Epidermal Growth Factor Receptor-deficient Mice Have Delayed Primary Endochondral Ossification Because of Defective Osteoclast Recruitment

The epidermal growth factor receptor (EGFR) and its ligands function in diverse cellular functions including cell proliferation, differentiation, motility, and survival. EGFR signaling is important for the development of many tissues, including skin, lungs, intestines, and the craniofacial skeleton. We have now determined the role of EGFR signaling in endochondral ossification. We analyzed long bone development in EGFR-deficient mice. EGFR deficiency caused delayed primary ossification of the cartilage anlage and delayed osteoclast and osteoblast recruitment. Ossification of the growth plates was also abnormal resulting in an expanded area of growth plate hypertrophic cartilage and few bony trabeculae. The delayed osteoclast recruitment was not because of inadequate expression of matrix metalloproteinases, including matrix metalloproteinase-9, which have previously been shown to be important for osteoclast recruitment. EGFR was expressed by osteoclasts, suggesting that EGFR ligands may act directly to affect the formation and/or function of these cells. EGFR signaling regulated osteoclast formation. Inhibition of EGFR tyrosine kinase activity decreased the generation of osteoclasts from cultured bone marrow cells. The epidermal growth factor receptor (EGFR) and its ligands function in diverse cellular functions including cell proliferation, differentiation, motility, and survival. EGFR signaling is important for the development of many tissues, including skin, lungs, intestines, and the craniofacial skeleton. We have now determined the role of EGFR signaling in endochondral ossification. We analyzed long bone development in EGFR-deficient mice. EGFR deficiency caused delayed primary ossification of the cartilage anlage and delayed osteoclast and osteoblast recruitment. Ossification of the growth plates was also abnormal resulting in an expanded area of growth plate hypertrophic cartilage and few bony trabeculae. The delayed osteoclast recruitment was not because of inadequate expression of matrix metalloproteinases, including matrix metalloproteinase-9, which have previously been shown to be important for osteoclast recruitment. EGFR was expressed by osteoclasts, suggesting that EGFR ligands may act directly to affect the formation and/or function of these cells. EGFR signaling regulated osteoclast formation. Inhibition of EGFR tyrosine kinase activity decreased the generation of osteoclasts from cultured bone marrow cells. Skeletal elements develop by two distinct mechanisms: intramembranous and endochondral ossification (1Caplan A.I. Ciba Found. Symp. 1988; 136: 3021Google Scholar). Endochondral ossification is a process by which a cartilaginous template is first formed and then replaced by bone. During embryogenesis, condensations of mesenchymal cells form, within which chondrocytes develop, proliferate, and differentiate to form a cartilage template that contains distinct zones of resting, proliferative, and hypertrophic chondrocytes. The proliferation and differentiation of chondrocytes within the cartilage template are spatially ordered, with proliferating cells at the two ends of the template and progressively more mature cells forming hypertrophic cartilage in the middle. Hypertrophic chondrocytes secrete a specialized extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; EGFR, epidermal growth factor receptor; M-CSF, monocyte-colony stimulating factor; MEM, minimal essential medium; MMP, matrix metalloproteinase; TRAP, tartrate-resistant acid phosphatase; RT, reverse transcriptase; HC, hypertrophic cartilage; VEGF, vascular endothelial growth factor; E, embryonic day. containing collagen X, which becomes calcified. Endochondral ossification begins with the invasion of the calcified hypertrophic cartilage by blood vessels, accompanied by osteoclasts and osteoblasts (primary ossification center). The function of osteoclasts is to remove the hypertrophic cartilage ECM and that of osteoblasts is to replace it with bone ECM. Longitudinal bone growth is accomplished by the continuing proliferation and maturation of chondrocytes at the ends of the cartilage template (the growth plates) to form more hypertrophic cartilage and its continual removal and replacement by bone (growth plate ossification or formation of primary spongiosa). Normal endochondral bone development requires the exquisite coordination of hypertrophic cartilage formation, vascular invasion, and the development and function of osteoclasts and osteoblasts (2Olsen B.R. Reginato A.M. Wang W. Annu. Rev. Cell Dev. Biol. 2000; 16: 191-220Crossref PubMed Scopus (773) Google Scholar, 3Karsenty G. Wagner E.F. Dev. 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Simmons H.A. Sandberg A.L. Canalis E. Endocrinology. 1980; 107: 270-273Crossref PubMed Scopus (138) Google Scholar). In this study, we showed that impaired recruitment of osteoclasts contributed to the impaired endochondral bone formation in EGFR-deficient mice and that EGFR signaling is necessary for osteoclast formation from bone marrow progenitors. Reagents—AG1478 was purchased from Calbiochem (San Diego, CA). Mouse macrophage colony stimulating factor (mouse M-CSF) was purchased from R 376: 337-341Crossref PubMed Scopus (875) Google Scholar). and were by for the for the were and the of the is as mice were at and the were and the long for In long from from are or were by of P.J. Chin J.R. Shum L. Slavkin H.C. Shuler C.F. Derynck R. Werb Z. Nat. Genet. 1999; 22: 69-73Crossref PubMed Scopus (219) Google Scholar). were in in and in for at to for were not were with using a from to the by the In to matrix and were used to using a from In was as previously U. G. J.A. H. and in Scholar). were with for at and with in and in a at the were with to a of at in for and with and Analyses—The long were from and and and in and were by and of the was using the of were to and containing were two in at for then in at were then and in and of and marrow cells were from mice by a from a previously K. J. Cell Sci. 2000; PubMed Google Scholar). mice were by and were of The ends of the bone were and bone marrow cells were a cell by at of the bone using a The cell was a cell and by at at were then in containing fetal in a cell at a of and at in The the cells were and a Ficoll-Hypaque The cell was from the and with The cells were in containing fetal bovine and in a plate at of these growth factor and and or at and were The was of osteoclast formation was by of tartrate-resistant acid osteoclast cells as osteoclast were for activity using a acid from for or cells were with with in for and to the by the the osteoclasts in well were the with as cells containing at The results were expressed as of was also the to the by the The of the and of cells in of were as previously L. J. Cell Sci. 1995; Google Scholar). of EGFR in by from of osteoclast and from and embryonic were using to the by the and in of of the was by at EGFR expression was determined using reverse was to a containing reverse and of EGFR in a of The EGFR of the mouse EGFR and the reverse The was at for was for of for for for were analyzed a from cultured osteoclasts, and embryonic were as was a to and then by were for at and then with a EGFR at were at a of in and then to at The EGFR used was by at the of L. J. Res. 1999; PubMed Scopus Google Scholar). The contains of the rat EGFR Cell marrow cells were as and in plates at a of with with fetal bovine M-CSF, or of and were to the were then at in the of cells was by the of a was to of in well and the cells were at in for of in was then to well and the was determined by at were used for and were was marrow cells were and cultured in the of and in with fetal osteoclasts form, the was to to with with fetal and or was the were to remove in and were were with a and analyzed using were in and of were and the the of in the of the in the area was as the was using the Endochondral Ossification and Hypertrophic mice within the first because of P.J. Warburton D. Bu D. Zhao J.S. Berger J.E. Minoo P. Koivisto T. Allen L. Dobbs L. Werb Z. Derynck R. Dev. 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In or the primary area was and many long in the were a few in bone formation and the in bone were also in mice and We that primary ossification of the cartilage and the ossification of the growth plates of the long are impaired in the mice. in the of growth retardation in the mice and that for a few to are growth we not mice not a bone the EGFR and were used in the results that are to as Hypertrophic determined the in endochondral ossification. A in primary ossification of the hypertrophic cartilage anlage is the recruitment of are the and in the the bone and differentiate cells that are the of osteoclasts G. Wagner E.F. Dev. Cell. 2002; 2: 389-406Abstract Full Text Full Text PDF PubMed Scopus (1198) Google Scholar). the with blood and the of Pedersen B. K. T. Werb Z. J. Cell Biol. 2000; PubMed Scopus Google Scholar). 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We of these was of EGFR of from bone marrow cells cultured in the of and RANK ligand with or showed in the expression of and with not the of osteoclast formation by EGFR signaling not to be these the of osteoclast formation by was because of an cell proliferation, we for cell proliferation in the bone marrow cell bone marrow cells were and cultured for in the of and RANK ligand with or with in a in of decreased cell that of EGFR signaling by to decreased cell may in for the in osteoclast formation caused by by EGFR EGFR signaling osteoclast we osteoclast activity is in the of the EGFR tyrosine kinase from bone marrow cells were to form and to in the of or for of the of showed that were in the area and osteoclasts EGFR signaling not to bone In this study we have the function of EGFR signaling in endochondral bone formation by long bone development in mice. showed that in mice primary ossification of the calcified hypertrophic cartilage anlage was delayed and was with a in the recruitment of osteoclasts and of the primary was also abnormal to of growth plate and decreased bone The in osteoclast recruitment be because of inadequate formation of osteoclasts resulting from EGFR EGFR for Normal and Endochondral in endochondral ossification is the invasion of the calcified hypertrophic cartilage in the of the cartilage of calcified is accompanied by the recruitment of osteoclasts and We that EGFR deficiency in delayed recruitment of osteoclasts calcified primary ossification. However, this was consistent with the that the of EGFR signaling formation of osteoclasts from cells the bone a for osteoclasts to the of osteoclasts calcified may also be osteoclast recruitment the as mice at ossification of growth plate was not as by the zones of the growth plates may be because of the of caused by the in primary ossification, and the of the of osteoclasts to the and ossification in the be in the growth plate ossification may also be abnormal because of abnormal formation of growth plate or in its A recent study also growth plate in the mice (19Sibilia M. Wagner B. Hoebertz A. Elliott C. Marino S. Jochum W. Wagner E.F. Development. 2003; 130: 4515-4525Crossref PubMed Scopus (114) Google Scholar). The expression of EGFR in in growth plate chondrocyte proliferation, and that EGFR regulated hypertrophic chondrocyte the the removal and ossification of growth plate may also be at a resulting in of growth plate We expression of EGFR mature osteoclasts, suggesting that these cells directly to EGFR The function of EGFR in osteoclasts is not it may be to EGFR ligands have been to bone in and in in bone (20Raisz L.G. Simmons H.A. Sandberg A.L. Canalis E. Endocrinology. 1980; 107: 270-273Crossref PubMed Scopus (138) Google Scholar, R.A. Derynck R. J. 1985; PubMed Scopus Google Scholar, P.J. M. J. J. Google Scholar). are necessary for the recruitment of osteoclasts calcified hypertrophic cartilage primary endochondral ossification Pedersen B. K. T. Werb Z. J. Cell Biol. 2000; PubMed Scopus Google Scholar). The craniofacial and of mice may be because of in activity P.J. Chin J.R. Shum L. Slavkin H.C. Shuler C.F. Derynck R. Werb Z. Nat. Genet. 1999; 22: 69-73Crossref PubMed Scopus (219) Google Scholar, 16Kheradmand F. Rishi K. Werb Z. J. Cell Sci. 2002; 115: 839-848PubMed Google Scholar). We that and was to be the primary for the delayed recruitment of osteoclasts However, this not EGFR of ECM that are important in osteoclast In because with Pedersen B. K. T. Werb Z. J. Cell Biol. 2000; PubMed Scopus Google Scholar), the delayed recruitment of osteoclasts ECM and the in endothelial growth factor is important for of and osteoclast recruitment calcified hypertrophic cartilage of the Pedersen B. K. T. Werb Z. J. Cell Biol. 2000; PubMed Scopus Google Scholar, A.M. J. Werb Z. N. Nat. 1999; PubMed Scopus Google Scholar). We in expression in by in that the delayed osteoclast recruitment is not because of deficiency in However, because may the of VEGF, the in osteoclast recruitment may an in activity to decreased VEGF, which to in and recruitment of EGFR to bone is decreased in the may be because of a in the recruitment of osteoblasts primary ossification. However, to be osteoblasts were in the primary are at the cartilage bone that formation of osteoblasts in the may be that and/or function may be of the mice showed that osteoblast differentiation and proliferation were regulated M. R. I. C. L. C.F. R.A. G. L. J. Cell Biol. 2002; PubMed Scopus Google Scholar). (19Sibilia M. Wagner B. Hoebertz A. Elliott C. Marino S. Jochum W. Wagner E.F. Development. 2003; 130: 4515-4525Crossref PubMed Scopus (114) Google that primary osteoblast showed decreased proliferation and differentiation as by to form bone in (19Sibilia M. Wagner B. Hoebertz A. Elliott C. Marino S. Jochum W. Wagner E.F. Development. 2003; 130: 4515-4525Crossref PubMed Scopus (114) Google Scholar). the decreased bone in the mice was because of decreased osteoblast A of EGFR signaling osteoblasts is by that EGFR was expressed in osteoblasts in (17Martineau-Doize B. Lai W.H. Warshawsky H. Bergeron J.J. Endocrinology. 1988; 123: 841-858Crossref PubMed Scopus (58) Google Scholar, C. Thesleff I. A. Res. 1995; PubMed Scopus Google Scholar), and that EGF osteoblast proliferation in M. PubMed Scopus Google Scholar, J. L. G. N. R. 1995; 16: PubMed Scopus Google Scholar). EGFR for the of have two essential for and Nat. Rev. Genet. 2003; PubMed Scopus Google Scholar). is necessary for the generation of the cell and for differentiation We that of osteoclast formation from bone marrow cells in the of and was in the of the EGFR tyrosine kinase is consistent with suggesting a role for EGFR ligands in osteoclast formation. of transforming growth or EGF the formation of osteoclasts from cultured bone marrow cells N. B.R. J. Derynck R. J. PubMed Scopus Google Scholar). In bone marrow cell of EGF or transforming growth osteoclast formation. may be because were of EGFR ligands in these or that the EGFR was by signaling A. Zwick E. Prenzel N. M. Ullrich A. 2001; PubMed Scopus Google Scholar). The role of EGFR signaling in osteoclast formation may be a osteoclast to proliferation or EGFR ligands may act bone marrow cells to of or that in act in a osteoclast to growth and are to these two