteoclast recruitment during bone development. We that addition of antagonists of RANKL and VEGF, osteoprotegerin and to reduced recruitment of osteoclasts in ex vivo cultures of embryonic both RANKL and VEGF induced osteoclast recruitment in the Boyden invasion We cytokine activation of the in cultures of purified osteoclasts and that RANKL and VEGF, as well as M-CSF, the ERK1/2, but to of the ERK1/2 that ERK1/2 is in osteoclast recruitment in embryonic we that RANKL and VEGF an for osteoclast migration is of the ERK1/2 we that RANKL-induced bone resorption is of ERK1/2 the important in the by the various and to at murine VEGF, M-CSF, and protein from protein was from was from toward ERK1/2, and by Cell The MAP kinase PD98059 and from of and cells in the T. T. N. N. S. T. A. N. T. J. Bone Miner. Res. PubMed Scopus Google Scholar). by from mice and in cells by the and of and with the and for in and osteoclasts as T. R. A. G. S. H. J. N. A. S. G. J. J. PubMed Scopus Google Scholar). from mice and through a The cells on a for The cells at the in and for in in the of M-CSF and RANKL modified from T. Foged N.T. Delaisse J.M. J. Miner. Res. 13: PubMed Scopus Google Scholar). The in modified Boyden with The with of at a of cells from by with and to The cells by and the with a The in the in The in and the cells cultured for with as The cells in and a In where the to for the addition of the inhibitor and cytokines to activity of the inhibitor on of the was as the of that has through the to the of the with the of in the Bone resorption was with well established (2.Blavier L. Delaisse J.M. J. Cell Sci. 1995; 108: 3649-3659PubMed Google Scholar), on with of at the of the The as a and the from of tibia from was used for and the as a The bone cultured on in of with and for the in and the in the The cultured for with of release the in the tissue was by and by The of the bone was expressed as the of osteoclasts for in to the activation of induced by the of and cytokines in the was followed by with the cytokines for various as in the with the osteoclasts in followed by in modified and inhibitor from Cell for at at of protein and on The to a from The of the protein was by with in was blocked by of the in for followed by with the at and a for at the from of the migration was the The on the release was a RANKL and VEGF their in and RANKL and VEGF have been important but roles in the development of We an and role for the two growth factors as of the osteoclast recruitment study the of RANKL and VEGF on osteoclast we used the modified Boyden with a RANKL was to the we a in invasion from the through the matrix into the activation was at RANKL of in an of of the of RANKL to the not showing that RANKL invasion in a chemotactic of of completely the RANKL-induced the of we not in osteoclast between the and not We have previously that VEGF 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). we a of invasion the VEGF with at where the invasion is by with the to addition of VEGF to the not in a showing that VEGF also invasion in a chemotactic of completely invasion was VEGF receptors of which both Flt-1 and have been 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: PubMed Scopus Google Scholar, Vu T.H. J. Werb Z. N. Med. 1999; 5: PubMed Scopus Google Scholar, M. T. T. S. T. T. K. M. 2000; PubMed Scopus Google Scholar). of the ligand growth to the modified Boyden in invasion to addition of VEGF, at least a role of Flt-1 in the M-CSF has previously been as a 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, L. J. Cell Biol. PubMed Scopus Google with M-CSF in the modified Boyden have been In M-CSF as a invasion to with RANKL and VEGF with activation with of at of M-CSF and is a known of endothelial cell migration and N. B. M. T. N. R. R. J. 1999; PubMed Scopus Google Scholar, L. L. L. G. M. G. Biochem. Biophys. Res. Commun. 1999; PubMed Scopus Google Scholar). on cell and remain to be We the of on and osteoclast invasion through in the modified Boyden completely VEGF and was on it is specific for VEGF We not in osteoclast between the and not of cells with for was for to its not with in endothelial cell migration N. B. M. T. N. R. R. J. 1999; PubMed Scopus Google Scholar). of RANKL and VEGF from murine an model for osteoclast recruitment during bone development. are from osteoclast precursors are present in the surrounding the calcified of the primitive long During the osteoclast precursors and begin invasion through the bone into the marrow In contrast, in from mature osteoclasts are present on the bone surface of the marrow and recruitment is for resorption (2.Blavier L. Delaisse J.M. J. Cell Sci. 1995; 108: 3649-3659PubMed Google Scholar, J. Bone Miner. Res. PubMed Scopus Google Scholar). In to RANKL a role in osteoclast recruitment during bone we the of in the two bone of of to of the release from the showing that RANKL a role in bone development Although it not be that part of this is to the of on we the of osteoclasts as well as the of the by not that recruitment and not only is We also that the of the tibia to of the control K. N. S. K. M. K. N. H. T. K. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). We have previously that addition of a VEGF receptor to a in osteoclast recruitment in the (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). we although not as as addition of osteoclast recruitment in this an important role of VEGF in bone development. we that not inhibit release in the tibia model, that of VEGF not resorption, but only recruitment. VEGF, and M-CSF MAP ERK1/2 and are in several cellular such as cell and We that and osteoclast recruitment In to have cell of cell such as cells we activation in osteoclasts of of RANKL to activation of ERK1/2 activation was the of ERK1/2 protein was for the activation of was two VEGF ERK1/2 activation with to of RANKL VEGF not to activation of from T. H. H. A. T. H. K. S. J. Cell Biol. 2000; PubMed Scopus Google and L. J. Cell Biol. PubMed Scopus Google Scholar, M-CSF with a activation followed by a in the activation as previously T. H. H. A. T. H. K. S. J. Cell Biol. 2000; PubMed Scopus Google Scholar). the kinetic of M-CSF from of RANKL and VEGF. on activation was we that ERK1/2 with a kinetic pattern to VEGF that Flt-1 is for the ERK1/2 activation by VEGF. of ERK1/2 by PD98059 in study the ERK1/2 activation is in osteoclast we used which the ERK1/2, but not the MAP kinase L. Sci. S. A. 1995; PubMed Scopus Google Scholar, A. J. Biol. 1995; PubMed Scopus Google Scholar), in the modified Boyden of PD98059 completely both and invasion in the modified Boyden and the osteoclast not that ERK1/2 play an important role in osteoclast recruitment induced by RANKL and VEGF. PD98059 on the of we of PD98059 on invasion that signal are likely to this that of osteoclast invasion PD98059 RANKL and invasion we PD98059 in to Although as from T. H. H. A. T. H. K. S. J. Cell Biol. 2000; PubMed Scopus Google Scholar), reduced of the at M-CSF invasion through the and PD98059 on this activation not show that M-CSF is a control for by osteoclast of ERK1/2 by PD98059 in Bone ERK1/2 activation a role in osteoclast recruitment in long bones, we the of PD98059 in the ex vivo bone of the activation of ERK1/2 by PD98059 to a in release from cultured embryonic from showing that the mechanism regulating invasion of osteoclasts into the marrow cavity to some on ERK1/2 of the inhibitor was in cultures of that resorption is of ERK1/2 of PD98059 on Bone The in the that of embryonic tibia by osteoclasts is of In the addition of is used to resorption in the tibia the is to RANKL at least to some the resorption, although the of cytokines be that only part of the which RANKL on osteoclasts are through In to PD98059 resorption, we RANKL in the tibia and as in RANKL induced of the to the as VEGF M-CSF was able to of the is in with with the of to inhibit and for M-CSF Cecchini M.G. R. Fleisch H. PubMed Scopus Google Scholar). shown in addition of PD98059 to tibia cultures not inhibit the that bone resorption is of ERK1/2 The of an osteoclast a of The mature osteoclast is from the hematopoietically derived Along the of the osteoclast is recruited to the future site of resorption where at least one of bone is the osteoclast in to bone formation to on the bone surface (reviewed in Ref. Exp. 1999; PubMed Scopus Google Scholar). The that the recruitment of the osteoclasts to their future site of resorption are of incorrect regulation of these is likely to be in of embryonic metatarsals, where resorption is on osteoclast invasion into the marrow a model to study the processes osteoclast recruitment (2.Blavier L. Delaisse J.M. J. Cell Sci. 1995; 108: 3649-3659PubMed Google Scholar, 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, J. Bone Miner. Res. PubMed Scopus Google Scholar, Cecchini M.G. R. Fleisch H. PubMed Scopus Google Scholar). the factors of cell recruitment are the In this we have the role of RANKL and VEGF, two cytokines known to be present in the immediate bone environment M.F. Cermelli S. Cancedda R. Descalzi C.F. J. Cell Sci. 2000; 113: 59-69PubMed Google Scholar, Vu T.H. J. Werb Z. N. Med. 1999; 5: PubMed Scopus Google Scholar, T. R. A. G. S. H. J. N. A. S. G. J. J. PubMed Scopus Google Scholar), on osteoclast recruitment. We show that both cytokines as on the osteoclast in a with to and signal pathway. Furthermore, RANKL and VEGF osteoclast recruitment through a differing from that of a well chemoattractant for osteoclasts. we that bone resorption induced by RANKL a with the chemotactic properties of the The by which to migrate to the future resorption where they with to mature osteoclast resorption, remains the role in bone of the that present at the future site of resorption, a homing which the osteoclast by of the cells are of the not remains an the molecular of the mediators is particular cytokines to their during bone development. its properties expressed the T. R. A. G. S. H. J. N. A. S. G. J. J. PubMed Scopus Google Scholar), is a of the osteoclast pathway. have shown that RANKL be from the surface of the cells and into the surrounding environment L. R. H. J. J. Biol. 1999; PubMed Scopus Google Scholar). In addition, the of RANKL has been shown to its T. R. A. G. S. H. J. N. A. S. G. J. J. PubMed Scopus Google Scholar). VEGF is expressed by and in the of M.F. Cermelli S. Cancedda R. Descalzi C.F. J. Cell Sci. 2000; 113: 59-69PubMed Google Scholar, Vu T.H. J. Werb Z. N. Med. 1999; 5: PubMed Scopus Google Scholar). Furthermore, VEGF was previously in osteoclast differentiation (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: PubMed Scopus Google and resorptive activity M. T. T. S. T. T. K. M. 2000; PubMed Scopus Google Scholar). cytokines are in a potential to play a role in osteoclast recruitment. The receptor for RANKL is expressed on the surface of precursors as well as T. T. T. K. T. T. J. Exp. Med. 1999; 190: PubMed Scopus Google Scholar, H. A. G. L. R. L. T. S. S. G. Sci. S. A. 1999; PubMed Scopus Google Scholar). the two VEGF and are expressed by 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: PubMed Scopus Google Scholar, Vu T.H. J. Werb Z. N. Med. 1999; 5: PubMed Scopus Google Scholar, M. T. T. S. T. T. K. M. 2000; PubMed Scopus Google Scholar). that Flt-1 is the VEGF receptor 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: PubMed Scopus Google Scholar). In the modified we that both RANKL and VEGF able to osteoclast invasion a of the cytokines with to that of the that the Flt-1 ligand induced invasion in a to VEGF (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: PubMed Scopus Google that Flt-1 is the VEGF receptor in osteoclasts. The of these chemotactic properties was in the ex vivo model of osteoclast recruitment where antagonists of and blocked of the calcified matrix in the marrow we have previously shown that Flt-1 invasion of osteoclast into the marrow cavity in long (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). it is well known that RANKL osteoclast differentiation and bone resorption this is to the study to a direct chemotactic and potential role of RANKL in osteoclast recruitment. are by a study by S. G. M. H. S. J. Cell Biol. 1999; PubMed Scopus Google Scholar), of by osteoclasts cultured on bone in the of RANKL with cultures We that the of the by was to an of the osteoclasts. The that antagonists of VEGF release in the model where bone resorption is on osteoclast invasion into the marrow but not in the tibia model where osteoclasts are present on the bone surface that the role of VEGF is to recruitment. In contrast, RANKL is in both recruitment and activation of resorption. The matrix metalloproteinases have been shown to play important roles in osteoclast recruitment and invasion (2.Blavier L. Delaisse J.M. J. Cell Sci. 1995; 108: 3649-3659PubMed Google Scholar, 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, T. Foged N.T. Delaisse J.M. J. Miner. Res. 13: PubMed Scopus Google Scholar, T. M. Foged N.T. Delaisse J.M. J. Cell Sci. 1997; PubMed Google Scholar), although the mechanism of action remains We have previously to an action of and VEGF in osteoclast recruitment on the role of as well as its to VEGF to the (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, G. R. G. Vu T.H. T. K. K. S. Werb Z. Cell Biol. 2000; PubMed Scopus Google Scholar). RANKL was from the cell at least in vitro L. R. H. J. J. Biol. 1999; PubMed Scopus Google Scholar). L. R. H. J. J. 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H. A. T. H. K. S. J. Cell Biol. 2000; PubMed Scopus Google Scholar, N. M. H. 1999; PubMed Scopus Google Scholar, L. J. Cell Biol. PubMed Scopus Google Scholar). the role of the ERK1/2 activation in osteoclasts has not been we that RANKL and VEGF ERK1/2 activation in and that this activation is for recruitment of the osteoclasts to the future site of resorption. In contrast, we that M-CSF a to recruitment. at the of in M-CSF induced osteoclast M.F. J. Bone Miner. Res. PubMed Scopus Google that migration of osteoclasts is on ERK1/2 activity M.F. J. Bone Miner. Res. PubMed Scopus Google Scholar). In contrast, L. J. Cell Biol. PubMed Scopus Google that activation of murine osteoclast by M-CSF was on a of the and the but of and The for this remains to be as by L. J. Cell Biol. PubMed Scopus Google it on the used for the likely factor is the of the osteoclast, used and and we used murine In addition, although not to cell M-CSF ERK1/2 activation in murine osteoclasts by these with a study demonstrating that RANKL activation N. M. H. 1999; PubMed Scopus Google Scholar), we that and ERK1/2 activation and cell migration in osteoclast be on The role of RANKL in bone resorption is well and as RANKL may well in the tibia cultures, as from both K. N. S. K. M. K. N. H. T. K. Biochem. Biophys. Res. Commun. PubMed Scopus Google and that the PD98059 not inhibit the RANKL induced bone resorption in that RANKL induced osteoclast recruitment in of from RANKL induced bone resorption. Although both VEGF and M-CSF have been to be able to bone resorption, of the cytokines was able to for the of in In we that the of functions of VEGF and RANKL during bone development is to include osteoclast recruitment through chemotactic Furthermore, their chemotactic properties a which in from that of that RANKL for its activation of bone degradation and its chemotactic properties toward the osteoclast, is in