EGF-like Ligands Stimulate Osteoclastogenesis by Regulating Expression of Osteoclast Regulatory Factors by Osteoblasts

Abstract

Epidermal growth factor (EGF)-like ligands and their receptors constitute one of the most important signaling networks functioning in normal tissue development and cancer biology. Recent in vivo mouse models suggest this signaling network plays an essential role in bone metabolism. Using a coculture system containing bone marrow macrophage and osteoblastic cells, here we report that EGF-like ligands stimulate osteoclastogenesis by acting on osteoblastic cells. This stimulation is not a direct effect because osteoclasts do not express functional EGF receptors (EGFRs). Further studies reveal that EGF-like ligands strongly regulate the expression of two secreted osteoclast regulatory factors in osteoblasts by decreasing osteoprotegerin (OPG) expression and increasing monocyte chemoattractant protein 1 (MCP1) expression in an EGFR-dependent manner and consequently stimulate TRAP-positive osteoclast formation. Addition of exogenous OPG completely inhibited osteoclast formation stimulated by EGF-like ligands, while addition of a neutralizing antibody against MCP-1 exhibited partial inhibition. Coculture with bone metastatic breast cancer MDA-MB-231 cells had similar effects on the expression of OPG and MCP1 in the osteoblastic cells, and those effects could be partially abolished by the EGFR inhibitor PD153035. Because a high percentage of human carcinomas express EGF-like ligands, our findings suggest a novel mechanism for osteolytic lesions caused by cancer cells metastasizing to bone. Epidermal growth factor (EGF)-like ligands and their receptors constitute one of the most important signaling networks functioning in normal tissue development and cancer biology. Recent in vivo mouse models suggest this signaling network plays an essential role in bone metabolism. Using a coculture system containing bone marrow macrophage and osteoblastic cells, here we report that EGF-like ligands stimulate osteoclastogenesis by acting on osteoblastic cells. This stimulation is not a direct effect because osteoclasts do not express functional EGF receptors (EGFRs). Further studies reveal that EGF-like ligands strongly regulate the expression of two secreted osteoclast regulatory factors in osteoblasts by decreasing osteoprotegerin (OPG) expression and increasing monocyte chemoattractant protein 1 (MCP1) expression in an EGFR-dependent manner and consequently stimulate TRAP-positive osteoclast formation. Addition of exogenous OPG completely inhibited osteoclast formation stimulated by EGF-like ligands, while addition of a neutralizing antibody against MCP-1 exhibited partial inhibition. Coculture with bone metastatic breast cancer MDA-MB-231 cells had similar effects on the expression of OPG and MCP1 in the osteoblastic cells, and those effects could be partially abolished by the EGFR inhibitor PD153035. Because a high percentage of human carcinomas express EGF-like ligands, our findings suggest a novel mechanism for osteolytic lesions caused by cancer cells metastasizing to bone. The adult human skeleton continuously undergoes remodeling, namely, being resorbed by osteoclasts and renewed by osteoblasts. The maintenance of the skeleton requires the coordinated activity and constant generation of these cells. Osteoblasts are derived from mesenchymal stromal stem cells and, along with their precursors, are the major sources for regulating osteoclast formation by producing several cytokines, including receptor activator of NF-κB ligand (RANKL), 2The abbreviations used are:RANKLreceptor activator of nuclear factor-κB ligandEGFepidermalgrowthfactorHB-EGFheparinbinding-EGFEGFREGF receptorOPGosteoprotegerinM-CSFmacrophage colony-stimulating factorMCP1monocyte chemoattractant protein 1PI 3-kinasephosphoinositide 3-kinasePMAphorbol 12-myristate 13-acetateELISAenzyme-linked immunosorbent assayBMMbone marrow macrophagesTGFtransforming growth factorTRAPtartrate-resistant acid phosphatase 2The abbreviations used are:RANKLreceptor activator of nuclear factor-κB ligandEGFepidermalgrowthfactorHB-EGFheparinbinding-EGFEGFREGF receptorOPGosteoprotegerinM-CSFmacrophage colony-stimulating factorMCP1monocyte chemoattractant protein 1PI 3-kinasephosphoinositide 3-kinasePMAphorbol 12-myristate 13-acetateELISAenzyme-linked immunosorbent assayBMMbone marrow macrophagesTGFtransforming growth factorTRAPtartrate-resistant acid phosphatase osteoprotegerin (OPG), and macrophage colony-stimulating factor (M-CSF). RANKL is the key mediator for osteoclast formation and activation (1Kong Y.Y. Yoshida H. Sarosi I. Tan H.L. Timms E. Capparelli C. Morony S. Oliveira-dos-Santos A.J. Van G. Itie A. Khoo W. Wake-ham A. Dunstan C.R. Lacey D.L. Mak T.W. Boyle W.J. Penninger J.M. Nature. 1999; 397: 315-323Crossref PubMed Scopus (2799) Google Scholar, 2Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3500) Google Scholar). OPG acts as a soluble decoy receptor by blocking the interaction of RANKL with its receptor (RANK) on osteoclasts, thereby inhibiting osteoclastogenesis (2Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. 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Chem., in press. receptor activator of nuclear factor-κB ligand epidermalgrowthfactor heparinbinding-EGF EGF receptor osteoprotegerin macrophage colony-stimulating factor monocyte chemoattractant protein 1 phosphoinositide 3-kinase phorbol 12-myristate 13-acetate enzyme-linked immunosorbent assay bone marrow macrophages transforming growth factor tartrate-resistant acid phosphatase receptor activator of nuclear factor-κB ligand epidermalgrowthfactor heparinbinding-EGF EGF receptor osteoprotegerin macrophage colony-stimulating factor monocyte chemoattractant protein 1 phosphoinositide 3-kinase phorbol 12-myristate 13-acetate enzyme-linked immunosorbent assay bone marrow macrophages transforming growth factor tartrate-resistant acid phosphatase The epidermal growth factor (EGF)-like ligands and their cognate receptors constitute one of the best-studied signaling networks. This network modulates cell functions in a variety of ways, including proliferation, survival, adhesion, migration, and differentiation. There are four distinct receptors in this family: EGF receptor (EGFR, also known as ErbB-1/HER1), ErbB-2 (HER2), ErbB-3 (HER3), and ErbB-4 (HER4). The EGF-like ligands consist of EGF, amphiregulin, and transforming growth factor α (TGFα), which only bind to the EGFR, and heparin-binding EGF (HB-EGF), betacellulin, and epiregulin, which can bind to both the EGFR and ErbB4. The EGFR is a receptor-tyrosine kinase. Upon ligand binding, it undergoes dimerization and phosphorylation at tyrosine residues in its intracellular domain, thus activating several important cellular signal transduction pathways, such as Ras-Raf-MAP-kinase, PI3-kinase-Akt, and PLCγ-PKC pathways etc (reviewed in Ref. 8Yarden Y. Eur. J. Cancer. 2001; 37: S3-S8Abstract Full Text Full Text PDF PubMed Google Scholar). Epidermal growth factor receptor signaling contributes to bone metabolism by affecting both osteoblasts and osteoclasts. Although most EGFR-null mice die within the first postnatal week, surviving animals display craniofacial alterations and cleft palate (9Miettinen 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 (218) Google Scholar). Overexpression of EGF-like ligands in mouse, such as betacellulin, EGF, and TGFα, all resulted in stunted growth (10Sandgren E.P. Luetteke N.C. Qiu T.H. Palmiter R.D. Brinster R.L. Lee D.C. Mol. Cell. Biol. 1993; 13: 320-330Crossref PubMed Scopus (128) Google Scholar, 11Schneider M.R. Dahlhoff M. Herbach N. Renner-Mueller I. Dalke C. Puk O. Graw J. Wanke R. Wolf E. Endocrinology. 2005; 146: 5237-5246Crossref PubMed Scopus (49) Google Scholar, 12Chan S.Y. Wong R.W. J. Biol. Chem. 2000; 275: 38693-38698Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Histology studies revealed there is abnormal overproliferation of osteoblasts in EGF transgenic mice (12Chan S.Y. Wong R.W. J. Biol. Chem. 2000; 275: 38693-38698Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). On the other hand, mice humanized for the EGFR (the endogenous mouse EGFR gene was replaced by human EGFR cDNA) exhibit low EGFR activity in bone and display accelerated osteoblast differentiation (13Sibilia M. Wagner B. Hoebertz A. Elliott C. Marino S. Jochum W. Wagner E.F. Development. 2003; 130: 4515-4525Crossref PubMed Scopus (112) Google Scholar). have shown that mice bone mice L. J. L. Lee D.C. E. N.C. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). with these in vivo in cell suggest that EGF and stimulate of cells their differentiation osteoblastic cells L. J. L. Lee D.C. E. N.C. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar, R. H. Y. S. M. M. N. M. Endocrinology. PubMed Scopus Google Scholar, M. M. K. T. H. T. K. PubMed Scopus Google Scholar, N.C. M. PubMed Scopus Google Scholar). these effects on EGF and have the ability to strongly stimulate bone in mouse and human marrow J. S. J. PubMed Scopus Google Scholar, N. J. Derynck R. J. PubMed Scopus Google Scholar, J. M. S. Derynck R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google these growth factors in regulating osteoclastogenesis and bone of EGFR-null mice revealed that EGFR of the and osteoclast and osteoblast Further studies have that osteoclastic express the EGFR K. H. Chin J.R. Werb Z. T.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the mechanism of EGF-like ligands stimulate bone is is the for cancer cells, such as and The in osteolytic osteoblastic all in osteolytic the osteoclast formation and activity are stimulated by The is that protein secreted by cells, plays a major role in this by acting on osteoblasts in bone to the expression of RANKL (reviewed in Ref. W. J. Biol. 2005; PubMed Scopus Google Scholar). The EGFR plays in the of human studies suggest that the of express at in several EGF-like The of EGF-like ligands and their receptors on cells are essential for growth and (reviewed in Ref. N. C. L. M. M.R. A. 2005; PubMed Scopus Google Scholar). the of EGF-like ligands to bone by osteoclasts. we report that EGF-like ligands strongly stimulate osteoclast formation in the coculture of osteoblastic cells and bone marrow macrophages the precursors for osteoclasts, by regulating the expression of OPG and MCP-1 in osteoblastic cells. Because coculture with bone metastatic breast cancer MDA-MB-231 cells had similar effects on the expression of OPG and MCP1 in the osteoblastic cells, and those effects could be partially abolished by EGFR we that EGF-like ligands, similar to secreted by cells, to osteolytic lesions in bone human EGF, TGFα, amphiregulin, from was from secreted RANKL human and neutralizing antibody against mouse MCP-1 from and phorbol 12-myristate 13-acetate from against RANKL EGFR, and used for cells D. K. K. G. J. 1999; PubMed Scopus Google Scholar, G. Y. P. G. Mol. 1997; PubMed Scopus Google in growth with and cells in growth osteoblast differentiation with at a of osteoclastic bone marrow cells from and of a mouse, in coculture with and in a and at in The the cells and at a of cells are as the osteoclast precursors, and in the of and M-CSF for with a at to osteoclasts. the of M-CSF not these cells cells, which osteoclastic cells addition of bone marrow osteoblastic bone marrow cells from and of mice and at a of in growth to differentiation on and coculture of cells and osteoclasts, cells on in coculture On on of cells with addition of to osteoclast formation. and cells for to TRAP-positive osteoclastic cells on coculture of osteoblastic cells and osteoclasts, osteoblastic cells as On on of osteoblastic cells along with was and with one on cancer cell MDA-MB-231 was from and in coculture coculture and cells, and MDA-MB-231 cells at the of in coculture at in the in the of was on with a at The was by and osteoclast for activity a acid phosphatase from from cell was was used to was on a The used for the are in the was used as an for mouse the coculture of and MDA-MB-231 cells in the mouse was used as an because its only the mouse gene and not the human and expression was not by EGF-like ligands not EGF and in and with mouse with EGF in containing 1 for at with cells in and in a cells in as and of to the and with at a of in coculture and M-CSF to coculture and from cells for for of OPG and protein in the mouse OPG and human from are expressed as of with all being at the EGF-like in of and the ability of osteoblasts to osteoclast we mouse with cells. is a cell which undergoes osteoblastic differentiation in the of there TRAP-positive osteoclastic cells in the coculture system in the of exogenous RANKL addition of EGF-like ligands, such as EGF, TGFα, and amphiregulin, strongly stimulated formation of TRAP-positive osteoclastic cells in this coculture system four that there and in expression in the EGF and this we this coculture mouse bone marrow osteoblastic cells of cells. our it for the bone marrow osteoblastic cells to bone as shown by and to express osteoblastic such as bone and phosphatase not this the osteoblastic cells with from and the at and that there TRAP-positive osteoclastic cells in the coculture system in the coculture EGF-like ligands exhibited similar effects not The indicate a in osteoclastogenesis in the coculture system EGF-like ligands are that exogenous RANKL is for this because both cells and osteoblastic cells not express low of endogenous RANKL and those are not to osteoclast formation. EGF-like on effects of EGF-like ligands on the osteoblast and osteoclast coculture could be caused by two EGF-like ligands have direct effects on osteoclastic cells, these have effects on osteoclastic cells by acting osteoblastic cells. the first we osteoclastic cells express functional EGF report that osteoclastic cells express EGFR K. H. Chin J.R. Werb Z. T.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). with bone marrow osteoclastic cells cells, a cell also the of EGFR at low not EGFR protein be by we used ligand to the of functional EGFR in osteoclasts. cells with we in cells, cells by with osteoclastic cells by with both M-CSF and the of EGF not the with the cells, the with these cells is most to cells, an osteoblastic cell used as a strongly to cells, an osteoblastic cell and this was by that osteoclasts functional EGF we ligand with cells on cells to for we of binding, in the cells, osteoclastic cells the that both and osteoclastic cells exhibit low of EGF namely, that EGF-like ligand had effects on osteoclast formation gene such as and it that EGF-like ligands have direct effects on osteoclasts. EGFR was in cells at both and protein and cells also strongly and this was because it was completely inhibited by addition of EGF while and that EGFR expression in cells, suggest that cells bind of as the cells that this not indicate that and cells express a similar of EGFR cell because are to receptor it is to cell of osteoblasts to in the for osteoclasts do not bind while cells is that osteoclasts express EGF-like ligands and have their EGFR both osteoclasts and cells express low and of EGF-like ligands these that cells, not osteoclasts, express functional EGFR and EGF-like ligands have effects on osteoclastic cells by acting osteoblastic cells. The of functional EGFR in osteoblasts in osteoblastic cell such as osteoblastic cell N.C. M. PubMed Scopus Google and in vivo osteoblasts in and C. I. A. PubMed Scopus Google Scholar). EGF-like the of in EGF-like ligands regulate osteoclast formation from cells of differentiation and used to mouse for with one on shown in and multinucleated osteoclasts in and had osteoclasts in and of had assay that osteoclasts with resorbed on that in and that exogenous RANKL was in this because cells do not express RANKL and most RANKL in a This that EGF expression of secreted in cells, and those factors have the ability to regulate EGF-like OPG and MCP1 on RANKL in secreted factors are known to osteoclast such as and we EGF expression of those in cells. Using we the of OPG cell differentiation in the in the of EGF Addition of acid strongly osteoblastic differentiation of cells and bone are in the we cells expressed OPG and EGF completely abolished this shown in cells in growth expressed similar of OPG the of cells in differentiation expressed of OPG at at at cells at in growth and these in OPG completely abolished by addition of by OPG protein in the by cells in growth only secreted OPG in the osteoblastic cells secreted high of OPG at and EGF strongly inhibited OPG to and at and from EGF, other EGF-like ligands, TGFα, and amphiregulin, also exhibited effects on OPG Because OPG is a decoy receptor for a in OPG expression by EGF-like ligands to an in osteoclast formation. also RANKL expression in cells. RANKL was expressed at low (the to is and acid its expression not reveal RANKL protein in cell of cells with while the RANKL protein the that EGF-like ligands mainly regulate not expression in these osteoblastic cells. This is also with our that EGF osteoclastogenesis by regulating secreted factors from osteoblastic cells while RANKL is a addition to we also the of and in cells EGF that both MCP1 and M-CSF expression by EGF MCP1 expression was the and M-CSF expression was not EGF not and M-CSF are for EGF-like ligands in osteoblastic cells, we their expression in mouse bone marrow osteoblastic of osteoblastic with EGF-like ligands inhibited OPG expression both at the and protein EGF-like ligands also MCP1 expression in osteoblastic cells we that these ligands had effect on M-CSF expression not that of M-CSF by EGF not be a EGF-like OPG and MCP1 in an EGFR-dependent receptors can to the EGFR is the receptor in the of OPG and MCP1 expression by EGF-like ligands, we tyrosine inhibitor in the of cells. shown in completely abolished the effects of on OPG and MCP1 a that and not EGFR Y. Eur. J. Cancer. 2001; 37: S3-S8Abstract Full Text Full Text PDF PubMed Google had effect on OPG and MCP1 expression in cells. suggest that of OPG and MCP1 requires EGFR tyrosine of by EGF-like OPG and MCP1 of OPG and MCP1 expression in osteoblasts by EGF-like ligands in stimulation of osteoclastogenesis in the coculture we the OPG by exogenous OPG MCP1 by neutralizing antibody to the of and cells. to our EGF stimulated osteoclast formation in the coculture while there TRAP-positive cells in the of EGF Addition of exogenous OPG completely EGF in TRAP-positive cells the of OPG used in this is which is the OPG in cells of differentiation addition of neutralizing antibody in the coculture not completely the of TRAP-positive cells in the coculture with the of the it that EGF stimulation of osteoclast formation in the coculture system is mainly inhibiting OPG and partially EGFR to of OPG and MCP1 in by MDA-MB-231 ligands are expressed in most human Because osteolytic bone strongly stimulate osteoclast we that EGF-like ligands expressed by metastasizing cells to this by acting on osteoblasts and regulating their expression of OPG and MCP1 to stimulate osteoclast formation. this we the effects of MDA-MB-231 cells on the osteoclastogenesis stimulated by osteoblasts. MDA-MB-231 cells because these breast cancer cells have bone metastatic osteolytic revealed that these cells express high of TGFα, and low of EGF and also used to in the from MDA-MB-231 cells of caused of the and its to the of in a was used for A. J. J. Biol. Chem. Full Text PDF PubMed Google and had effect on expression not we and in the shown in similar to TGFα, MDA-MB-231 cells stimulate TRAP-positive osteoclast formation. the we MDA-MB-231 cells regulate OPG and MCP1 expression in cells and this is the EGFR this we MDA-MB-231 cells with cells for Because MDA-MB-231 cells derived from human cancer tissue and cells derived from mouse we that mouse OPG and MCP1 not human OPG and revealed that the of MDA-MB-231 cells strongly inhibited mouse OPG expression and mouse MCP1 expression by cells a inhibitor of the EGFR tyrosine was to the coculture to this is EGFR-dependent This partially of OPG expression in OPG in the of in the of The stimulation of MCP1 expression was also by addition of in MCP1 in the of in the of effect on EGF-like ligand in MDA-MB-231 OPG and MCP1 expression in cells not the of EGFR signaling in of osteoclast regulatory factors in osteoblasts by breast cancer cells and also indicate that other signaling pathways are in by from MDA-MB-231 from MDA-MB-231 and addition of 1 from MDA-MB-231 that was with for 1 1 from MDA-MB-231 that was with for 1 from MDA-MB-231 that was with for 1 from MDA-MB-231 from MDA-MB-231 and addition of from MDA-MB-231 that was with for 1 from MDA-MB-231 that was with for 1 from MDA-MB-231 that was with for 1 in a this we that EGF-like ligands have the ability to strongly stimulate osteoclastogenesis by regulating OPG and MCP1 expression in osteoblastic cells in an EGFR-dependent also that cancer cells that can to bone this EGFR signaling to osteoclast formation. these suggest a novel mechanism for osteolytic lesions caused by cancer bone Because of a of EGFR as we that this is of EGF and have as bone mouse EGF is on and is inhibited by J. M. S. Derynck R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R.D. Derynck R. J. PubMed Scopus Google Scholar). in bone EGF is not inhibited by R.D. Derynck R. J. PubMed Scopus Google Scholar, E. Endocrinology. PubMed Scopus Google Scholar). EGF shown to endogenous in the cell of the used in this K. M. T. S. N. T. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The is the of osteoclastogenesis in bone. other bone and cytokines, such as and osteoclastogenesis stimulation of RANKL and of OPG expression in cells E. 2000; PubMed Scopus Google Scholar). that EGF-like ligands OPG have effect on RANKL expression in osteoblastic cells and in stimulation of osteoclast formation. is that EGF OPG the our also that MCP1 partially the effect of the EGF-like MCP1 was as an important factor acting on to stimulate osteoclast formation and activity in the of RANKL (5Kim M.S. Day C.J. Morrison N.A. J. Biol. 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