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• W2158698452 abstract "Communication between bone-depositing osteoblasts and bone-resorbing osteoclasts is required for bone development and homeostasis. Here, we identify EBF2, a member of the early B cell factor (EBF) family of transcription factors that is expressed in osteoblast progenitors, as a regulator of osteoclast differentiation. We find that mice homozygous for a targeted inactivation of Ebf2 show reduced bone mass and an increase in the number of osteoclasts. These defects are accompanied by a marked downregulation of the osteoprotegerin (Opg) gene, encoding a RANK decoy receptor. EBF2 binds to sequences in the Opg promoter and transactivates the Opg promoter in synergy with the Wnt-responsive LEF1/TCF:β-catenin pathway. Taken together, these data identify EBF2 as a regulator of RANK-RANKL signaling and osteoblast-dependent differentiation of osteoclasts. Communication between bone-depositing osteoblasts and bone-resorbing osteoclasts is required for bone development and homeostasis. Here, we identify EBF2, a member of the early B cell factor (EBF) family of transcription factors that is expressed in osteoblast progenitors, as a regulator of osteoclast differentiation. We find that mice homozygous for a targeted inactivation of Ebf2 show reduced bone mass and an increase in the number of osteoclasts. These defects are accompanied by a marked downregulation of the osteoprotegerin (Opg) gene, encoding a RANK decoy receptor. EBF2 binds to sequences in the Opg promoter and transactivates the Opg promoter in synergy with the Wnt-responsive LEF1/TCF:β-catenin pathway. Taken together, these data identify EBF2 as a regulator of RANK-RANKL signaling and osteoblast-dependent differentiation of osteoclasts. The bones of vertebrates are formed and maintained by the interplay of different cell types. The bone-forming osteoblasts, which share a mesenchymal precursor with chondrocytes, secrete the bone extracellular matrix (ECM), whereas osteoclasts, which differentiate from hematopoietic progenitors of the myelomonocytic cell lineage, degrade the bone matrix (Karsenty and Wagner, 2002Karsenty G. Wagner E.F. Reaching a genetic and molecular understanding of skeletal development.Dev. Cell. 2002; 4: 389-406Abstract Full Text Full Text PDF Scopus (1196) Google Scholar, Teitelbaum and Ross, 2003Teitelbaum S.L. Ross F.P. Genetic regulation of osteoclast development and function.Nat. Rev. Genet. 2003; 4: 638-649Crossref PubMed Scopus (1306) Google Scholar, Baron, 2004Baron R. Arming the osteoclast.Nat. Med. 2004; 10: 458-460Crossref Scopus (44) Google Scholar). During development, bone formation is initiated by the condensation of mesenchymal cells that differentiate along the chondrocytic pathway to form cartilage, which is subsequently replaced by bone, generated by osteoblasts in a process termed endochondral ossification. Alternatively, condensed mesenchymal cells can differentiate directly into osteoblasts to form intramembranous skeletal elements (de Crombrugghe et al., 2001de Crombrugghe B. Lefebvre V. Nakashima K. Regulatory mechanisms in the pathways of cartilage and bone formation.Curr. Opin. Cell Biol. 2001; 13: 721-727Crossref PubMed Scopus (403) Google Scholar, Olsen et al., 2000Olsen B.R. Reginato A.M. Wang W. Bone development.Annu. Rev. Cell Dev. Biol. 2000; 16: 191-220Crossref PubMed Scopus (772) Google Scholar). The formation and maintenance of bones involves a fine balance between osteoblasts and osteoclasts, whereby osteoblasts regulate the differentiation of osteoclast precursors into terminally differentiated cells (Takahashi et al., 1988Takahashi N. Akatsu T. Udagawa N. Sasaki T. Yamaguchi A. Moseley J.M. Martin T.J. Suda T. Osteoblastic cells are involved in osteoclast formation.Endocrinology. 1988; 123: 2600-2602Crossref PubMed Scopus (851) Google Scholar). Changes in the numbers and/or activities of osteoblasts and osteoclasts can result in an increase of bone mass, due to a failure of bone resorption (osteopetrosis) or increased bone production (osteosclerosis), or, alternatively, in a loss of bone mass, termed osteopenia or osteoporosis (Teitelbaum, 2000Teitelbaum S.L. Bone resorption by osteoclasts.Science. 2000; 289: 1504-1508Crossref PubMed Scopus (3075) Google Scholar). The communication between osteoblasts and osteoclasts is governed by at least two signaling pathways. The macrophage colony-stimulating factor (M-CSF) is secreted from osteoblasts and provides a survival signal to osteoclast precursors and osteoclasts (Lagasse and Weissman, 1997Lagasse E. Weissman I.L. Enforced expression of Bcl-2 in monocytes rescues macrophages and partially reverses osteopetrosis in op/op mice.Cell. 1997; 89: 1021-1031Abstract Full Text Full Text PDF Scopus (262) Google Scholar, Tsurukai et al., 2000Tsurukai T. Udagawa N. Matsuzaki K. Takahashi N. Suda T. Roles of macrophage-colony stimulating factor and osteoclast differentiation factor in osteoclastogenesis.J. Bone Miner. Metab. 2000; 18: 177-184Crossref Scopus (93) Google Scholar, Yoshida et al., 1990Yoshida H. Hayashi S. Kunisada T. Ogawa M. Nishikawa S. Okamura H. Sudo T. Shultz L.D. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene.Nature. 1990; 345: 442-444Crossref PubMed Scopus (1523) Google Scholar). In addition, the tumor necrosis factor-related receptor activator of NF-κB ligand (RANKL), which is displayed on the cell surface of preosteoblasts and osteoblasts, regulates the differentiation of preosteoclasts by the interaction with the receptor activator of NF-κB (RANK) (Dougall et al., 1999Dougall W.C. Glaccum M. Charrier K. Rohrbach K. Brasel K. De Smedt T. Daro E. Smith J. Tometsko M.E. Maliszewski C.R. et al.RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1202) Google Scholar, Kong et al., 1999Kong Y.Y. Yoshida H. Sarosi I. Tan H.L. Timms E. Capparelli C. Morony S. Oliveira-dos-Santos A.J. Van G. Itie A. et al.OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.Nature. 1999; 397: 315-323Crossref PubMed Scopus (2863) Google Scholar, Lacey et al., 1998Lacey D.L. Timms E. Tan H.L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. et al.Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4623) Google Scholar, Yasuda et al., 1998Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. et al.Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL.Proc. Natl. Acad. Sci. USA. 1998; 95: 3597-3602Crossref PubMed Scopus (3569) Google Scholar). Signaling by RANKL can be modulated by the decoy receptor Osteoprotegerin (OPG), which binds RANKL and is secreted from osteoblasts and several other cell types (Simonet et al., 1997Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.S. Luthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. et al.Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4340) Google Scholar). The role of OPG in regulating osteoclast differentiation and bone homeostasis was shown by targeted gene inactivation of the Opg gene, which results in enhanced osteoclast formation and osteoporosis (Bucay et al., 1998Bucay N. Sarosi I. Dunstan C.R. Morony S. Tarpley J. Capparelli C. Scully S. Tan H.L. Xu W. Lacey D.L. et al.Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification.Genes Dev. 1998; 12: 1260-1268Crossref PubMed Scopus (2128) Google Scholar), and by the transgenic overexpression of Opg, which results in osteopetrosis (Simonet et al., 1997Simonet W.S. Lacey D.L. Dunstan C.R. Kelley M. Chang M.S. Luthy R. Nguyen H.Q. Wooden S. Bennett L. Boone T. et al.Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4340) Google Scholar). Several transcription factors have been shown to regulate the differentiation and function of osteoblasts and osteoclasts. Differentiation of osteoblasts, which can be monitored by the expression of markers such as alkaline phosphatase, bone sialoprotein, and osteocalcin, is regulated by the transcription factors Runx2/Cbfa1 and Osterix. Runx2 is expressed at the onset of osteoblast differentiation (Ducy et al., 1997Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation.Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3638) Google Scholar), and targeted disruption of this gene results in a complete absence of osteoblasts (Komori et al., 1997Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. et al.Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts.Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3643) Google Scholar, Otto et al., 1997Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. et al.Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2410) Google Scholar). Likewise, the genetic inactivation of Osterix leads to an arrest of osteoblast differentiation (Nakashima et al., 2002Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation.Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2789) Google Scholar). In addition, the transcription factors ATF4 and TCF1 regulate the function of osteoblasts, which includes the differentiation of osteoclasts (Glass et al., 2005Glass 2nd, D.A. Bialek P. Ahn J.D. Starbuck M. Patel M.S. Clevers H. Taketo M.M. Long F. McMahon A.P. Lang R.A. Karsenty G. Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation.Dev. Cell. 2005; 8: 751-764Abstract Full Text Full Text PDF PubMed Scopus (1266) Google Scholar). This process is also regulated by transcription factors that act in the monocyte/myeloid lineage, such as Pu.1 (Tondravi et al., 1997Tondravi M.M. McKercher S.R. Anderson K. Erdmann J.M. Quiroz M. Maki R. Teitelbaum S.L. Osteopetrosis in mice lacking haematopoietic transcription factor PU.1.Nature. 1997; 386: 81-84Crossref Scopus (452) Google Scholar), NFATc, NF-κB, and the AP1 proteins c-fos and c-jun, which function downstream of RANKL signaling (Kenner et al., 2004Kenner L. Hoebertz A. Beil T. Keon N. Karreth F. Eferl R. Scheuch H. Szremska A. Amling M. Schorpp-Kistner M. et al.Mice lacking JunB are osteopenic due to cell-autonomous osteoblast and osteoclast defects.J. Cell Biol. 2004; 164: 613-623Crossref Scopus (172) Google Scholar, Takayanagi et al., 2002aTakayanagi H. Kim S. Koga T. Nishina H. Isshiki M. Yoshida H. Saiura A. Isobe M. Yokochi T. Inoue J. et al.Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts.Dev. Cell. 2002; 3: 889-901Abstract Full Text Full Text PDF PubMed Scopus (1986) Google Scholar, Takayanagi et al., 2002bTakayanagi H. Kim S. Matsuo K. Suzuki H. Suzuki T. Sato K. Yokochi T. Oda H. Nakamura K. Ida N. et al.RANKL maintains bone homeostasis through c-Fos-dependent induction of interferon-β.Nature. 2002; 416: 744-749Crossref PubMed Scopus (592) Google Scholar). Although the role of the RANK-RANKL signaling pathway in the osteoblast-mediated differentiation of osteoclasts has been well established, our insight into the transcriptional control mechanisms that operate upstream of this signaling pathway is limited. Transcription factors of the “early B cell factor” (EBF) family bind to DNA as homo- or heterodimers through a zinc finger domain and a helix-loop-helix-like dimerization domain (Dubois and Vincent, 2001Dubois L. Vincent A. The COE—Collier/Olf1/EBF—transcription factors: structural conservation and diversity of developmental functions.Mech. Dev. 2001; 108: 3-12Crossref Scopus (117) Google Scholar). The founding member of this family, EBF1, is expressed in B lymphocytes, adipocytes, and neuronal cells, and targeted inactivation of the Ebf1 gene results in a block of early B cell differentiation (Hagman et al., 1993Hagman J. Belanger C. Travis A. Turck C.W. Grosschedl R. Cloning and functional characterization of early B-cell factor, a regulator of lymphocyte-specific gene expression.Genes Dev. 1993; 7: 760-773Crossref Scopus (234) Google Scholar, Lin and Grosschedl, 1995Lin H. Grosschedl R. Failure of B-cell differentiation in mice lacking the transcription factor EBF.Nature. 1995; 376: 263-267Crossref PubMed Scopus (542) Google Scholar). Other family members, including EBF2 (O/E3) and EBF3 (O/E2), are expressed in adipocytes, neurons, and several other cell types (Garel et al., 1997Garel S. Marin F. Mattei M.G. Vesque C. Vincent A. Charnay P. Family of Ebf/Olf-1-related genes potentially involved in neuronal differentiation and regional specification in the central nervous system.Dev. Dyn. 1997; 210: 191-205Crossref PubMed Scopus (149) Google Scholar, Malgaretti et al., 1997Malgaretti N. Pozzoli O. Bosetti A. Corradi A. Ciarmatori S. Panigada M. Bianchi M.E. Martinez S. Consalez G.G. Mmot1, a new helix-loop-helix transcription factor gene displaying a sharp expression boundary in the embryonic mouse brain.J. Biol. Chem. 1997; 272: 17632-17639Crossref Scopus (53) Google Scholar, Mella et al., 2004Mella S. Soula C. Morello D. Crozatier M. Vincent A. Expression patterns of the coe/ebf transcription factor genes during chicken and mouse limb development.Gene Expr. Patterns. 2004; 4: 537-542Crossref Scopus (23) Google Scholar, Wang et al., 2004Wang S.S. Lewcock J.W. Feinstein P. Mombaerts P. Reed R.R. Genetic disruptions of O/E2 and O/E3 genes reveal involvement in olfactory receptor neuron projection.Development. 2004; 131: 1377-1388Crossref PubMed Scopus (105) Google Scholar). EBF proteins can act in a redundant manner, but some functions have been attributed to specific members of the family (Corradi et al., 2003Corradi A. Croci L. Broccoli V. Zecchini S. Previtali S. Wurst W. Amadio S. Maggi R. Quattrini A. Consalez G.G. Hypogonadotropic hypogonadism and peripheral neuropathy in Ebf2-null mice.Development. 2003; 130: 401-410Crossref PubMed Scopus (83) Google Scholar, Wang et al., 2004Wang S.S. Lewcock J.W. Feinstein P. Mombaerts P. Reed R.R. Genetic disruptions of O/E2 and O/E3 genes reveal involvement in olfactory receptor neuron projection.Development. 2004; 131: 1377-1388Crossref PubMed Scopus (105) Google Scholar). Ebf2 is expressed in the embryonic central nervous system (Malgaretti et al., 1997Malgaretti N. Pozzoli O. Bosetti A. Corradi A. Ciarmatori S. Panigada M. Bianchi M.E. Martinez S. Consalez G.G. Mmot1, a new helix-loop-helix transcription factor gene displaying a sharp expression boundary in the embryonic mouse brain.J. Biol. Chem. 1997; 272: 17632-17639Crossref Scopus (53) Google Scholar), and targeted gene inactivation of Ebf2 has revealed an important function in peripheral nerve morphogenesis, the migration of neurons that produce gonadotropin-releasing hormone, and projections of olfactory neurons (Corradi et al., 2003Corradi A. Croci L. Broccoli V. Zecchini S. Previtali S. Wurst W. Amadio S. Maggi R. Quattrini A. Consalez G.G. Hypogonadotropic hypogonadism and peripheral neuropathy in Ebf2-null mice.Development. 2003; 130: 401-410Crossref PubMed Scopus (83) Google Scholar, Wang et al., 2004Wang S.S. Lewcock J.W. Feinstein P. Mombaerts P. Reed R.R. Genetic disruptions of O/E2 and O/E3 genes reveal involvement in olfactory receptor neuron projection.Development. 2004; 131: 1377-1388Crossref PubMed Scopus (105) Google Scholar). However, the role of EBF2 in other developmental processes has not been studied. In this study, we show that EBF2 is also expressed in osteoblastic progenitors and regulates osteoclast differentiation by activating the gene encoding the RANK decoy receptor OPG. With the aim of examining the developmental expression pattern of EBF2 in more detail, we analyzed mice that carry an in-frame insertion of the bacterial LacZ gene immediately downstream of the translation initiation site of the Ebf2 gene (Corradi et al., 2003Corradi A. Croci L. Broccoli V. Zecchini S. Previtali S. Wurst W. Amadio S. Maggi R. Quattrini A. Consalez G.G. Hypogonadotropic hypogonadism and peripheral neuropathy in Ebf2-null mice.Development. 2003; 130: 401-410Crossref PubMed Scopus (83) Google Scholar). Whole-mount staining for β-galactosidase activity at different developmental stages showed that EBF2-lacZ is initially expressed in the first and second branchial arches at E9, in somites, specifically the forming sclerotomes, at E10 and E10.5, as well as in dorsal root ganglia at E12.5 (Figure S1; see the Supplemental Data available with this article online). Cryosections of E16.5 embryos revealed that EBF2 is expressed in bone-forming areas and adipose tissue (Figure 1A). Moreover, EBF2 is expressed in specific neural tissues, as described previously (Corradi et al., 2003Corradi A. Croci L. Broccoli V. Zecchini S. Previtali S. Wurst W. Amadio S. Maggi R. Quattrini A. Consalez G.G. Hypogonadotropic hypogonadism and peripheral neuropathy in Ebf2-null mice.Development. 2003; 130: 401-410Crossref PubMed Scopus (83) Google Scholar). A similar expression pattern was observed in homozygous mutant embryos, indicating that the inactivation of the Ebf2 gene does not result in a loss of EBF2-expressing cells (Figure 1B). In bone-forming areas, EBF2 is expressed along the mesenchymal condensations at E14.5 (Figure 1C), in the perichondrium, and in cells invading the cartilagenous structures at E16.5 (Figure 1D). In situ hybridization of tibias of E18.5 embryos to detect Ebf2 transcripts indicated that individual Ebf2-expressing cells are scattered throughout the trabecular/cancellous bone area of wild-type mice, whereas no Ebf2 transcripts can be detected in the corresponding areas of Ebf2−/− mice, which were used as a negative control (Figures 1E and 1F). Various cell types, including osteoblasts and macrophages, are attached to the bone. To characterize the identity of the EBF2-expressing cells in more detail, we isolated adherent and nonadherent bone marrow cells from long bones of Ebf2+/− neonatal mice, by using collagenase and dispase, incubated the cells with anti-CD45 antibody, and loaded the cells with the fluorogenic β-galactosidase substrate FDG. In flow cytometry analysis, approximately 8% of the gated nonhematopoietic cells were positive for LacZ activity, and all of the CD45-positive hematopoietic cells were negative for EBF2-lacZ (Figure 1G and data not shown). Real-time RT-PCR analysis indicated that the EBF2-lacZ-expressing cells contain high levels of Runx2 and low levels of Sox9 transcripts (Figure 1H). These cells also contained low levels of transcripts of alkaline phosphatase and bone sialoprotein, but no detectable transcripts of Osterix and osteocalcin. Moreover, EBF2-lacZ-expressing cells contained transcripts of the chondrocyte marker collagen2a1, but lacked transcripts of other markers of chondrocytes (Figure S2A). We also analyzed the expression of EBF2 in sorted chondrocytes and in differentiated osteoclasts. Neither chondrocytes nor osteoclasts were found to express Ebf2 (Figure S2A; data not shown). Finally, we examined the presence of EBF2-expressing cells in calvarial bone preparations of E18.5 Ebf2+/− mice by FDG loading and flow cytometry. We found that 2%–3% of the cells were positive for EBF2-lacZ (Figure S2B). Based on the expression of Runx2, the absence of osteocalcin transcripts, and the presence of low levels of Sox9 and collagen2a1 transcripts, EBF2-expressing cells appear to represent immature osteoblastic cells. With the aim of further defining the expression of Ebf2 in osteoblastic cells, we performed in vitro differentiation experiments with calvarial cells from newborn wild-type mice (Bellows et al., 1990Bellows C.G. Heersche J.N. Aubin J.E. Determination of the capacity for proliferation and differentiation of osteoprogenitor cells in the presence and absence of dexamethasone.Dev. Biol. 1990; 140: 132-138Crossref Scopus (192) Google Scholar). In this experiment, we placed calvarial cells of E18.5 wild-type mice into culture for 3 days and thereafter differentiated the cells into mature osteoblasts by the addition of β-glycerophosphate and ascorbic acid. Quantitative RT-PCR analysis of three independent cultures indicated that the expression of Ebf2 is induced ∼5-fold within 2 days, but declines after 4 days, when transcripts of Osterix and osteocalcin are detected at high levels (Figure 2A). Ebf3 showed a similar profile of expression, albeit at a lower level, whereas transcripts of Ebf1 accumulate in a pattern that resembles more that of Osterix. Taken together, these data suggest that Ebf2 is expressed in immature osteoblastic cells that may include osteochondroprogenitor cells (Eames et al., 2004Eames B.F. Sharpe P.T. Helms J.A. Hierarchy revealed in the specification of three skeletal fates by Sox9 and Runx2.Dev. Biol. 2004; 274: 188-200Crossref PubMed Scopus (108) Google Scholar). Moreover, EBF2 may be partially redundant with other members of the EBF family of transcription factors. To examine the consequences of a loss of EBF2 function on skeletal development, we performed alcian blue and alizarin red staining of skeletons at E18.5. No obvious differences in the morphology of bones and cartilages were detected between wild-type and Ebf2−/− mice (Figure 2B). Von Kossa staining and in situ hybridizations with probes to detect markers for osteoblasts (bone sialoprotein, collagen1a1) or chondrocytes (collagen2a1) did not reveal any significant changes in mutant embryos (Figure 2C). Moreover, histomorphometric analysis indicated that the cancellous bone area (B.Ar./T.Ar.) in the distal femurs of E18.5 wild-type and Ebf2−/− embryos is similar (Figure 2D). However, quantitative RT-PCR assays to detect transcripts encoding regulators of osteoblast differentiation or components of the extracellular matrix indicated that the expression of bone sialoprotein and osteocalcin is decreased modestly, but reproducibly, in directly sorted Ebf2−/− osteoblastic cells, relative to Ebf2+/− osteoblasts (Figure 2E). Thus, the lack of EBF2 may result in a small defect in the terminal differentiation of osteoblasts. To analyze the bones of older mice, we used mice at 3 weeks of age, when the dwarfism and impaired viability of Ebf2−/− mice is less severe than in adult mice (Corradi et al., 2003Corradi A. Croci L. Broccoli V. Zecchini S. Previtali S. Wurst W. Amadio S. Maggi R. Quattrini A. Consalez G.G. Hypogonadotropic hypogonadism and peripheral neuropathy in Ebf2-null mice.Development. 2003; 130: 401-410Crossref PubMed Scopus (83) Google Scholar). At 3 weeks of age, the size and weight of the Ebf2−/− mice are reduced by 22% and 43%, respectively, relative to wild-type mice. X-ray analysis and histology of the tibia at low resolution showed that tibial length in EBF2-deficient mice is decreased by ∼19% relative to wild-type mice (Figures 3A and 3B). Von Kossa staining of femurs of wild-type and Ebf2−/− mice revealed a cancellous and cortical bone osteopenia in the mutant mice (Figure 3C). The cortical thickness of the femur midshaft, measured by histomorphometry, was reduced by 65% in mutant mice (136 ± 17 μm versus 61 ± 8 μm, wild-type versus Ebf2−/−; n = 4–5). High-power micrographs of cortical bone in the midshaft region showed areas of osteoclastic endocortical bone resorption in mutant, but not wild-type, mice (Figure 3C). To quantify the effects of cortical thinning on bone biomechanics, we performed three-point bending tests of femurs from wild-type and Ebf2−/− mice. We found an ∼3-fold decrease in ultimate force in Ebf2−/− mice (Figure 3D). Von Kossa staining also indicated that the cancellous bone area (B.Ar/T.Ar) and the width of the growth plate of Ebf2−/− mice are reduced, whereas the number of adipocytes is increased, as shown for the proximal tibial metaphysis (Figure 3E). Osteoblasts could be detected in normal numbers in histological sections (data not shown), and the rate of cancellous bone formation, as measured by calcein fluorochrome labeling, showed no significant difference between wild-type and Ebf2−/− mice (0.99 ± 0.22 μm2/μm/day in wild-type versus 1.18 ± 0.18 μm2/μm/day in Ebf2−/− mice; n = 4–5). Furthermore, quantitative RT-PCR analysis of the expression of osteoblastic marker genes in cells isolated from the tibia of 3-week-old wild-type and EBF2-deficient mice did not reveal any significant differences (Figure S2C). Finally, van Gieson staining of femurs showed a similar density and direction of collagen fibers in Ebf2+/+ and Ebf2−/− mice (Figures S2D and S2E). However, we detected an increase in the number of TRAP-positive osteoclasts in EBF2-deficient mice (Figure 3G). Quantification of these defects by bone histomorphometry indicated that the trabecular bone area of the tibia is reduced by 49% relative to wild-type mice (Figure 3F). These findings were confirmed by peripheral quantitative computer tomography (pQCT), showing that the bone mineral density (BMD) of both the distal metaphysis and shaft of the femur of Ebf2−/− mice is diminished by 40% (Figure 3F; Table S1). Finally, the osteoclast perimeter (Oc.Pm/B.Pm), a measurement of the surface of bone covered by osteoclasts, was increased by 47%, and the urinary excretion of the collagen degradation product deoxypyridinoline (DPD), a whole body marker for bone resorption, was elevated by 50% in the mutant mice (Figure 3H). Taken together, these data indicate that the osteopenia in EBF2-deficient mice, which is more pronounced than can be accounted for by the dwarfism, is due to an increased bone resorption and enhanced osteoclastogenesis. The observed increase in the number of osteoclasts in EBF2-deficient mice prompted us to examine the expression of components of the RANK-RANKL signaling pathway that regulate communication between osteoblasts and osteoclasts. In a first step, we examined the expression of the Rankl and Opg genes by immunohistochemistry on tibia bones from Ebf2+/+ and Ebf2−/− E18.5 embryos (Figures 4A and 4B). Expression of OPG and RANKL proteins could be detected in cells adjacent to trabecular bone. Relative to wild-type mice, however, the expression of OPG appeared weaker, and that of RANKL appeared stronger, in Ebf2−/− mice. To confirm the putative changes in the expression of these components of the RANK-RANKL signaling pathway, we purified osteoblasts from the calvariae of heterozygous and homozygous mutant newborn mice by sorting for β-galactosidase activity, expanded the cells in culture, and analyzed the expression of putative EBF2 target genes by RT-PCR. In EBF2-deficient osteoblasts, the number of Opg transcripts was markedly reduced, whereas the number of Rankl transcripts was increased (Figure 4C). No significant changes in the expression of Runx2 and Pparγ were observed (Figure 4C). With the aim of quantifying these effects and extending the observations to cells that have not been cultured in vitro, we performed quantitative RT-PCR to detect transcripts in heterozygous and homozygous EBF2-expressing osteoblastic cells that have been sorted directly after isolation. In homozygous mutant cells, the expression of Opg was decreased by a factor of 14, and the expression of Rankl was increased by a factor of 5 (Figure 4D). The increase in osteoclast numbers in the bones of EBF2-deficient mice and the downregulation of Opg expression in Ebf2−/− osteoblasts raised questions as to whether these defects result in a decrease in the serum levels of OPG and whether the defects are osteoblast autonomous. Although OPG is expressed by a variety of cell types, the levels of OPG in the serum of 3-week-old Ebf2−/− mice were reduced by 41% relative to wild-type mice (Figure 4E). We also examined whether the defects in osteoclastogenesis can be recapitulated in vitro and overcome by the addition of recombinant OPG. Toward this end, we incubated wild-type bone marrow cells with macrophage colony-stimulating factor (M-CSF) to enrich for osteoclast precursors, and we cultured these cells on osteoblastic cells from heterozygous and homozygous mutant mice that had been sorted for the expression of EBF2-lacZ. Osteoblast-mediated differentiation of the bone marrow cells into osteoclasts was monitored by TRAP staining. The number of osteoclasts in the coculture with Ebf2−/− osteoblasts was 57% higher than in the coculture with Ebf2+/− osteoblasts (Figure 4F). As a control, no difference in the generation of osteoclasts was observed with EBF2-deficient bone marrow cells compared to wild-type bone marrow cells. If the increase in the differentiation of osteoclasts is dependent on the decrease of OPG expression in Ebf2−/− osteoblasts, it should be possible to reverse the defect by the addition of exogenous OPG protein. Therefore, we added increasing amounts of recombinant OPG to the bone marrow cells being cultured with Ebf2+/− or Ebf2−/− osteoblasts. In both cocultures, the numbers of TRAP-positive osteoclasts were reduced in a dose-dependent manner, although more exogenous OPG is needed in the Ebf2−/− cultures to achieve a similar block in oste" @default.
• W2158698452 created "2016-06-24" @default.
• W2158698452 creator A5022682784 @default.
• W2158698452 creator A5024176454 @default.
• W2158698452 creator A5047486023 @default.
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• W2158698452 date "2005-12-01" @default.
• W2158698452 modified "2023-10-12" @default.
• W2158698452 title "EBF2 Regulates Osteoblast-Dependent Differentiation of Osteoclasts" @default.
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