Critical Regulation of Bone Morphogenetic Protein-induced Osteoblastic Differentiation by Delta1/Jagged1-activated Notch1 Signaling

Abstract

Functional involvement of the Notch pathway in osteoblastic differentiation has been previously investigated using the truncated intracellular domain, which mimics Notch signaling by interacting with the DNA-binding protein CBF-1. However, it is unclear whether Notch ligands Delta1 and Jagged1 also induce an identical cellular response in osteoblastic differentiation. We have shown that both Delta1 and Jagged1 were expressed concomitantly with Notch1 in maturating osteoblastic cells during bone regeneration and that overexpressed and immobilized recombinant Delta1 and Jagged1 alone did not alter the differentiated state of MC3T3-E1 and C2C12 cells. However, they augmented bone morphogenetic protein-2 (BMP2)-induced alkaline phosphatase activity and the expression of several differentiation markers, except for osteocalcin, and ultimately enhanced calcified nodule and in vivo ectopic bone formation of MC3T3-E1. In addition, both ligands transmitted signal through the CBF-1-dependent pathway and stimulated the expression of HES-1, a direct target of Notch pathway. To test the necessity of Notch signaling in BMP2-induced differentiation, Notch signaling was inhibited by the dominant negative extracellular domain of Notch1, specific inhibitor, or small interference RNA. These treatments decreased alkaline phosphatase activity as well as the expression of other differentiation markers and inhibited the promoter activity of Id-1, a target gene of the BMP pathway. These results indicate the functional redundancy between Delta1 and Jagged1 in osteoblastic differentiation whereby Delta1/Jagged1-activated Notch1 enhances BMP2-induced differentiation through the identical signaling pathway. Furthermore, our data also suggest that functional Notch signaling is essential not only for BMP2-induced osteoblast differentiation but also for BMP signaling itself. Functional involvement of the Notch pathway in osteoblastic differentiation has been previously investigated using the truncated intracellular domain, which mimics Notch signaling by interacting with the DNA-binding protein CBF-1. However, it is unclear whether Notch ligands Delta1 and Jagged1 also induce an identical cellular response in osteoblastic differentiation. We have shown that both Delta1 and Jagged1 were expressed concomitantly with Notch1 in maturating osteoblastic cells during bone regeneration and that overexpressed and immobilized recombinant Delta1 and Jagged1 alone did not alter the differentiated state of MC3T3-E1 and C2C12 cells. However, they augmented bone morphogenetic protein-2 (BMP2)-induced alkaline phosphatase activity and the expression of several differentiation markers, except for osteocalcin, and ultimately enhanced calcified nodule and in vivo ectopic bone formation of MC3T3-E1. In addition, both ligands transmitted signal through the CBF-1-dependent pathway and stimulated the expression of HES-1, a direct target of Notch pathway. To test the necessity of Notch signaling in BMP2-induced differentiation, Notch signaling was inhibited by the dominant negative extracellular domain of Notch1, specific inhibitor, or small interference RNA. These treatments decreased alkaline phosphatase activity as well as the expression of other differentiation markers and inhibited the promoter activity of Id-1, a target gene of the BMP pathway. These results indicate the functional redundancy between Delta1 and Jagged1 in osteoblastic differentiation whereby Delta1/Jagged1-activated Notch1 enhances BMP2-induced differentiation through the identical signaling pathway. Furthermore, our data also suggest that functional Notch signaling is essential not only for BMP2-induced osteoblast differentiation but also for BMP signaling itself. Notch signaling pathway is highly conserved beyond species and plays a critical role in a variety of cellular functions, including cell proliferation, differentiation, and apoptosis (1Hansson E.M. Lendahl U. Chapman G. Semin. Cancer Biol. 2004; 14: 320-328Crossref PubMed Scopus (186) Google Scholar). To date, four Notch receptors (Notch1–4) and five of their ligands (Delta1, -3, -4 and Jagged1, -2) have been identified in mammalians; all are single-span transmembrane polypeptides that act by cell-to-cell contact (2Baron M. Semin. Cell Dev. Biol. 2003; 14: 113-119Crossref PubMed Scopus (275) Google Scholar). Notch receptors contain 36 epidermal growth factor-like repeats and 3 cysteine-rich Notch/LIN-12 repeats in a large extracellular domain and 6 tandem cdc10/ankyrin repeats, a glutamine-rich domain, and a PEST sequence in an intracellular domain. Notch ligands also contain epidermal growth factor repeats in the extracellular domain in addition to a unique cysteine-rich N-terminal region referred to as the Delta:Serrate:LAG2 (DSL) domain, but their intracellular domains are small and poorly conserved. The interaction of Notch with the ligands induces the nuclear translocation of the intracellular domain of Notch as a result of proteolytic cleavage at the juxtamembrane portion. In the nuclei, the intracellular domain interacts with CSL DNA-binding proteins, including CBF1/RBP-J, and transactivates target genes such as hairy enhancer of split-1 (HES-1) 1The abbreviations used are: HES, hairy/enhancer of split; BMP2, bone morphogenetic protein-2; ALP, alkaline phosphatase; EGF, epidermal growth factor; Col1α, type 1 collagen; TGFβ, transforming growth factor-β; siRNA, small interference RNA; NICD, intracellular domain of Notch1; DN-Notch, dominant negative NOTCH. and HES-5. In an alternative pathway, Notch receptors have also been reported to transmit signal through CSL-independent pathways by interacting with other signaling molecules, such as mitogen-activated protein kinase, Src, and nuclear factor κB (3Small D. Kovalenko D. Kacer D. Liaw L. Landriscina M. Di Serio C. Prudovsky I. Maciag T. J. Biol. Chem. 2000; 19: 32022-32030Google Scholar, 4Small D. Kovalenko D. Soldi R. Mandinova A. Kolev V. Trifonova R. Bagala C. Kacer D. Battelli C. Liaw L. Prudovsky I. Maciag T. J. Biol. Chem. 2003; 278: 16405-16413Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 5Cheng P. Zlobin A. Volgina V. Gottipati S. Osborne B. Simel E.J. Miele L. Gabrilovich D.I. J. Immunol. 2001; 167: 4458-4467Crossref PubMed Scopus (181) Google Scholar). The Notch pathway has been demonstrated to be critical for a variety of developmental processes (1Hansson E.M. Lendahl U. Chapman G. Semin. Cancer Biol. 2004; 14: 320-328Crossref PubMed Scopus (186) Google Scholar), but its functional role in bone development has been poorly understood as mice deficient in Notch1, Jagged1, or Delta1 die at an early embryonic stage before bone formation due to impaired somatogenesis and/or vascularization (6Swiatek P.J. Lindsell C.E. del Amo F.F. Weinmaster G. Gridley T. Genes Dev. 1991; 113: 707-719Google Scholar, 7Hrabe de Angelis M. McIntyre II, J. Gossler A. Nature. 1997; 386: 717-721Crossref PubMed Scopus (547) Google Scholar, 8Xue Y. Gao X. Lindsell C.E. Norton C.R. Chang B. Hicks C. Gendron-Maguire M. Rand E.B. Weinmaster G. Gridley T. Hum. Mol. Genet. 1999; 8: 723-730Crossref PubMed Scopus (641) Google Scholar). In contrast, the in vitro potential of Notch pathway in osteoclastogenesis and osteoblastogenesis has been investigated in several reports. Notch activation reduces the surface expression of c-Fms, which is a receptor for macrophage colony-stimulating factor, in osteoclast precursor cells and enhances the expression of osteoprotegerin in stromal cells, which results in the down-regulation of osteoclastogenesis (9Yamada T. Yamazaki H. Yamane T. Yoshino M. Okuyama H. Tsuneto M. Kurino T. Hayashi S. Sakano S. Blood. 2003; 101: 2227-2234Crossref PubMed Scopus (105) Google Scholar). However, controversial results have been obtained with respect to osteoblastic differentiation. Continuous expression of the intracellular domain of Notch1 (NICD), believed to act as a constitutive active form by retrovirus system or by an ordinary stable transfection study, inhibits bone morphogenic protein (BMP) 2-induced osteoblast differentiation in osteoblast precursor cells, including MC3T3-E1 (10Sciaudone M. Gazzerro E. Priest L. Delany A.M. Canalis E. Endocrinology. 2003; 144: 5631-5639Crossref PubMed Scopus (169) Google Scholar, 11Shindo K. Kawashima N. Sakamoto K. Yamaguchi A. Umezawa A. Takagi M. Katsube K. Suda H. Exp. Cell Res. 2003; 290: 370-380Crossref PubMed Scopus (63) Google Scholar). In contrast, the transient expression of NICD by the adenovirus system in MC3T3-E1 cells leads to an enhanced bone mineral deposition (12Tezuka K. Yasuda M. Watanabe N. Morimura N. Kuroda K. Miyatani S. Hozumi N. J. Bone Miner. Res. 2002; 17: 231-239Crossref PubMed Scopus (178) Google Scholar). However, despite these results, it is critical to determine whether Notch ligands Delta and Jagged have an identical function in osteoblastic differentiation because these ligands have been shown to exert distinct activity in certain cells (13Amsen D. Blander J.M. Lee G.R. Tanigaki K. Honjo T. Flavell R.A. Cell. 2004; 117: 515-526Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar, 14Trifonova R. Small D. Kacer D. Kovalenko D. Kolev V. Mandinova A. Soldi R. Liaw L. Prudovsky I. Maciag T. J. Biol. Chem. 2004; 279: 13285-13288Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Moreover, it is also unclear whether Notch signaling itself is required for the osteoblastic differentiation induced by BMP2. The interaction between Notch and BMP has been demonstrated to be crucial for endothelial cell migration and myogenic differentiation (15Dahlqvist C. Blokzijl A. Chapman G. Falk A. Dannaeus K. Ibanez C.F. Lendahl U. Development. 2003; 130: 6089-6099Crossref PubMed Scopus (210) Google Scholar, 16Itoh F. Itoh S. Goumans M.J. Valdimarsdottir G. Iso T. Dotto G.P. Hamamori Y. Kedes L. Kato M. Dijke EMBO J. 2004; 23: 541-551Crossref PubMed Scopus (199) Google Scholar). To clarify the functional involvement of Delta/Jagged-Notch signaling pathway in osteoblastic differentiation, we first confirmed the expression of Notch, Delta, and Jagged by immunohistochemistry in an in vivo bone regeneration model. We then addressed the functional difference between Delta1 and Jagged1 by transient transfection study and by using recombinant proteins in MC3T3-E1 and C2C12 cells. We also clarified the essential role of Notch1 signaling in BMP2-induced osteoblastic differentiation by inhibiting Notch signaling with the dominant negative form of Notch1, a specific inhibitor, and siRNA. Bone Regeneration Model and Immunostaining—Bone defects (1.2-mm diameter) were created in the diaphysis of unilateral femurs of 8-week-old C57BL6/J mice using a dental drill with copious saline irrigation. Notch expression was analyzed after 5, 10, and 15 days of operation. For isolation of RNA, the femur was dissected into 8-mm-long pieces containing the bone defect using a bone saw. For immunohistochemistry, the animals were perfusion-fixed by 4% paraformaldehyde in phosphate-buffered saline, decalcified with 10% ethylenediaminotetraacetic acid, and embedded in paraffin. The deparaffinized sections were with in for by 10% for 1 The were with for Notch1 and Delta1 or Jagged1 for at These were by and and by as previously T. K. Yamaguchi A. H. S. T. J. Bone Miner. Res. 2003; PubMed Scopus Google Scholar). To the active form of Notch1, the was with and was by with as a for the of Jagged1 and Delta1 D. Kovalenko D. Soldi R. Mandinova A. Kolev V. Trifonova R. Bagala C. Kacer D. Battelli C. Liaw L. Prudovsky I. Maciag T. J. Biol. Chem. 2003; 278: 16405-16413Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, B. T. M. M. L. D. Itoh A. Sakano S. M. Blood. 2001; PubMed Scopus Google Scholar). of type and dominant negative form K. S. K. S. H. Honjo T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google and NICD D. Kovalenko D. Soldi R. Mandinova A. Kolev V. Trifonova R. Bagala C. Kacer D. Battelli C. Liaw L. Prudovsky I. Maciag T. J. Biol. Chem. 2003; 278: 16405-16413Abstract Full Text Full Text PDF PubMed Scopus (54) Google contain and contain the promoter region of the gene and of the the K. T. A. K. Y. T. Y. K. T. A. Yamaguchi A. 2003; PubMed Scopus Google Scholar). The four tandem of the the promoter T. M. T. Suda T. N. R. Genes 2002; PubMed Scopus Google Scholar). the to promoter of the Cell and and C2C12 cells were obtained and MC3T3-E1 were in essential containing 10% and C2C12 cells were in containing 10% and the osteoblastic cells were as previously Y. H. S. T. K. Gao Y. Yamaguchi A. PubMed Scopus Google and in the cells in bone were obtained femurs as of the was with to the of the cells were with or BMP2, which was by activity was after transfection by the The processes of and the have also been previously (9Yamada T. Yamazaki H. Yamane T. Yoshino M. Okuyama H. Tsuneto M. Kurino T. Hayashi S. Sakano S. Blood. 2003; 101: 2227-2234Crossref PubMed Scopus (105) Google Scholar). and of activity was by using and as previously K. T. A. K. Y. T. Y. K. T. A. Yamaguchi A. 2003; PubMed Scopus Google Scholar). The of in the was by In Bone cells in were into by a that was with 1 of BMP2, and/or The was then into the region of mice as previously K. T. A. K. Y. T. Y. K. T. A. Yamaguchi A. 2003; PubMed Scopus Google and to and after and specific for Notch1 was into MC3T3-E1 and C2C12 cells at the of 1 with to the The for Notch1 were as of these the and for and The sequence was used as the the cells were with or recombinant and to or For the cells were in a of and a and the was by and to a Notch1 expression was by and was 1 of using and and the expression was by the system and the are The of was at of and by Notch during Bone receptors and their ligands have been demonstrated to in certain osteoblastic cells, but their in vivo expression To clarify of Jagged1, and Notch1 was using an femur a bone defect in the to Notch1 was in However, expression was in the cells that into the bone defect days after and Delta1 was also and demonstrated between Notch1 and with Jagged1 10, the bone was in a that both Notch1 and Jagged1 in the the surface of the using that the active form of Notch1 Lee Nature. 2003; PubMed Scopus Google that the Notch1 signal is in the and the bone defect demonstrated that the expression of Notch1, and Jagged1 was after the drill was These results indicate that the Notch1 signal is in cells during bone regeneration of Delta1 and and by of Notch1, and Jagged in MC3T3-E1 and C2C12 to the functional role of Notch signaling in osteoblast differentiation, the expression of -3, and Jagged1, was in cell and in the of and bone osteoblastic cells. demonstrated that the expression of several such as and was or in several cells, other molecules, including Notch1, and Jagged1, were expressed well in the cells the functional of Notch activation in osteoblast differentiation is not we to the functional of Delta1 and For we first a transient transfection study using the expression of NICD and of Delta1 and Jagged1 in the MC3T3-E1 and C2C12 cells. that of these alone did not activity but enhanced BMP2-induced activation in both cell the promoter that Delta1 and Jagged1 the promoter activity in the of In contrast, BMP2-induced of osteocalcin, for osteoblast differentiation, was through both protein and by a and These results suggest that of Delta1 and Jagged1 are with and in Bone ligands are transmembrane proteins and act by cell-to-cell To that recombinant Delta1 and Jagged1 also identical as demonstrated by transient expression study and to test whether is MC3T3-E1 and C2C12 cells were with recombinant Delta1 or Jagged1 the recombinant proteins were in or immobilized the The result that for BMP2, an in BMP2-induced activity was in the of immobilized Delta1 and Jagged1 but not in the of Delta1 and Jagged1 in the In addition, of expression was also in the immobilized The expression of osteoblastic differentiation markers was investigated by in C2C12 cells. In addition to ALP, of type 1 and were by Jagged1 did not and decreased results were obtained with Delta1 not However, 3 of in the of and in enhanced in the of Delta1 and Jagged1 with of and We also the in vivo potential of Delta1 and Jagged1 for bone a MC3T3-E1 cells with Jagged1, and/or was into the region of mice for The results that Delta1 or Jagged1 alone did not but enhanced ectopic bone formation induced by The results that of bone be in both and Delta1 or Jagged1 with alone These results indicate that both Delta1 and Jagged1 to be immobilized and act as for osteoblastic differentiation, is in an early stage of differentiation. CBF-1-dependent and Notch1 through CSL DNA-binding and type or dominant negative was with or Delta1 or Jagged1 to the of for Notch The results that alone is to BMP2-induced activation and that activity and by were in the expression of and not In addition, both Delta1 and Jagged1 induced the expression of HES-1, a target gene of Notch, through the CBF-1-dependent pathway and these results suggest that is in the osteoblast differentiation induced by Delta1 and of Notch1 by and signaling has been demonstrated to a crucial role for cell by interacting with BMP signaling F. Itoh S. Goumans M.J. Valdimarsdottir G. Iso T. Dotto G.P. Hamamori Y. Kedes L. Kato M. Dijke EMBO J. 2004; 23: 541-551Crossref PubMed Scopus (199) Google Scholar, T. K. T. Res. 2003; PubMed Scopus Google Scholar). To whether Notch1 signaling is required for BMP2-induced osteoblastic differentiation, we the extracellular domain of Notch1 as a dominant negative form and of and with in a of activity induced by 5, and also BMP2-induced expression using promoter demonstrated that is by also demonstrated that the expression of ALP, osteocalcin, and were by to and of the These results suggest that Notch1 signaling is for osteoblast differentiation. of Notch1 by the role of Notch pathway, distinct small interference which were the region of Notch1, were into the cells. The transfection of was not of cell of MC3T3-E1 cells specific of Notch1 to the of the of Notch1 in a of BMP2-induced activity as well as expression and by the However, the expression of osteoblastic differentiation markers were not with the promoter activity except for osteocalcin, the expression of which was decreased The of ALP, and was of the RNA. these results indicate that Notch1 signaling is essential for BMP2-induced osteoblast differentiation. between Notch and BMP interference of signaling pathway between Notch and growth has been previously demonstrated F. Itoh S. Goumans M.J. Valdimarsdottir G. Iso T. Dotto G.P. Hamamori Y. Kedes L. Kato M. Dijke EMBO J. 2004; 23: 541-551Crossref PubMed Scopus (199) Google Scholar, T. K. T. Res. 2003; PubMed Scopus Google Scholar, A. C. E. Falk A. A. Lendahl U. Ibanez C.F. J. Cell Biol. 2003; PubMed Scopus Google Scholar). To study a of of BMP we first whether Notch activation BMP For the expression and of Id-1, a target gene of BMP were in cells with immobilized Delta1 and Jagged1 in the or of BMP2. However, the results that and promoter activity induced by not be by Notch activation and In addition, it was by that Notch1 activation did not alter the expression of such as and not These data suggest that the of activity by Notch1 is not due to the activation of BMP we investigated a by which the of the Notch signal leads to a in BMP with the expression of In addition, the transient transfection of and in the of promoter activity and these data indicate that the of BMP2-induced osteoblastic differentiation by Notch is at due to BMP Notch receptors are to an role in and are required for the of including and (2Baron M. Semin. Cell Dev. Biol. 2003; 14: 113-119Crossref PubMed Scopus (275) Google Scholar). the expression of in bone a study has demonstrated that Notch1 is expressed in the and in the during N. Y. K. Miyatani S. Morimura N. Yasuda M. R. Kuroda K. Y. Hozumi N. K. J. Bone Miner. 2003; PubMed Scopus Google Scholar). study, it that Notch1, and Jagged1 are expressed in osteoblast precursor cells as well as in during bone regeneration and that Notch1 is in these cells. These results suggest that in addition to the of cell J.M. E. P. Nature. 2003; PubMed Scopus Google Scholar), Notch signaling plays an role in the of cells to the osteoblastic cell Moreover, the expression of Notch1, and Jagged1 to a of an and/or of a cell-to-cell signaling that is by a in the development of the system N. S. P. 1999; PubMed Scopus Google Scholar). is functional difference between Delta and Jagged in a of these ligands distinct expression in a during development G. G. Development. 1991; 113: PubMed Google Scholar, C.E. J. G. Gossler A. Weinmaster G. Mol. Cell. 8: PubMed Scopus Google Scholar). that specific distinct a study has demonstrated the role of Delta and Jagged in the of (13Amsen D. Blander J.M. Lee G.R. Tanigaki K. Honjo T. Flavell R.A. Cell. 2004; 117: 515-526Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar). an extracellular Notch, which in leads to specific of Jagged P. Zlobin A. Volgina V. Gottipati S. Osborne B. Simel E.J. Miele L. Gabrilovich D.I. J. Immunol. 2001; 167: 4458-4467Crossref PubMed Scopus (181) Google Scholar). In study, we demonstrated NICD, Delta1 and Jagged1 identical activity in the of osteoblastic differentiation. Delta1 or Jagged1 alone is to differentiated these ligands BMP2-induced expression of several differentiation markers, such as ALP, and Col1α, in the In addition, Delta1 and Jagged1 are of bone mineral deposition and BMP2-induced ectopic bone Furthermore, it that both Delta1 and Jagged1 transmit through and the results have been demonstrated by (12Tezuka K. Yasuda M. Watanabe N. Morimura N. Kuroda K. Miyatani S. Hozumi N. J. Bone Miner. Res. 2002; 17: 231-239Crossref PubMed Scopus (178) Google Scholar), the adenovirus system to expression is to be in an of differentiation Notch1 is (12Tezuka K. Yasuda M. Watanabe N. Morimura N. Kuroda K. Miyatani S. Hozumi N. J. Bone Miner. Res. 2002; 17: 231-239Crossref PubMed Scopus (178) Google Scholar, D. A. Weinmaster G. Development. 1999; PubMed Google Scholar). In Notch signaling is to differentiation a differentiation or to the cell to in an state (1Hansson E.M. Lendahl U. Chapman G. Semin. Cancer Biol. 2004; 14: 320-328Crossref PubMed Scopus (186) Google Scholar). the expression of Delta1 and Jagged1 in vivo bone these results suggest that is a functional redundancy between Delta1 and Jagged1 and that these ligands direct cells to the differentiated through identical signaling However, results have been reported a retrovirus or an ordinary stable transfection study in which NICD osteoblastic differentiation of stromal cell and MC3T3-E1 and cells (10Sciaudone M. Gazzerro E. Priest L. Delany A.M. Canalis E. Endocrinology. 2003; 144: 5631-5639Crossref PubMed Scopus (169) Google Scholar, 11Shindo K. Kawashima N. Sakamoto K. Yamaguchi A. Umezawa A. Takagi M. Katsube K. Suda H. Exp. Cell Res. 2003; 290: 370-380Crossref PubMed Scopus (63) Google Scholar). of difference is that the of Notch activation the of Notch transient and activation of the Notch pathway osteoblastic differentiation as shown in our study, activation to the of osteoblastic be to the functional difference of Notch signaling in osteoblastic differentiation. In study, we demonstrated that the of Notch signaling by or leads to a in and Furthermore, the of expression of Notch1 by also in a in osteoblastic differentiation with the in differentiation markers, such as and In expression was at the is not with the results of the study, it that a functional Notch signal be essential for expression through a distinct with other these results indicate that constitutive Notch1 signaling is critical for BMP2-induced differentiation. In it has been demonstrated that the of Notch signal through and of of C2C12 cells (15Dahlqvist C. Blokzijl A. Chapman G. Falk A. Dannaeus K. Ibanez C.F. Lendahl U. Development. 2003; 130: 6089-6099Crossref PubMed Scopus (210) Google Scholar), that functional Notch signaling be to for differentiation. To a of the interaction between Notch and BMP signaling in osteoblast differentiation, we the interaction between BMP and Notch have that Notch and pathways such as and or target gene F. Itoh S. Goumans M.J. Valdimarsdottir G. Iso T. Dotto G.P. Hamamori Y. Kedes L. Kato M. Dijke EMBO J. 2004; 23: 541-551Crossref PubMed Scopus (199) Google Scholar, T. K. T. Res. 2003; PubMed Scopus Google Scholar, A. C. E. Falk A. A. Lendahl U. Ibanez C.F. J. Cell Biol. 2003; PubMed Scopus Google Scholar). In Notch pathway, NICD interacts with and the including and in the activation of Notch target genes such as and F. Itoh S. Goumans M.J. Valdimarsdottir G. Iso T. Dotto G.P. Hamamori Y. Kedes L. Kato M. Dijke EMBO J. 2004; 23: 541-551Crossref PubMed Scopus (199) Google Scholar, T. K. T. Res. 2003; PubMed Scopus Google Scholar). In our study, Notch1 did not BMP signaling to also did not induce the expression of gene These results indicate the of other interaction between BMP and is that other Notch target genes such as and that be in C2C12 cells with response to J.M. E.J. Res. 2004; PubMed Scopus Google be in the interaction between and Notch in osteoblast differentiation, target gene of both Notch and as negative N. D. D. M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). our data that the of Notch signaling or expression leads to a in promoter that Notch signaling is required for BMP signaling itself and that BMP signaling of Notch as previously in the regeneration in B. J.M. Dev. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). However, it is that Notch signaling be critical for pathway as because activity using and was also inhibited by and in our cells. are to the that Notch to the In our results that both Delta1 and Jagged1 transmit through the pathway and induce osteoblastic differentiation by with In addition, functional Notch1 activation is essential not only for osteoblast differentiation but also for BMP signaling itself. results suggest a potential for the signaling pathway Delta1 and Jagged1 to gene expression in bone regeneration as well as We T. Honjo for the expression R. for T. for and T. Maciag for dominant negative and constitutive active Notch1 and Jagged1 expression We also K. K. and K. for