Structural Coupling of Smad and Runx2 for Execution of the BMP2 Osteogenic Signal

Abstract

Two regulatory pathways, bone morphogenetic protein (BMP)/transforming growth factor-β (TGFβ) and the transcription factor RUNX2, are required for bone formation in vivo. Here we show the interdependent requirement of these pathways to induce an osteogenic program. A panel of Runx2 deletion and point mutants was used to examine RUNX2-SMAD protein-protein interaction and the biological consequences on BMP2-induced osteogenic signaling determined in Runx2 null cells. These cells do not respond to BMP2 signal in the absence of Runx2. We established that a triple mutation in the C-terminal domain of RUNX2, HTY (426-428), disrupts the RUNX2-SMAD interaction, is deficient in its ability to integrate the BMP2/TGFβ signal on promoter reporter assays, and is only marginally functional in promoting early stages of osteoblast differentiation. Furthermore, the HTY mutation overlaps the unique nuclear matrix targeting signal of Runx factors and exhibits reduced subnuclear targeting. Thus, formation of a RUNX2-SMAD osteogenic complex and subnuclear targeting are structurally and functionally inseparable. Our results establish the critical residues of RUNX2 for execution and completion of BMP2 signaling for osteoblastogenesis through a mechanism that requires RUNX2-SMAD transcriptional activity. Two regulatory pathways, bone morphogenetic protein (BMP)/transforming growth factor-β (TGFβ) and the transcription factor RUNX2, are required for bone formation in vivo. Here we show the interdependent requirement of these pathways to induce an osteogenic program. A panel of Runx2 deletion and point mutants was used to examine RUNX2-SMAD protein-protein interaction and the biological consequences on BMP2-induced osteogenic signaling determined in Runx2 null cells. These cells do not respond to BMP2 signal in the absence of Runx2. We established that a triple mutation in the C-terminal domain of RUNX2, HTY (426-428), disrupts the RUNX2-SMAD interaction, is deficient in its ability to integrate the BMP2/TGFβ signal on promoter reporter assays, and is only marginally functional in promoting early stages of osteoblast differentiation. Furthermore, the HTY mutation overlaps the unique nuclear matrix targeting signal of Runx factors and exhibits reduced subnuclear targeting. Thus, formation of a RUNX2-SMAD osteogenic complex and subnuclear targeting are structurally and functionally inseparable. Our results establish the critical residues of RUNX2 for execution and completion of BMP2 signaling for osteoblastogenesis through a mechanism that requires RUNX2-SMAD transcriptional activity. Skeletal development and bone formation require coordinated activities of multiple signaling pathways that include bone morphogenetic protein 2 (BMP2) 5The abbreviations used are:BMPbone morphogenetic proteinTGFβtransforming growth factor-βRunx2runt-related transcription factor 2RHDrunt homology domainSMIDSMAD interacting domainALPalkaline phosphataseNMTSnuclear matrix targeting signalαMEMα minimal essential mediumHAhemagglutininm.o.i.multiplicity of infectionWTwild type. and transforming growth factor-β (TGFβ). Transduction of these signals results in the activation of target genes that are essential for bone development. Specific receptor-regulated SMADs (R-SMADs) serve as substrates for the BMP and TGFβ/activin/Nodal receptors. SMAD-1, -2, -3, and -5 transduce, whereas Smad-4 serves as a common partner for all R-SMADs to provide the DNA binding property (1Liu F. Hata A. Baker J.C. Doody J. Carcamo J. Harland R.M. Massague J. 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RUNX2 point mutants and with a the Runx2 as the common an whereas the for for for for and for the for for for for and for with the common an and for and as to a the the the with and the with are the as The the with and the A was to RUNX2 was used as and the are as for The of and the of all the by an and C-terminal deletion of RUNX2 are F. Pratap J. Ito K. Ito Y. Stein J.L. van Wijnen A.J. Stein G.S. Lian J.B. Javed A. J. Cell. 2005; PubMed Scopus Google Scholar). The and RUNX2 by the Runx2 by a common and a common an with and of mutation and the of the of the by DNA The and transcriptional activity of all in cells. The with and cells with the DNA for and by The with to its and cells to The cells by and was by and used for The multiplicity of was used for and point mutants of Runx2 to an of and in Runx2 null cells and and promoter reporter assays, cells in with of Runx2 of of the Runx promoter with and of reporter in medium by the DNA with of and for The in of complete medium and on cells. with with and for an in the absence of with TGFβ was to in reporter and activity was determined a with null cells a of cells well of a with the Runx2 and control in cells in by of complete in the absence of BMP2 for an Two infection cells with osteogenic and and cells for of in in for and was control and Runx2 null cells of in the and absence of with and with to to the was with to DNA and a was used to of was to to and of was by for the to in The used for S. R. Pratap J. R. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Javed A. J. Cell. Biochem. 2007; PubMed Scopus Google Scholar). with and in the absence of BMP2 for and in and in in for with the all for the and The was to by the of and the was and to the The for development was The with and cells an and cells a of cells with of RUNX2 and BMP2 whereas for TGFβ Cell in and by for and was of by for and of the was as with of Runx2 and on a for with and with and by on a with Runx2 and and with and of and cells in with of and Runx2 and as for of nuclear as A. Stein J.L. L. Lian J.B. Stein G.S. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). of nuclear protein determined by and and a Runx binding was used for and are A. Stein J.L. L. Lian J.B. Stein G.S. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). and of the is as A. Stein J.L. L. Lian J.B. Stein G.S. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). and RUNX2 with for and a to for cells a of on in with of Runx2 and for BMP2 was for in as as A. B. S. J.-Y. J. S. Stein J.L. Lian J.B. van Wijnen A.J. Stein G.S. J. Cell Sci. 2000; PubMed Google Scholar). RUNX2 was by a and SMAD with a a of used and a of a with a and with of in was A with and with was used cells with of proteins Cell with in was and of Runx2 and SMAD and The was and HTY of ten cells that are Specific the RUNX2-SMAD to the that a critical of RUNX2 in bone formation and in the of osteoblast on its interaction with SMAD proteins in to BMP2 and TGFβ We by C-terminal deletion studies the of a in RUNX2, overlaps the well characterized nuclear matrix targeting signal as in We the and HTY residues that the of and 2 in the These residues are and L. B. Javed A. J.-Y. S. Lian J.B. van Wijnen A.J. Stein J.L. Stein G.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google the and C-terminal of the RUNX2 and studies a interaction of with the triple of residues in the HTY mutation in 2 in complete of The mutation of results in complete of the RUNX2-SMAD interaction, to the RUNX2, is as control the mutation and on SMAD of and RUNX2 proteins are in RUNX2 interaction with the and studies not Thus, show the HTY in 2 of the L. B. Javed A. J.-Y. S. Lian J.B. van Wijnen A.J. Stein J.L. Stein G.S. J. Biol. Full Text Full Text PDF PubMed Scopus Google is required for formation of a RUNX2-SMAD the critical the RUNX2 HTY for SMAD interaction, we a panel of RUNX2 residues of the HTY and in We the and in of the RUNX2 proteins proteins and a nuclear to the RUNX2 Thus, of HTY with not the nuclear of the RUNX2 studies that with protein is of residues are of the RUNX2-SMAD interaction is only all are to deletion of the domain of RUNX2 A and The of all and are the minimal required for formation of the cells with of and Runx2 in the of of BMP2 and with RUNX2 as HTY and RUNX2 protein to of RUNX2-SMAD complex formation is of is only for HTY RUNX2 cells with of and HTY Runx2 in the of TGFβ BMP2 and for in cells with of proteins for of RUNX2 and SMAD by and a is The the of for and and HTY and We the consequences of HTY mutation on in of RUNX2-SMAD by wild HTY protein is the by the is in its ability to with protein an a of is RUNX2 protein and and HTY protein exhibits a of only and with and HTY mutation disrupts and in with these establish the HTY as a minimal point that is required for formation of the RUNX2-SMAD regulatory complex in to Mutations in of the on Gene the functional consequences of disruption of the RUNX2-SMAD interaction, we the ability of mutants to RUNX2 target DNA binding of of the of the HTY with cells with the Runx2 with an Runx cells lack RUNX2 RUNX2 DNA binding of and proteins RUNX2 proteins DNA binding activities results with in and and RUNX2 proteins not with their DNA binding in transcriptional activation of a promoter reporter was and all RUNX2 proteins promoter activity to with the RUNX2-SMAD transcriptional activation was a promoter that DNA binding sites for Runx and to TGFβ in the of with the HTY RUNX2 reduced TGFβ whereas a complete was for RUNX2 TGFβ of HTY and proteins is with of RUNX2-SMAD these that mutation of the HTY in RUNX2 not DNA binding transcriptional activation of the regulatory signal for target of the RUNX2-SMAD BMP2-induced to the and and of cells are with bone morphogenetic protein interaction of BMP SMADs with RUNX2 a of the BMP2 signal the that osteoblast is on the RUNX2-SMAD we a biological a characterized Runx2 null (15Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.-H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3678) Google Scholar, S. R. Pratap J. R. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Javed A. J. Cell. Biochem. 2007; PubMed Scopus Google Scholar). These cells with RUNX2 proteins and for in the absence of BMP2 as in of and RUNX2 proteins are for an to by 2 of the bone early of in osteogenic was determined by and Runx2 null cells are to in to BMP2 with control as by the lack of activity in control cells. of RUNX2 RUNX2 protein to RUNX2 subnuclear targeting S.K. A.J. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Proc. Natl. Acad. Sci., U. S. A. 2002; PubMed Scopus Google by infection the osteoblast with activity A in activity was was to BMP2 cells with HTY RUNX2 of activity in the absence of BMP2 These are not to in to the of protein as and not We do a in with HTY with BMP2 not these The and RUNX2 of osteogenic and BMP signal by commitment of Runx2 null cells to osteoblast was for Runx2 null cells in with of Runx2 and and control of a on the and their with and and for early alkaline phosphatase was as of are activity and BMP2 signal only in and cells. cells with in the and absence of BMP2 in osteogenic and the for of the early for osteoblasts by These suggest HTY RUNX2 protein exhibits DNA binding and transcriptional activation in assays, of SMAD interaction its to induce osteogenic as well as in of of the BMP2 osteogenic signal in a biological The RUNX2-SMAD for the of the of the HTY RUNX2 protein with to stages of osteoblast the osteogenic of the Runx2 null cells by of and osteoblast genes is to DNA a in all with of growth of not and the RUNX2 protein a activation of bone and to the for RUNX2 target genes is reduced to with the HTY to early The RUNX2 the and to RUNX2 target genes that differentiation. Runx2 null cells only through stages of osteoblast with RUNX2 protein that a RUNX2-SMAD we do that the proteins and characterized as proteins that to activation of genes for promoting osteoblast of RUNX2 S. M. Javed A. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. J. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, G. M. M. L. B. Karsenty G. R. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google are by and all RUNX2 These results their osteoblast is not on RUNX2 activities provide that the RUNX2-SMAD interaction is essential for a regulatory signal the BMP2/TGFβ for osteoblast of the osteogenic genes was in to BMP2 A was in the of BMP2 for the and BMP2 is not on RUNX2-SMAD The BMP and for bone RUNX2 target BMP2 is by Runx2 HTY proteins for bone matrix RUNX2, and the transcription factors in cells is by BMP2 N. S. M. K. Dev. Biol. 2007; PubMed Scopus Google Scholar, S. M. B. Montecino M. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Mol. Cell. Biol. 2007; PubMed Scopus Google the of BMP2 BMP2 of osteogenic for target are by and RUNX2 SMAD BMP2 mediated is for and Runx2. We that for on the BMP2 for was in of the BMP2 signal for all genes was reduced in HTY and RUNX2 mutants SMAD interactions are The of a of by on was to as in these that residues in RUNX2 a critical for formation of RUNX2-SMAD regulatory is essential for activation of that osteoblast differentiation. results establish RUNX2 as a point that is required for execution and completion of signaling in Our is the of a critical requirement for RUNX2-SMAD interaction to the osteogenic signal that to osteoblast differentiation. RUNX2 characterized as an essential transcription factor for bone formation and and as osteogenic A. K. K. 2006; PubMed Scopus Google Scholar, B. M. 2000; Google Scholar, J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J. E. C. J. Res. 2004; 19: PubMed Google the of these factors for promoting osteoblast is established We a mechanism by BMP signaling is to an osteogenic signal through the formation of RUNX2-SMAD transcriptional regulatory This mechanism was characterized by of a requirement for mutation of residues to the RUNX2-SMAD and functional This HTY RUNX2 was to osteoblast of Runx2 null cells. The consequences of the HTY RUNX2 mutation on osteoblast of the Runx2 null are to as as that of the RUNX2 with deletion of the The of these mutants in an that the a mineralized skeleton to the of SMAD interaction with RUNX2 J.-Y. Pratap J. Javed A. Zaidi S.K. Xing L. Balint E. Dalamangas S. Boyce B. van Wijnen A.J. Lian J.B. Stein J.L. Jones S.N. Stein G.S. Proc. Natl. Acad. Sci., U. S. A. 2001; PubMed Scopus Google Scholar). The HTY RUNX2 mediates a reduced activation of target and in a the mutants DNA and a BMP2 to transcriptional activity of HTY A of studies is that RUNX2 in the absence of BMP2 is for complete to the osteoblast in the absence of This only established in an early osteogenic lineage of the Runx2 The of a of the Runx2 null mice with a and biological S. R. Pratap J. R. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Javed A. J. Cell. Biochem. 2007; PubMed Scopus Google Scholar). of Runx2 null by BMP2 is on Runx2. These are to cells the (15Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.-H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3678) Google Scholar, Pratap J. Javed A. B. Y. van Wijnen A. Montecino M. Stein G.S. Stein J.L. Lian J.B. J. Cell. Biochem. 2005; PubMed Scopus Google Scholar). a the Runx2 null that respond to BMP2 signal of Runx2 T. Gao Y. K. T. K. T. Y. K. T. Yamaguchi A. J. Cell. 2007; PubMed Scopus Google Scholar). Our to studies with and RUNX2 proteins to the and of the RUNX2 and BMP osteogenic is to that of osteogenic factors and transcription that in these cells are not to the BMP2 for the complete of osteoblast differentiation. the RUNX2 HTY mutation not only disrupts formation of the RUNX2-SMAD complex is to the of RUNX2-SMAD for of the BMP RUNX2 is with the nuclear by a nuclear matrix targeting SMADs the in to a BMP is a requirement for and to RUNX2 to the BMP2 signal to activation of RUNX2 target genes S.K. A.J. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Proc. Natl. Acad. Sci., U. S. A. 2002; PubMed Scopus Google Scholar). of transcriptional regulatory their in subnuclear domains is a of control for several RUNX2 proteins that in the C-terminal J.B. Javed A. Zaidi S.K. Lengner C. Montecino M. van Wijnen A.J. Stein J.L. Stein G.S. Crit. Rev. Eukaryot. Gene Expr. 2004; 14: 1-41Crossref PubMed Google Scholar, 10Schroeder T.M. Jensen E.D. Westendorf J.J. Birth Defects Res. C. Embryo. Today. 2005; 75: 213-225Crossref PubMed Scopus (249) Google the studies establish that the osteogenic of BMP2 are for by RUNX2-SMAD interactions and suggest that these interactions to the in bone in human and studies provide that RUNX2 not require BMP2 for of osteoblast BMP2 requires RUNX2 for of osteoblast differentiation. The of SMAD by the HTY mutation with the of to with proteins in subnuclear a critical structural requirement for RUNX2-SMAD interactions in subnuclear as for signaling to the control of osteoblast differentiation. and established in skeletogenesis A. K. K. 2006; PubMed Scopus Google Scholar). We that the RUNX2-SMAD interaction is a mechanism for of the osteogenic activity of the BMP that the development of and and 2003; PubMed Scopus Google Scholar, M. G. C. Massague J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: PubMed Scopus Google Scholar, S. M. H. M. T. Nakashima K. A. T. Mol. Cell. Biol. 2007; PubMed Scopus Google Scholar, Y. P.B. Y. H. J. Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). of skeletal lineage cells requires coordinated activity of RUNX2 and in the absence of the RUNX2 osteogenic signaling of BMP pathways is