Extracellular Matrix-mediated Signaling by Dentin Phosphophoryn Involves Activation of the Smad Pathway Independent of Bone Morphogenetic Protein

Abstract

Cells have ingenious mechanisms for interpreting complex signals from their external microenvironment. Previously, we have shown that phosphophoryn (PP) regulates the expression of bone/dentin marker genes via the integrin/MAPK signaling pathway (Jadlowiec, J., Koch, H., Zhang, X., Campbell, P. G., Seyedain, M., and Sfeir, C. (2004) J. Biol. Chem. 279, 53323–53330). We hypothesize that other signaling pathways important for mineralized tissue morphogenesis such as the Smad pathway could be involved in PP signaling. We determined activation of the Smad pathway in human adult mesenchymal stem cells following treatment with recombinant PP (rPP). We observed that PP enhanced phosphorylation of Smad1 within 30 min and Smad1 translocation to the nucleus within 1 h. PP up-regulated the expression of Smad1 target genes, Smad6, Dlx5, and Runx2. The timing of PP activation of Smad1 implies this is a direct effect; however, we also investigated the possible involvement of bone morphogenetic proteins in PP stimulation of the Smad pathway. PP was shown to up-regulate Bmp-2 gene expression 12 h post-treatment with PP, which is much later than initial detection of Smad1 phosphorylation at 30 min. Furthermore, addition of Noggin did not block Smad1 phosphorylation by PP. We propose that PP could signal via the Smad pathway by either directly stimulating the phosphorylation of Smad1 via integrins or other mechanisms. These might include integrin/bone morphogenetic protein receptor interactions or involvement of PP with other growth factors leading to the modulation of intracellular signaling. It is noteworthy that a non-transforming growth factor-β family member activates the Smad pathway. The role of PP in regulating the Smad pathway raises very interesting questions regarding the role of PP during bone and tooth development. Cells have ingenious mechanisms for interpreting complex signals from their external microenvironment. Previously, we have shown that phosphophoryn (PP) regulates the expression of bone/dentin marker genes via the integrin/MAPK signaling pathway (Jadlowiec, J., Koch, H., Zhang, X., Campbell, P. G., Seyedain, M., and Sfeir, C. (2004) J. Biol. Chem. 279, 53323–53330). We hypothesize that other signaling pathways important for mineralized tissue morphogenesis such as the Smad pathway could be involved in PP signaling. We determined activation of the Smad pathway in human adult mesenchymal stem cells following treatment with recombinant PP (rPP). We observed that PP enhanced phosphorylation of Smad1 within 30 min and Smad1 translocation to the nucleus within 1 h. PP up-regulated the expression of Smad1 target genes, Smad6, Dlx5, and Runx2. The timing of PP activation of Smad1 implies this is a direct effect; however, we also investigated the possible involvement of bone morphogenetic proteins in PP stimulation of the Smad pathway. PP was shown to up-regulate Bmp-2 gene expression 12 h post-treatment with PP, which is much later than initial detection of Smad1 phosphorylation at 30 min. Furthermore, addition of Noggin did not block Smad1 phosphorylation by PP. We propose that PP could signal via the Smad pathway by either directly stimulating the phosphorylation of Smad1 via integrins or other mechanisms. These might include integrin/bone morphogenetic protein receptor interactions or involvement of PP with other growth factors leading to the modulation of intracellular signaling. It is noteworthy that a non-transforming growth factor-β family member activates the Smad pathway. The role of PP in regulating the Smad pathway raises very interesting questions regarding the role of PP during bone and tooth development. The ECM relays complex signals from the microenvironment to cells. This directs proliferation, differentiation, and apoptosis for proper tissue development and remodeling. During formation of mineralized tissues such as bone and dentin, the cell utilizes several intracellular signaling pathways representing an interconnected network of proteins with the goal to decipher cues from the ECM. We focused our current work on the role of the non-collagenous proteins of dentin such as phosphophoryn (PP) 2The abbreviations used are: PP, phosphophoryn; rPP, recombinant PP;MAPK, mitogen-activated protein kinase; TGF, transforming growth factor; BMP, bone morphogenetic protein; hMSC, human adult mesenchymal stem cells; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; qPCR, quantitative real-time PCR. 2The abbreviations used are: PP, phosphophoryn; rPP, recombinant PP;MAPK, mitogen-activated protein kinase; TGF, transforming growth factor; BMP, bone morphogenetic protein; hMSC, human adult mesenchymal stem cells; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; qPCR, quantitative real-time PCR. in cell signaling and differentiation. We have determined the involvement of the MAPK pathway in PP's signaling (1Jadlowiec J. Koch H. Zhang X. Campbell P.G. Seyedain M. Sfeir C. J. Biol. Chem. 2004; 279: 53323-53330Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). In the present study, our hypothesis is that the Smad pathway might be involved since many of the genes up-regulated by PP are critical to bone differentiation. The Smad pathway is one such pathway important in signal interpretation from the ECM during osteogenesis/dentinogenesis. Smad proteins are intracellular signaling molecules that are activated by members of the transforming growth factor (TGF-β) family of growth factors which includes the bone morphogenetic proteins (BMPs). Smads are the initial responders to receptor activation of the TGF-β family and have been studied as transcriptional activators of cell differentiation. There are three types of Smads: receptor-regulated (R-Smads), common mediator (Co-Smads), and inhibitory (I-Smads) (2Souchelnytskyi S. Moustakas A. Heldin C.H. Trends Cell Biol. 2002; 12: 304-307Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Signaling initiates with ligand binding to type I and type II receptor serine/threonine kinases on the cell surface. The type II receptor phosphorylates the type I receptor which then phosphorylates the R-Smads (Smad1, Smad2, Smad3, Smad5, and Smad8) within the C-terminal MH2 domain of Smad1/2. Following phosphorylation of the R-Smads (Smad1, Smad5, and Smad8 primarily by BMPs and Smad2 and Smad3 primarily by TGF-β1), heteromeric complexes with Smad4 are formed and translocate to the nucleus where they regulate transcription of target genes together with other nuclear cofactors. Several excellent reviews on Smad signaling are available in the literature and discuss the caveats and specifics of Smad signaling (2Souchelnytskyi S. Moustakas A. Heldin C.H. Trends Cell Biol. 2002; 12: 304-307Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 3Shi Y. Massague J. 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Chem. 2002; 277: 5330-5338Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). The Smad pathway can be influenced by factors other than TGF-β/BMP via the MAPK pathway (25Pera E.M. Ikeda A. Eivers E. De Robertis E.M. Genes Dev. 2003; 17: 3023-3028Crossref PubMed Scopus (338) Google Scholar, 26Kretzschmar M. Doody J. Massague J. Nature. 1997; 389: 618-622Crossref PubMed Scopus (772) Google Scholar). Smad activity may be a point of convergence from complex interconnected signaling networks and could help interpret the diverse messages from the ECM into cell fate decisions (26Kretzschmar M. Doody J. Massague J. Nature. 1997; 389: 618-622Crossref PubMed Scopus (772) Google Scholar). We have recently reported that a non-collagenous protein, PP, has a signaling role in mesenchymal stem cells, osteoblasts (MC3T3-E1), and fibroblasts (NIH3T3) cells (1Jadlowiec J. Koch H. Zhang X. Campbell P.G. Seyedain M. Sfeir C. J. Biol. Chem. 2004; 279: 53323-53330Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). PP is localized in dentin and bone and is a regulator of matrix mineralization (27Qin C. Brunn J.C. Cadena E. Ridall A. H. H. N. J. Res. 2002; PubMed Scopus Google Scholar). PP is highly and of many of where could be as as A. K. Sfeir C. A. J. J. PubMed Scopus Google Scholar). work has that PP activates many of the target genes as BMP-2, and (1Jadlowiec J. Koch H. Zhang X. Campbell P.G. Seyedain M. Sfeir C. J. Biol. Chem. 2004; 279: 53323-53330Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). We that PP activation of the MAPK pathway (1Jadlowiec J. Koch H. Zhang X. Campbell P.G. Seyedain M. Sfeir C. J. Biol. Chem. 2004; 279: 53323-53330Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). We that BMP-2 and PP might be to of the pathway and this could signaling In the present study, we that the Smad pathway is by PP. our we human adult mesenchymal stem cells to recombinant PP and determined the of Smad1 nuclear and transcriptional We that PP regulates the Smad pathway and this may be of from stem cell mesenchymal cell growth and from and to the to the to mesenchymal stem cell and BMP-2 was from was from protein and inhibitors from and I from real-time and and from target gene and from DNA Smad1 was from was from Cell Signaling from was from other from Cell and in at with For and cells in for h to treatment with either or For the of and was In our this was for activation of signal pathways (1Jadlowiec J. Koch H. Zhang X. Campbell P.G. Seyedain M. Sfeir C. J. Biol. Chem. 2004; 279: 53323-53330Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). was in in our protein where the was and was as (1Jadlowiec J. Koch H. Zhang X. Campbell P.G. Seyedain M. Sfeir C. J. Biol. Chem. 2004; 279: 53323-53330Abstract Full Text Full Text PDF PubMed Scopus (131) Google and is in with of Smad1 in as Cells in with either or was the Cells for 30 min and and h. Cells on in in the of The cells then and protein The at at h to of protein and to and with Smad1 by by of and to was of was as on Cells then with either or for and 12 h. at Cells with and in for min. cells in and then with in for min. Cells then with and in a in for 30 min. Cells then for 1 h in in Cells then three in and with a for 1 h. Cells three with and a with on and and a in in to Cells then in with or for 30 min and and h. was with I quantitative real-time was as (1Jadlowiec J. Koch H. Zhang X. Campbell P.G. Seyedain M. Sfeir C. J. Biol. Chem. 2004; 279: 53323-53330Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). for target gene and are shown in expression as with for point on the Scholar). for treatment for the at the point as the target genes to the for and in a of BMP-2 as the of Noggin to BMP-2 activity in hMSC, cells with or for 1 h in the of Noggin been to the and 30 min at to addition to the cells. Smad1 signaling by PP involved BMP-2, cells with PP and Noggin for 30 min and and h. Cell and for by as at three and one is reported as the of three treatment For the of was from three For treatment and target genes the of did not of by was to treatment was PP that the Smad a pathway of was involved in as PP was shown to up-regulate several bone/dentin marker genes (1Jadlowiec J. Koch H. Zhang X. Campbell P.G. Seyedain M. Sfeir C. J. Biol. Chem. 2004; 279: 53323-53330Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). we the activation of the Smad pathway by of the phosphorylation of the BMP-2 on Smad1 nuclear and transcriptional activity following treatment of with PP. We an in Smad1 within 30 min of treatment BMP-2, is a of cells enhanced for at h with The of for activation to be which was with stimulation with was primarily in cells in nuclear translocation within 1 h of treatment with either or of is shown in the nuclear in with for 1 of by for h and then with as or for or 12 h. of in A. of in G. in the was for with for PP Smad1 the activation of Smad pathway during PP signaling we expression of direct target genes of the Smad transcription complex Smad6, Dlx5, and real-time PCR. In hMSC, and gene expression was enhanced following treatment with in gene expression was observed 12 h following stimulation with This to h and then to by h. gene expression was at h following treatment and to at h For genes, gene expression a to however, in expression and for to at and to at We have reported that is up-regulated in by PP h of (1Jadlowiec J. Koch H. Zhang X. Campbell P.G. Seyedain M. Sfeir C. J. Biol. Chem. 2004; 279: 53323-53330Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). In the present study, we have that is up-regulated also at 12 h and is at this to h a of to at h. was not by treatment with this of not BMP-2 treatment within h not which is with our (1Jadlowiec J. Koch H. Zhang X. Campbell P.G. Seyedain M. Sfeir C. J. Biol. Chem. 2004; 279: 53323-53330Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, A.B. Hollinger J.O. Campbell P.G. J. Cell Biochem. 2005; 95: 518-528Crossref PubMed Scopus (134) Google Scholar). In has been shown that of gene expression can expression of H. J.A. Campbell P.G. Cell Dev. 2005; Scholar, J. D. J. C. Campbell P. 2005; PubMed Scopus (24) Google Scholar). BMP-2 by PP activates the Smad pathway via BMP-2, for Bmp-2 gene expression was on with PP In hMSC, Bmp-2 gene expression at to h following Bmp-2 gene expression was enhanced at 12 h and h following treatment with BMP-2 gene expression to h This that PP directly activates the Smad pathway of BMP during to 12 PP of BMP-2 an of Smad1 activation in hMSC, we did not that BMP be directly involved in initial Smad1 activation to timing of activation 30 and that BMP-2 gene expression is not up-regulated at h we the that BMP binding to receptor could be for Smad activation by PP. we BMP binding to receptor by addition of a receptor of BMP signaling. activation of the Smad pathway involved BMP-2 receptor we that Noggin with such a We the of Noggin on of in of Noggin the of to In the of PP, the activation was not in the of Noggin This that PP activates the Smad pathway in a that is of that a protein from the TGF-β family the Smad pathway was We have determined the role of PP in signaling in human mesenchymal stem cells via the MAPK pathway. In the present study, we have the involvement of the Smad pathway in signaling. The current the to the of which direct involvement of non-collagenous proteins of the ECM during of tissue development, and N.S. Tissue Res. 2003; 44: PubMed Google Scholar). enhanced phosphorylation and nuclear translocation of Smad1 with expression of Smad target an of did not block phosphorylation of Smad1 in with PP. BMP-2 receptor binding not to be for of Smad1 by PP. This to that PP not Smad1 by stimulating or BMP-2 or by BMP-2 PP activated Smad1 within 30 min and was at for h. Furthermore, PP did not up-regulate Bmp-2 gene expression h activation of and of Noggin to PP signaling a direct the that PP activates the Smad pathway of on the (1Jadlowiec J. Koch H. Zhang X. Campbell P.G. Seyedain M. Sfeir C. J. Biol. Chem. 2004; 279: 53323-53330Abstract Full Text Full Text PDF PubMed Scopus (131) Google and in this we propose a for PP signaling in is the phosphorylation of Smad1 important for of the also translocate into the nucleus as a complex with the Smad1 nuclear translocation following PP in the the Smad complex was and the expression of Smad1 target genes Smad6, Dlx5, and Runx2. this with of Bmp-2 gene of Noggin to activation by PP this of is an inhibitory Smad and a direct target of signaling. of activation by Smad1 phosphorylation by the BMP we have shown that PP also PP activates as a to block stimulation of Smad and MAPK has been shown to p38 signaling by directly with N. R. H. K. T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). was activated by PP, not that BMP-2 is not the of for of the Smad pathway by PP. It that PP activation of the Smad pathway a as BMP-2, with of the pathway activated from initial phosphorylation by PP at the as BMPs by nuclear translocation a and activation of the Smad target the Smad pathway is not from a BMP PP. It is at this of the and Smad1 may be involved with PP signaling and activation of Smad target genes and other pathways might be In our we have that PP activates the integrin/MAPK signaling (1Jadlowiec J. Koch H. Zhang X. Campbell P.G. Seyedain M. Sfeir C. J. Biol. Chem. 2004; 279: 53323-53330Abstract Full Text Full Text PDF PubMed Scopus (131) Google and this could to possible mechanisms by which PP the Smad pathway. signaling could Smad transcriptional For example, when is by Smad transcriptional activity is in the of BMP Y. Y. M. S. M. K. T. J. Bone Miner. Res. 2001; PubMed Scopus Google Scholar). a integrins and growth factors pathways could be influenced by ECM PP the Smad pathway via this an integrins and BMP or other growth factor These questions are in of the many for the intracellular signaling pathways by ECM (25Pera E.M. Ikeda A. Eivers E. De Robertis E.M. Genes Dev. 2003; 17: 3023-3028Crossref PubMed Scopus (338) Google Scholar, J. A. P. Genes Dev. 2004; PubMed Scopus Google Scholar, J. Genes Dev. 2003; 17: PubMed Scopus Google Scholar, E. Science. 1999; PubMed Scopus Google which are to mineralized tissue The MAPK pathway can the Smad pathway by Smad nuclear the Smad and MAPK pathways was by (26Kretzschmar M. Doody J. Massague J. Nature. 1997; 389: 618-622Crossref PubMed Scopus (772) Google Scholar). The reported that BMP-2 and signals which on Smad1 (26Kretzschmar M. Doody J. Massague J. Nature. 1997; 389: 618-622Crossref PubMed Scopus (772) Google Scholar). of Erk kinases by growth factor growth and growth factor II shown to nuclear translocation of Smads and BMP signaling in Xenopus (25Pera E.M. Ikeda A. Eivers E. De Robertis E.M. Genes Dev. 2003; 17: 3023-3028Crossref PubMed Scopus (338) Google Scholar). this was that Smad1 phosphorylation signals at within the protein and the the of Smad1 in the and Smad1 is important in the transcriptional activity of many genes such as J.L. U. S. A. 2002; PubMed Scopus Google the signaling network of Smads and MAPK and their be investigated to of the for pathways in the and for and is possible that signals from integrins and growth factor could in the nucleus to differentiation Y. Y. M. S. M. K. T. J. Bone Miner. Res. 2001; PubMed Scopus Google Scholar). This is in a the of the growth which was shown to be for differentiation F. S. M. X. X. H. M. K. C. G. Nature. 2005; PubMed Scopus Google Scholar). phosphorylates and regulates the activity of an important transcription factor during differentiation X. K. P. S. T. L. S. P. A. G. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In was recently shown that the transcription factors and differentiation activation of G. Jiang D. C. Y. H. M. X. G. Franceschi R.T. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus (199) Google Scholar). MAPK is also important for phosphorylation and activation of G. Jiang D. P. Benson M.D. K. G. Franceschi R.T. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google which could signaling from PP. The role of activation of MAPK and the to the Smad and other pathways and are not and MAPK and Smad signaling pathways are activated by PP and BMP-2, is possible that PP might to the signaling networks that are by the ECM and regulate bone/dentin These interesting questions to the involvement of signaling with to the MAPK and other signaling factors within the ECM. Cells pathways to decipher signals from the ECM into in cell BMP and PP the MAPK pathway and Smad pathway. is the Furthermore, is the of Smad pathway activation by PP in the of tissue For example, during tooth development many interactions are by BMP signaling the Smad pathway. We could a role of the matrix in the development of the tissue and a possible role in This involvement of non-collagenous matrix proteins in tissue morphogenesis is of During tooth development, PP is by and is localized the mineralization to E. PubMed Scopus Google Scholar, M. M. Biochem. J. PubMed Scopus Google Scholar, M. J. Cell Biol. PubMed Scopus Google Scholar). can a role for PP during the of tooth development where PP signals cells in the microenvironment to into PP might also a later role not in the of and growth also in the of the tooth PP might to signaling to the of ECM proteins during dentin formation and In of signaling pathways such as the Smad and MAPK pathways can of which are activated by PP or other growth factors within the such as In this PP might regulate of signaling differentiation or is This could be in the of tissue as as since the of dentin is that PP is highly C. A. Tissue Res. 1996; PubMed Scopus Google Scholar, C. A. J. Bone Miner. Res. PubMed Scopus Google Scholar, A. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google the role of the to signaling We are the role of phosphorylation of PP on signaling interesting is are the signaling which are activated by diverse into the is the of a of These questions important when the and signals for tissue work is focused on the that might and Smads during PP signaling and PP could in signaling of complex cues from the microenvironment to the cell during tissue development and We and for for the and of the and for of