Role of the α2-Integrin in Osteoblast-specific Gene Expression and Activation of the Osf2 Transcription Factor

Extracellular matrix molecules such as type I collagen are required for the adhesion, migration, proliferation, and differentiation of a number of cell types including osteoblasts. Matrix components often affect cell function by interacting with members of the integrin family of cell surface receptors. Previous work showed that collagen matrix synthesis, induced by addition of ascorbic acid to cells, precedes and is essential for the expression of osteoblast markers and induction of the osteocalcin promoter in murine MC3T3-E1 cells. This later response requires OSE2, the promoter element recognized by Osf2 (also called Cbfa1/AML3/PEBP2αA), a recently identified osteoblast-specific transcription factor. Osteoblasts express several integrins including α2β1 which is a major receptor for type I collagen. This paper examines the role of the α2-integrin subunit in osteocalcin promoter activation and osteoblast differentiation. Disruption of α2-integrin-ECM interactions with a blocking antibody or DGEA peptide containing the cell-binding domain of type I collagen blocked activation of the mouse osteocalcin gene 2 promoter by ascorbic acid as well as induction of endogenous osteocalcin mRNA and mineralization. Furthermore, anti-α2-integrin blocking antibody or peptide reduced ascorbic acid-dependent binding of Osf2 to OSE2 without affecting levels of transcription factor mRNA. Time course studies revealed that ascorbic acid-dependent binding of Osf2 to OSE2 preceded increases in osteocalcin and bone sialoprotein expression and this increase in Osf2 binding was not accompanied by comparable changes in levels of transcription factor mRNA or protein. Taken together, these studies demonstrate that an α2-integrin-collagen interaction is required for activation of Osf2 and induction of osteoblast-specific gene expression. Furthermore, matrix signals may regulate Osf2 through a post-translational pathway or via an accessory factor. Extracellular matrix molecules such as type I collagen are required for the adhesion, migration, proliferation, and differentiation of a number of cell types including osteoblasts. Matrix components often affect cell function by interacting with members of the integrin family of cell surface receptors. Previous work showed that collagen matrix synthesis, induced by addition of ascorbic acid to cells, precedes and is essential for the expression of osteoblast markers and induction of the osteocalcin promoter in murine MC3T3-E1 cells. This later response requires OSE2, the promoter element recognized by Osf2 (also called Cbfa1/AML3/PEBP2αA), a recently identified osteoblast-specific transcription factor. Osteoblasts express several integrins including α2β1 which is a major receptor for type I collagen. This paper examines the role of the α2-integrin subunit in osteocalcin promoter activation and osteoblast differentiation. Disruption of α2-integrin-ECM interactions with a blocking antibody or DGEA peptide containing the cell-binding domain of type I collagen blocked activation of the mouse osteocalcin gene 2 promoter by ascorbic acid as well as induction of endogenous osteocalcin mRNA and mineralization. Furthermore, anti-α2-integrin blocking antibody or peptide reduced ascorbic acid-dependent binding of Osf2 to OSE2 without affecting levels of transcription factor mRNA. Time course studies revealed that ascorbic acid-dependent binding of Osf2 to OSE2 preceded increases in osteocalcin and bone sialoprotein expression and this increase in Osf2 binding was not accompanied by comparable changes in levels of transcription factor mRNA or protein. Taken together, these studies demonstrate that an α2-integrin-collagen interaction is required for activation of Osf2 and induction of osteoblast-specific gene expression. Furthermore, matrix signals may regulate Osf2 through a post-translational pathway or via an accessory factor. extracellular matrix ascorbic acid osteocalcin osteoblast-specific factor 2 core binding factor α mouse osteocalcin gene 2 monoclonal antibody Dulbecco's α-modified Eagle's medium fetal bovine serum phosphate-buffered saline bone sialoprotein mitogen-activated protein kinase fluorescein isothiocyanate. As a cell primarily devoted to matrix production, the osteoblast must have the ability to monitor the composition of the extracellular matrix (ECM)1 it is secreting as well as adapt matrix composition to the changing mechanical needs of bone. Consistent with the concept that there is a dialogue between the osteoblast and its ECM, osteoblast precursors must secrete a collagenous matrix before they will differentiate. Inhibition of collagen synthesis by growing cells in the absence of ascorbic acid (AA) or through the use of specific inhibitors totally blocks osteoblast differentiation (1Gerstenfeld L.C. Chipman S.D. Glowacki J. Lian J.B. Dev. Biol. 1987; 122: 49-60Crossref PubMed Scopus (357) Google Scholar, 2Owen T.A. Aronow M. Shalhoub V. Barone L.M. Wilming L. Tassinari M.S. Kennedy M.B. Pockwinse S. Lian J.B. Stein G.S. J. Cell. Physiol. 1990; 143: 420-430Crossref PubMed Scopus (1377) Google Scholar, 3Franceschi R.T. Iyer B.S. J. Bone Miner. Res. 1992; 7: 235-246Crossref PubMed Scopus (479) Google Scholar, 4McCauley L.K. Koh A.J. Beecher C.A. Cui Y. Rosol T.J. Franceschi R.T. J. Cell. Biochem. 1996; 61: 638-647Crossref PubMed Scopus (81) Google Scholar, 5Ibaraki K. Termine J.D. Whitson S.W. Young M.F. J. Bone Miner. 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The present study was undertaken to evaluate the role of α2-integrin-type I collagen interactions in the control of osteoblast-specific transcription and differentiation. As will be shown, blocking integrin-type I collagen binding prevents activation of the OCN promoter by AA and suppresses binding of Osf2 to OSE2 DNA. Interestingly, the dramatic stimulation of Osf2 activity and osteoblast-specific gene expression by AA is not accompanied by comparable increases in transcription factor mRNA or protein suggesting that the ECM activates this factor through either a post-translational pathway or via an accessory factor. Tissue culture medium and fetal bovine serum from and from and from while the peptide was by the core of the of monoclonal antibodies to the mouse α2-integrin subunit or and α2-integrin antibody from was from was from Osf2 was in a peptide the of the Osf2 (20Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3646) Google This with members of the runt family of transcription factors either or and of course of Osf2 protein levels and binding of Osf2 cell extracts from the cell as control MC3T3-E1 and containing of protein for The was with Osf2 a of Osf2 protein cells in the presence or absence of AA for the cell extracts containing 2 of with Osf2 as in A. of cells as in and as a of nuclear of the major species is by This study of the murine MC3T3-E1 preosteoblast cell cells express levels of osteoblast and a ECM growth in cells a in the presence of AA and of a mouse osteocalcin gene 2 promoter gene that is induced by AA with a course that endogenous OCN mRNA (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. 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Res. 1995; PubMed Scopus Google Scholar). extracts and as (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar). 2 of of nuclear For Osf2 serum was with nuclear extracts for to addition of are as S.D. cell from a experiments and Previous work from this showed that interactions are for osteoblast differentiation and gene expression. Induction of osteoblast markers by AA is blocked by collagen synthesis inhibitors or of the ECM with R.T. Iyer B.S. Cui Y. J. Bone Miner. Res. 1994; 9: PubMed Scopus Google Scholar). In a AA-dependent promoter activation also requires collagen synthesis (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar). is known the of this in the experiments is that osteoblast differentiation requires an α2-integrin-type I collagen This integrin subunit is a of the a major binding site for that in osteoblasts D. Salter S. Dedhar S. Simpson R. J. Bone Miner. Res. 1993; 8: 527-533Crossref PubMed Scopus (230) Google Scholar). As in MC3T3-E1 cells the α2-integrin subunit as by both and of a α2-integrin mRNA with in culture not but not by AA an α2-integrin-type I collagen interaction is required for activation of the osteocalcin specific peptides and antibodies to this and related These experiments cells, a MC3T3-E1 containing of the promoter expression of As growth of these cells in the presence of AA OCN promoter activity as well as endogenous OCN and (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar). Induction of osteocalcin promoter activity was by a DGEA peptide 2 which is a of the domain of type I collagen J. Biol. Chem. Full Text PDF PubMed Google Scholar) or a specific α2-integrin blocking antibody 2 Inhibition was and specific in that a control peptide with the an peptide which the cell-binding domain of fibronectin and related the fibronectin cell-binding domain or an blocking antibody totally the for the DGEA peptide and α2-integrin antibody also to be and inhibitors of the for osteoblast differentiation 2 with inhibitors showed of such as cell or in the of culture of of of of In an showed that AA-dependent activation of the OCN promoter requires OSE2, the element in this promoter that is by Osf2. Furthermore, nuclear extracts from cells to OSE2 binding activity (presumably Osf2) (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar) (also see peptide or antibody blocking of α2-integrin function osteoblast differentiation and activity of the OCN it important to the for this integrin species in Osf2 activation as by its ability to OSE2 This and experiments MC3T3-E1 cells for in the presence or absence of the cells this also with AA (26Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar). for in the presence of AA and the peptides or antibodies before or nuclear extracts DGEA peptide or anti-α2-integrin antibody expression of endogenous OCN mRNA without affecting the Osf2 In binding of nuclear extracts to OSE2 was by both of these and antibody of The species nuclear extracts with OSE2 in Osf2 it was by an The of the was by that it was not to with the species between a cell nuclear and OSE2 (also see for information the of this cells and related mammalian runt domain that are also of binding the OSE2 core (21Levanon D. Negreanu V. Bernstein Y. Bar A. Avivi L. Groner Y. Genomics. 1994; 23: 425-432Crossref PubMed Scopus (381) Google Scholar). Taken together, these experiments demonstrate that AA-dependent induction of the OCN of MC3T3-E1 preosteoblast cells, and stimulation of Osf2 transcription factor binding to OSE2 an α2-integrin-collagen As of α2-integrin binding to collagen blocked osteoblast-specific gene expression and binding of Osf2 to OSE2, these not affect Osf2 mRNA Osf2 mRNA and protein levels are this that an signal increases the of Osf2 without affecting its This was in the experiments in and the between Osf2 mRNA and protein with OSE2 binding activity and osteoblast mRNA expression differentiation. As several required for induction of OCN and R.T. Iyer B.S. J. Bone Miner. Res. 1992; 7: 235-246Crossref PubMed Scopus (479) Google Scholar, G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar, R.T. Iyer B.S. Cui Y. J. Bone Miner. Res. 1994; 9: PubMed Scopus Google Scholar) The increase in was 2 while required for induction of the OCN mRNA. AA OCN and mRNA levels approximately Interestingly, levels of Osf2 mRNA by AA the and AA Osf2 mRNA to Osf2 protein levels in cell extracts In with a (18Banerjee C. McCabe L.R. Choi J.Y. Hiebert S.W. Stein J.L. Stein G.S. Lian J.B. J. Cell. Biochem. 1997; 66: 1-8Crossref PubMed Scopus (398) Google Scholar), antibody a major with species and this and was by the that signal was in extracts from the cells cells, cells, or cells As this antibody also to with the species in cells and AA the of Osf2 of the the of in control as by of was and for and In AA the of the and for this species and The of AA Osf2 mRNA and protein levels is to be with its ability to and binding of nuclear extracts to OSE2 as by OSE2 binding activity was as as AA before in Osf2 and to increase to In the dramatic increase in osteoblast mRNAs, OCN promoter and binding of nuclear extracts to OSE2 is not accompanied by changes in levels of the Osf2 transcription factor. These that AA increases binding and activity of Osf2 without major expression of this protein. This study the role of the α2-integrin a of one of the major collagen in Osf2 osteoblast-specific gene and differentiation. We either a specific α2-integrin blocking antibody or a DGEA peptide that the cell-binding domain of type I collagen to the interaction of cells with type I collagen via studies that an α2-integrin-collagen interaction is necessary for activation of the OCN osteoblast differentiation, and binding of Osf2 to OSE2 and that collagen signals increase binding of Osf2 to while the of this transcription factor. This is the study to a specific integrin species to the activation of osteoblast-specific transcription by Osf2. studies have for integrins in osteoblast differentiation. those integrin species interacting with type I collagen and the antibodies and peptides in the present and showed that binding of to type I is necessary for AA-dependent induction of alkaline phosphatase and down-regulation of transforming growth factor-β receptors in MC3T3-E1 cells (17Takeuchi Y. Nakayama K. Matsumoto T. J. Biol. Chem. 1996; 271: 3938-3944Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In a peptide containing the cell-binding domain of fibronectin was to both bone formation and in a culture J. Bone Miner. Res. 1994; 9: PubMed Scopus Google Scholar). culture of osteoblasts with antibodies or fibronectin peptides containing the cell-binding domain also to differentiation J. D. S. J. Cell Sci. 1996; PubMed Google Scholar). The present work is with the study by and (17Takeuchi Y. Nakayama K. Matsumoto T. J. Biol. Chem. 1996; 271: 3938-3944Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) which and collagen synthesis to osteoblast differentiation. In to for of either the cell-binding domain of fibronectin or the in MC3T3-E1 differentiation, the of to be for the DGEA peptide and possible for this is related to the of cell in these experiments of MC3T3-E1 cells that for ability to express osteoblast markers and to These cells are in that they express Osf2 they are not secreting an ECM or osteoblast markers in the absence of see and of ECM the later of osteoblast differentiation. In or precursors in of as well as cells. of the in the of osteoblast precursors may binding for cell growth or differentiation, but this is in cells. I interactions regulate including activation of T. J. L. A. J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), stimulation of collagen B.M. A.P. Cell. Full Text PDF PubMed Scopus Google Scholar), and formation R. J. Cell Sci. 1995; PubMed Google Scholar), in the pathway Integrins convey information the extracellular environment by as both a between the ECM and via the and and by as signal transduction molecules to to and pathways D. T. Cell Res. 1994; PubMed Scopus Google Scholar, Y. M. L. J. Cell Biol. 1997; PubMed Scopus Google Scholar, A.P. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). and Y. M. T. E. T. Matsumoto T. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) for the of focal adhesion kinase and in induction of alkaline an osteoblast differentiation Inhibition of of focal or of focal adhesion kinase mRNA AA-dependent activation of extracellular kinase and as well as stimulation of alkaline In expression of a alkaline phosphatase activity while of a alkaline phosphatase in the absence of it is not known these also the expression of osteoblast these that integrin activation of or related pathways is an important of the response to ECM. in this study that an is necessary for activation of the osteoblast-specific transcription and gene expression. Of particular interest, the dramatic increase in OCN promoter activity and Osf2 binding to OSE2 was not accompanied by changes in levels of transcription factor mRNA or protein. In of Osf2 by and that these factors the mRNA (20Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3646) Google Scholar, R. Ducy P. Karsenty G. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). are possible Osf2 binding to OSE2 be members of the runt domain family of transcription factors function as of α and subunits the of both subunits a site for Osf2 is one of three α subunits identified in and The in in the K. Yagi H. Bronson R.T. K. T. Deguchi K. Y. Kishimoto T. T. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). Thus, of or of the subunit is one for ECM of Osf2 a for by an In a and T. M. K. K. Y. K. K. Y. Y. T. H. Mol. Cell. Biol. 1996; 16: PubMed Scopus (137) Google Scholar) showed that is in a cell in vivo the of the In addition of also the Furthermore, was to the ability of the Of particular interest, these are in Osf2 (20Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3646) Google Scholar). As was in the present Osf2 three species with molecular which of this transcription factor Interestingly, AA to increase the of the species studies will be required to Osf2 be studies in this are to which of these possible interactions osteoblast-specific gene expression.