Coordinated Activation of Notch, Wnt, and Transforming Growth Factor-β Signaling Pathways in Bone Morphogenic Protein 2-induced Osteogenesis (2004)

To examine early events in osteoblast differentiation, we analyzed the expression of about 9,400 genes in the murine MC3T3 cell line, whose robust differentiation was documented cytochemically and molecularly. The cells were stimulated for 1 and 3 days with the osteogenic stimulus containing bone morphogenic protein 2. Total RNA was extracted and analyzed by Affymetrix GeneChip oligonucleotide arrays. A regulated expression of 394 known genes and 295 expressed sequence tags was detected. The sensitivity and reliability of detection by microarrays was shown by confirming the expression pattern for 20 genes by radioactive quantitative reverse transcription-PCR. Functional classification of regulated genes was performed, defining the groups of regulated growth factors, receptors, and transcription factors. The most interesting finding was concomitant activation of transforming growth factor-β, Wnt, and Notch signaling pathways, confirmed by strong up-regulation of their target genes by PCR. The transforming growth factor-β pathway is activated by stimulated production of the growth factor itself, while the exact mechanism of Wnt and Notch activation remains elusive. We showed that bone morphogenic protein 2 stimulated expression of Hey1, a direct Notch target gene, in mouse MC3T3 and C2C12 cells, in human mesenchymal cells, and in mouse calvaria. Small interfering RNA-mediated inhibition of Hey1 induction led to an increase in osteoblast matrix mineralization, suggesting that Hey1 is a negative regulator of osteoblast maturation. This negative regulation is apparently achieved via interaction with Runx2: Hey1 completely abrogated Runx2 transcriptional activity. These findings identify the Notch-Hey1 pathway as a negative regulator of osteoblast differentiation/maturation, which is a completely novel aspect of osteogenesis and could point to possible new targets for bone anabolic agents. To examine early events in osteoblast differentiation, we analyzed the expression of about 9,400 genes in the murine MC3T3 cell line, whose robust differentiation was documented cytochemically and molecularly. The cells were stimulated for 1 and 3 days with the osteogenic stimulus containing bone morphogenic protein 2. Total RNA was extracted and analyzed by Affymetrix GeneChip oligonucleotide arrays. A regulated expression of 394 known genes and 295 expressed sequence tags was detected. The sensitivity and reliability of detection by microarrays was shown by confirming the expression pattern for 20 genes by radioactive quantitative reverse transcription-PCR. Functional classification of regulated genes was performed, defining the groups of regulated growth factors, receptors, and transcription factors. The most interesting finding was concomitant activation of transforming growth factor-β, Wnt, and Notch signaling pathways, confirmed by strong up-regulation of their target genes by PCR. The transforming growth factor-β pathway is activated by stimulated production of the growth factor itself, while the exact mechanism of Wnt and Notch activation remains elusive. We showed that bone morphogenic protein 2 stimulated expression of Hey1, a direct Notch target gene, in mouse MC3T3 and C2C12 cells, in human mesenchymal cells, and in mouse calvaria. Small interfering RNA-mediated inhibition of Hey1 induction led to an increase in osteoblast matrix mineralization, suggesting that Hey1 is a negative regulator of osteoblast maturation. This negative regulation is apparently achieved via interaction with Runx2: Hey1 completely abrogated Runx2 transcriptional activity. These findings identify the Notch-Hey1 pathway as a negative regulator of osteoblast differentiation/maturation, which is a completely novel aspect of osteogenesis and could point to possible new targets for bone anabolic agents. Bone is a dynamic tissue that is constantly remodeled, i.e. degraded and renewed. These two processes are accomplished by two main types of bone cells: bone-forming osteoblasts of mesenchymal origin and bone-resorbing osteoclasts of hematopoietic origin (1Baron R. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 5th Ed. American Society for Bone and Mineral Research, Washington, D. C.2003: 1-9Google Scholar). A synchronized action of osteoblasts and osteoclasts enables balanced bone remodeling. If this balance is changed in a way that bone resorption exceeds bone formation, osteoporosis occurs, a disease prevalent in old age and characterized by bone loss and a high risk of fractures. Most treatments for osteoporosis available so far target osteoclasts and inhibit bone resorption. In osteoporosis, however, bone loss exceeds the degree of bone gain that can be restored by inhibitors of resorption. Therefore, there is a large need for anabolic agents that would accelerate osteoblast differentiation and promote bone formation. For that purpose, knowledge about molecular events involved in osteoblast differentiation is crucial. Previously osteoblast differentiation was examined mostly at the cellular or single gene level. Many external regulating factors are known, but critical molecular steps in osteoblast differentiation and bone formation are largely unknown. One key player was identified recently as the Runx2 transcription factor (2Karsenty G. Semin. Cell Dev. Biol. 2000; 11: 343-346Crossref PubMed Scopus (146) Google Scholar). Another transcription factor, Osterix (Osx), which cooperates with and is genetically downstream of Runx2, has also been identified (3Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2784) Google Scholar). However, more molecular players in osteoblast differentiation remain to be identified. In vivo, bone forming osteoblasts develop from mesenchymal precursors, and this can be in cellular osteogenesis were as and of characterized by an of genes PubMed Scopus Google Scholar). of osteoblast differentiation are and bone To examine osteoblast differentiation in a is the of cells or cell and which an differentiation To with differentiation of cell which are and be of the we a mouse cell line, This cell is also known for in R. D. Bone PubMed Scopus Google we a cell at a from a that the cells at their Cell Biol. PubMed Scopus Google Scholar). To that the cells also at the molecular we from this cell a which activated a gene and activated Runx2 and This of cells robust and differentiation We this cell to examine a pattern of gene expression early differentiation The and of was confirmed by detection of a of and genes known to be regulated we also novel of gene regulation in osteoblasts by defining activated signaling We the of of a transcription factor of the and of and of bone morphogenic protein expressed sequence Hey1, and of with quantitative radioactive reverse protein Runx2, transcription factor interfering transcription factor transforming growth Wnt, murine protein and in transforming growth early expression growth 2 factor and of bone morphogenic protein expressed sequence Hey1, and of with quantitative radioactive reverse protein Runx2, transcription factor interfering transcription factor transforming growth Wnt, murine protein and in transforming growth early expression growth 2 factor the expression of Hey1 in the and negative we could in osteogenesis and to a main osteoblast a transcription factor of the Cell cell was a of Cell Biol. PubMed Scopus Google Scholar). The was with gene, the of Runx2, by a for a in which was activated by the osteogenic stimulus The cells were in with and in The was in the For RNA and cells were on in of in 1 of and in 3 of were to for 3 days at in and stimulated with and 1 cells were stimulated with For cell were with and osteogenic factors days cells were with with for at and with For from was in of and of was to the for at were with and for or days cells were in with for at and with was and was and for at were to with and RNA were in the containing Total RNA was with and to the were and in For RNA were in a to two bone was in 1 of was and were for 1 1 of at the was and was of was to and were and for at by a at at The was RNA was with and the was with and in of RNA was and the to the was as by D. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of and of were to 1 of were for at and on were to 20 with a containing of a of and 1 20 of reverse was or to The reverse transcription was for 2 at and by for at were with and at 1 of or as a were in a of containing a of 1 of a 1 of and of and For of protein 1 and the in The was as of at of at for 1 at genes were at and which were at for 1 and at for 1 20 The was with a at for of were with and and analyzed on were to and by The on were by the For gene a was performed, and the of for the quantitative was the of The and in the to and the of in were as and and and and Runx2 and and and protein and in and and RNA and and and and and and and and and protein and transforming growth and and and Hey1, and and Osterix (Osx), and by shown in this are from with MC3T3 In the cells were at days 1 and and osteogenic stimulus at days 1 and Total RNA from was extracted and analyzed on oligonucleotide for we a gene and for and to that cells to the osteogenic were in the Affymetrix were that of with of oligonucleotide in These for genes characterized genes and expressed sequence were from to be from of cellular RNA as The were on the and the were to the were analyzed the and were as expressed in a were as but as or A at at was as the were to genes and were with but with that gene were as regulated by osteogenic stimulus their expression more from the at in at two of that expression for were that most of the genes their expression of the a degree of in the expression with the for the of 1 the for In to this that is a of the known genes as and were to a degree and expression of genes on the in in a new factors regulated differentiation of MC3T3 growth growth growth factor, growth protein growth protein growth protein growth growth factor growth factor growth factor in a new osteoblast genes for we that the of the differentiation is the the cells were the For was to a degree at 3 in 3 as with 1 and suggesting that the differentiation was in 3 of at 1 in 3 also that in this more genes are regulated at suggesting a differentiation in 1 and 2. of this we as regulated genes regulation in two of a that the of the differentiation expression from the which to was C2C12 cells were with and or for 1 and 3 Total RNA was extracted and were analyzed on the Affymetrix GeneChip arrays. mesenchymal cells from human bone from the of the bone of a were with and Total RNA was extracted and the were analyzed on the Affymetrix GeneChip was the by from 1 of to the For quantitative an sequence detection was were in a in a of of by Hey1, and 2 of to 20 of were as at at by of at and 1 at were in with of and expression two were from in at which an increase in a could be were from was as a Small RNA cells were on in 2 of was in a of 1 to the was and the of was was by of containing and the osteogenic RNA was or For containing and the osteogenic stimulus was changed was The sequence of Hey1 was with by The sequence of was with by and the and Hey1 were the expression and in a for Hey1 The containing the in of the gene was as a This expression was a from or been of the were in in of and for was to the for cell and were For the 20 of One was to the were for 20 at was at in a For the cells were with and was and for at the were for at and for 1 at 20 of was a 2 of was and was In to osteogenic stimulus and the of cells to as showed a strong increase in at 3 Runx2 protein were also by 3 the cells bone which by days a to osteogenic the of more of the the osteoblast is 2 the exact of osteoblast To the differentiation at the transcriptional we analyzed expression of a of molecular of osteoblast MC3T3 cells were stimulated with osteogenic stimulus for 1 and 3 days and with of osteoblast differentiation were transcription factors and Runx2, and matrix and of osteoblast were in to the osteogenic was at 1 and at was at and Runx2 was at and of osteoblast differentiation, were at For and high of expression were in cells and osteogenic MC3T3 cells are to the osteoblast high expression of matrix is of RNA and were in C2C12 and mouse cells were also as cellular to the MC3T3 cell However, expressed a of regulated and the was in MC3T3 cells We that MC3T3 cells are an for in gene expression an in osteoblast differentiation we to identify genes involved in this by a of gene MC3T3 cells were stimulated with osteogenic factors, and RNA was extracted at days and 3 from expression was analyzed on Affymetrix GeneChip microarrays about and genes with known To the for expression of osteoblast genes on microarrays was with the The GeneChip oligonucleotide for of osteoblast and for For a pattern of The from a are shown in 2. were for expressed as transcription factors and Runx2, a sensitivity of We that detection was to expression and regulation of osteoblast and be a to regulation of novel genes as To an the transcriptional events involved in the osteoblast differentiation, we genes whose expression changed with the osteogenic gene expression were and We a regulation in at two of the of 394 genes with known and of 295 In we on the genes with known This of genes was analyzed by a which groups genes to their regulation One genes were and genes were was a the gene expression in and in their We groups to the cellular of the and A large of regulated genes matrix and This is with the that osteoblasts are cells for production of the bone The regulation of genes involved in the cell and is with that the cells, to to to a degree to 3 days with the osteogenic stimulus This regulation is also with the of the cell and to differentiation We however, on groups of whose could to the differentiation growth factors, receptors, and transcription factors. growth factor genes to be regulated in osteoblasts but also novel genes the we known of osteoblast growth factor growth factor, growth factor, and of Bone R. of Bone of Bone 2002; PubMed Scopus Google Scholar). The most was regulation of of the and were while was are known and of the osteoblasts and bone and of but their regulation by osteogenic stimulus containing has been We shown that the osteogenic is for activation of The activation of the signaling pathway by the osteogenic stimulus was by the regulation of target the transcriptional the matrix protein the and the 2 Cell. 2000; PubMed Scopus Google PubMed Scopus Google Cell. 2000; PubMed Scopus Google Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). from the is a up-regulation of the which to a negative These a new on the growth factors in osteoblast of regulated genes of the Wnt, and Notch signaling and genes involved in signaling growth growth growth early growth transcription matrix signaling protein of genes and target of Wnt factor activation of signaling for and for and transcription Notch target of in a new we and known of osteoblast a of was at days 1 and was to a in osteoblast differentiation, this could be of PubMed Scopus Google Scholar). genes were and of which can differentiation or of bone-resorbing osteoclasts PubMed Scopus Google PubMed Scopus Google suggesting that osteoblasts a to genes in the for the of factors in osteoblast differentiation or as factor growth factor and 2002; PubMed Scopus Google Cell. Scopus Google PubMed Scopus Google of Bone PubMed Scopus Google Google PubMed Scopus Google Scholar). of the as the or the and been to a in The genes were an osteoblast and a for a that the signaling of of the main in was This with the of and in the that osteoblasts a to bone-resorbing osteoclasts and differentiation regulated differentiation of MC3T3 in factor growth factor growth factor cell A protein growth factor protein growth factor for and protein cell factor for and in a new Notch 1 and 3 were The of Notch signaling in osteoblast differentiation has been two that Notch signaling osteoblast differentiation K. K. Bone 2002; PubMed Scopus Google PubMed Scopus Google Scholar). and were also This is is an player in bone and has been identified as a high bone gene G. D. K. G. B. B. B. K. D. B. B. B. R. Cell. Full Text Full Text PDF PubMed Scopus Google B. K. X. R. B. X. G. R.R. 2002; Full Text Full Text PDF PubMed Scopus Google R. G. Cell Biol. 2002; PubMed Scopus Google Scholar). transcription factor of regulated genes is the the that transcription factors are we regulated transcription factor with we confirmed regulation for of genes by The regulation of a large of transcription factors that their regulation is for the osteoblast differentiation factors regulated differentiation of MC3T3 transcriptional transcriptional of Wnt genes and target of Wnt transcriptional transcription Notch target signaling target factor transcription early growth transcription signaling signaling factor activation of factor for RNA and of and a protein transcription factor growth factor 3 protein transcription transcription transcriptional in a new genes of the signaling were shown to be The and were at 1 of a negative for regulation of This is in with Bone PubMed Scopus Google Scholar). A was at that of signaling pathway as a of to regulated transcription factor genes an osteoblast genes that were identified as target genes in osteoblasts by and Bone PubMed Scopus Google were also to be regulated in a of transcription factor that is involved in osteoblast differentiation PubMed Scopus Google was at and transcriptional inhibitors shown to be direct target genes Biol. 2002; Full Text Full Text PDF PubMed Scopus Google were while of this gene was The transcription factors of the and are in Z. 2000; PubMed Scopus Google D. J.M. R. Full Text Full Text PDF PubMed Scopus Google Cell. Google Scholar). a known target gene and osteoblast differentiation and and known targets D. PubMed Scopus Google were in osteoblast differentiation Another of the was also and this to the of regulation of this These findings the of novel a Wnt pathway target gene was at is transcription factor Wnt signaling and was also at This is a novel and finding a of the Wnt pathway in osteoblast differentiation a Wnt and a high bone gene G. D. K. G. B. B. B. K. D. B. B. B. R. Cell. Full Text Full Text PDF PubMed Scopus Google B. K. X. R. B. X. G. R.R. 2002; Full Text Full Text PDF PubMed Scopus Google R. G. Cell Biol. 2002; PubMed Scopus Google Scholar). However, we a regulation of Wnt genes in in to a G. B. R. Bone PubMed Scopus Google Scholar). In of we up-regulation of and at 3 up-regulation of was confirmed by However, in the two were We that the Wnt pathway is but the exact mechanism of activation remains to be of the Wnt pathway are shown in of the most novel we point to a strong up-regulation of the Hey1 transcription factor at 1 and about at Hey1 to a of transcription factors which to a of transcription factors. The Hey1 has recently been and Hey1, and B. Dev. PubMed Scopus Google Scholar). with are to be direct targets of the Notch signaling pathway G. Cell. Biol. PubMed Scopus Google G. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). up-regulation of the Hey1 transcription factor that the Notch signaling pathway is activated in MC3T3 cells osteogenic of the Notch signaling pathway are shown in We a genes from the gene groups and their expression by an genes were growth factors and and and transcription factors and For the genes the expression by to the by and with osteoblast and we analyzed and confirmed the expression of 20 the of gene expression in In we on expression and of Notch target gene transcription factor To which of the osteogenic stimulus is for Hey1 we analyzed Hey1 MC3T3 cells with of the osteogenic stimulus A was days of the The that Hey1 is a gene, a at we analyzed Hey1 expression in osteoblast differentiation Hey1 is by in murine C2C12 cells, which a to differentiation or osteoblasts The induction was by at days 1 and 3 and about Hey1 expression was by microarrays in human mesenchymal cells stimulated by osteogenic stimulus containing The induction to We analyzed Hey1 expression in in mouse bone by PCR. was and RNA was and Hey1 were high in bone tissue to the in MC3T3 cells and This is from for Hey1 detection and is to of with a of Hey1 induction a of at of Hey1 was in MC3T3 cells by osteogenic in expression at as by We that Hey1 is by in mouse and human cells and is expressed in mouse of the as transcriptional Cell. PubMed Scopus Google and Hey1 differentiation Biol. Full Text Full Text PDF PubMed Scopus Google we the induction of Hey1 could inhibit osteogenic For this we Hey1 up-regulation with a for Hey1 Hey1 was at and this 3 Hey1 in to the we the of this Hey1 on osteoblast early genes However, genes and were by the by for This that Hey1 was involved in regulation of genes expressed early in Therefore, we the of Hey1 on events in differentiation, as bone formation and with osteogenic stimulus in MC3T3 The of MC3T3 cells with for Hey1 this mineralization, while a with These showed that Hey1 has an matrix by osteoblasts and that of Hey1 is to this The to the molecular of osteoblast to the inhibition of Hey1 Runx2 is a transcription factor that an in osteoblast differentiation, and interfering with with in in and D. R. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Many that osteoblast differentiation Runx2 G. Cell. PubMed Scopus Google Scholar). Therefore, we examined the of Hey1 on Runx2 transcriptional a gene by an containing in of a shown in expression of Runx2 stimulated transcription of the gene by the Runx2 gene that was by the which activation by Runx2 was with the containing of Hey1 with Runx2, however, completely abrogated transcription Runx2 showed that a on genes and that gene activity. expression of Runx2 and Hey1 in These that Hey1 bone matrix by osteoblasts by Runx2 activity. of in is a strong in new players in the osteoblast differentiation which is far from completely One to the molecular players in the osteoblast differentiation is to gene expression differentiation, and the of recently been Bone PubMed Scopus Google B. Cell Google 2002; PubMed Scopus Google Bone PubMed Scopus Google D. Cell. PubMed Scopus Google Scholar). These of osteoblast differentiation and differentiation new genes involved in the differentiation the of In this we identified 394 genes with known and 295 to be regulated osteoblast The of this are cellular and molecular of the osteoblast differentiation for analyzed by to to gene expression cell of regulated and and classification of regulated genes to their cellular the most interesting and novel finding we the concomitant regulation of genes of the Wnt, and Notch signaling we to that in the of cellular and of and to new of genes that are regulated osteogenesis and to to a more of showed that activation of the pathway by osteogenic stimulus and more by is achieved by the production of and and of by the up-regulation of target genes and for a for in osteoblast regulation is and of but of action are and in the to on of and cell differentiation J.M. Google Scholar). in osteoblasts Z. 2002; PubMed Scopus Google and differentiation 2000; PubMed Scopus Google Scholar). has been shown that of cells with the of to by to the Cell. Biol. PubMed Google Scholar). on the of the cells this or Cell. Biol. PubMed Google Scholar). Therefore, that the pathway a way to and the direct action of signaling on Wnt interesting finding was the activation of the Wnt pathway by osteogenic stimulus documented by up-regulation of two target transcription factors and the of the Wnt pathway in bone the finding that a for Wnt, is a high bone gene and is in G. D. K. G. B. B. B. K. D. B. B. B. R. Cell. Full Text Full Text PDF PubMed Scopus Google B. K. X. R. B. X. G. R.R. 2002; Full Text Full Text PDF PubMed Scopus Google R. G. Cell Biol. 2002; PubMed Scopus Google Scholar). we showed that was by the osteogenic was shown that activation of an early of osteoblast differentiation, on signaling and that and in Wnt pathway activation G. B. R. Bone PubMed Scopus Google Scholar). We also transcriptional up-regulation of Wnt however, were to and and this activation was in two of Therefore, we that this up-regulation be and that of Wnt pathway activation by need Notch and novel finding was a strong up-regulation of Hey1, a direct Notch target Notch signaling is an mechanism by to cell cell PubMed Scopus Google Scholar). the Notch pathway was to bone by that in the genes a Notch and a Notch signaling K. R. 2000; PubMed Scopus Google Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). was shown that of hematopoietic cells in bone is by activation of Notch pathway by a by to a interaction bone and bone J.M. PubMed Scopus Google Scholar). far there been two Notch signaling in of the and K. K. Bone 2002; PubMed Scopus Google PubMed Scopus Google Scholar). We that the Notch pathway is activated by osteogenic stimulus and on a strong up-regulation of Hey1 transcription However, we for the Notch and in MC3T3 cells that are expressed or Notch activation is achieved via R. Dev. Biol. 2000; PubMed Scopus Google a high could be for Another for of Notch activation is with gene a growth factor from the of tissue growth factor and gene B. Full Text Full Text PDF PubMed Scopus Google Scholar). was shown that with in C2C12 cells and to induction of of target Notch genes and K. R. B. K. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). This activation led to an inhibition of differentiation, an to the of we that expression is high in MC3T3 cells, and is in cells differentiation in with and A of gene activation on Wnt, and Notch is shown in is the of Notch pathway activation in cell of Notch differentiation of and cells and of hematopoietic cells K. R. B. K. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Z. J.M. G. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google D. G. PubMed Google Scholar). shown that and Notch signaling differentiation of and cells and that Hey1 is a player in this G. K. PubMed Scopus Google G. PubMed Scopus Google Scholar). in osteoblast cells that inhibition of Hey1 expression to matrix mineralization, a of their by Therefore, in the as the Notch pathway to a of cell of and osteogenesis that cells are this pathway to their the Notch pathway to a at the of the is the mechanism of action of One of the players in the osteoblast differentiation is the Runx2 transcription factor, which involved in osteoblast differentiation G. Cell. PubMed Scopus Google Scholar). two transcription factors were shown to with Runx2 and to inhibit osteoblast G. Dev. PubMed Scopus Google and K. X. D. K. G. Dev. Cell. Full Text Full Text PDF Scopus Google Scholar). that Hey1 can completely Runx2 transcriptional that Hey1 is a novel of In in this we identified a of genes that were regulated in differentiation and that were known for their in this or were The of activated genes showed that osteogenic in to the activation signaling of Wnt, and Notch we a negative of a Notch target gene, Hey1, in the osteogenic novel in osteoblast We in the of and in the of for in and the human mesenchymal cell and with and for and for gene and expression and for the in and a in for with