Scleraxis and NFATc Regulate the Expression of the Pro-α1(I) Collagen Gene in Tendon Fibroblasts

Abstract

The combinatorial action of separate cis-acting elements controls the cell-specific expression of type I collagen genes. In particular, we have shown that two short elements located between -3.2 and -2.3 kb and named TSE1 and TSE2 are needed for expression of the mouse COL1a1 gene in tendon fibroblasts. In this study, we analyzed the trans-acting factors binding to TSE1 and TSE2. Gel shift experiments showed that scleraxis (SCX), which is a basic helix-loop-helix transcription factor that is expressed selectively in tendon fibroblasts, binds TSE2, preferentially as a SCX/E47 heterodimer. In transfection experiments, overexpression of SCX and E47 strongly enhanced the activity of reporter constructs harboring either four copies of TSE2 cloned upstream of the COL1a1 minimal promoter or a 3.2-kb segment of the COL1a1 proximal promoter. Analysis of TSE1 showed that it contains a consensus binding site for NFATc transcription factors. This led us to show that the NFATc4 gene is expressed in tendons of developing mouse limbs and in TT-D6 cells, a cell line that has characteristics of tendon fibroblasts. In gel shift assays, TSE1 bound NFATc proteins present in nuclear extracts from TT-D6 cells. In transfection experiments, overexpression of NFATc transactivated a reporter construct harboring four copies of TSE1 cloned upstream of the COL1a1 minimal promoter. By contrast, inhibition of the nuclear translocation of NFATc proteins in TT-D6 cells strongly inhibited the expression of the COL1a1 gene. Taken together, these results suggest that SCX and NFATc4 cooperate to activate the COL1a1 gene specifically in tendon fibroblasts. The combinatorial action of separate cis-acting elements controls the cell-specific expression of type I collagen genes. In particular, we have shown that two short elements located between -3.2 and -2.3 kb and named TSE1 and TSE2 are needed for expression of the mouse COL1a1 gene in tendon fibroblasts. In this study, we analyzed the trans-acting factors binding to TSE1 and TSE2. Gel shift experiments showed that scleraxis (SCX), which is a basic helix-loop-helix transcription factor that is expressed selectively in tendon fibroblasts, binds TSE2, preferentially as a SCX/E47 heterodimer. In transfection experiments, overexpression of SCX and E47 strongly enhanced the activity of reporter constructs harboring either four copies of TSE2 cloned upstream of the COL1a1 minimal promoter or a 3.2-kb segment of the COL1a1 proximal promoter. Analysis of TSE1 showed that it contains a consensus binding site for NFATc transcription factors. This led us to show that the NFATc4 gene is expressed in tendons of developing mouse limbs and in TT-D6 cells, a cell line that has characteristics of tendon fibroblasts. In gel shift assays, TSE1 bound NFATc proteins present in nuclear extracts from TT-D6 cells. In transfection experiments, overexpression of NFATc transactivated a reporter construct harboring four copies of TSE1 cloned upstream of the COL1a1 minimal promoter. By contrast, inhibition of the nuclear translocation of NFATc proteins in TT-D6 cells strongly inhibited the expression of the COL1a1 gene. Taken together, these results suggest that SCX and NFATc4 cooperate to activate the COL1a1 gene specifically in tendon fibroblasts. Type I collagen is the most abundant extracellular matrix protein in vertebrates. It is composed of two α1 chains and one α2 chain that are coiled around each other in an uninterrupted triple helix. In the extracellular space, type I collagen molecules self-assemble into highly organized fibrils and then into fibers. The high tensile strength of these fibers is essential to provide mechanical strength to tissues and in particular to bone, tendon, and ligament. This is exemplified by the dramatic consequences of genetic diseases resulting from mutations in one of the genes encoding type I collagen, such as osteogenesis imperfecta or Ehlers-Danlos syndromes type VIIA and VIIB (1Myllyharju J. Kivirikko K.I. Ann. Med. 2001; 33: 7-21Crossref PubMed Scopus (552) Google Scholar). The hallmark of osteogenesis imperfecta is brittle bones, but the disease can also involve other tissues rich in type I collagen, such as ligaments, tendons, fascia, sclerae, and teeth. Ehlers-Danlos syndromes are characterized by skin hyperextensibility, vascular fragility, and increased ligament elasticity responsible for joint hypermobility. Although type I collagen is widely distributed throughout the body, it is produced by a discrete subset of cells, including fibroblasts, osteoblasts, and odontoblasts. Studies performed using transgenic mice harboring various segments of the mouse, rat, or human type I collagen promoters cloned upstream of a reporter gene have shown that the cell-specific expression of type I collagen genes is based on a modular organization of separate cell-specific cis-acting elements (2Pavlin D. Lichtler A.C. Bedalov A. Kream B.E. Harrison J.R. Thomas H.F. Gronowicz G.A. Clark S.H. Woody C.O. Rowe D.W. J. Cell Biol. 1992; 116: 227-236Crossref PubMed Scopus (123) Google Scholar, 3Liska D.J. Reed M.J. Sage E.H. Bornstein P. J. Cell Biol. 1994; 125: 695-704Crossref PubMed Scopus (60) Google Scholar, 4Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (212) Google Scholar, 5Bou-Gharios G. Garrett L.A. Rossert J. Niederreither K. Eberspaecher H. Smith C. Black C. Crombrugghe B. J. Cell Biol. 1996; 134: 1333-1344Crossref PubMed Scopus (135) Google Scholar). In particular, the segment of the mouse pro-α1(I) collagen (COL1a1) promoter located downstream of -2.3 kb induces high level expression of reporter genes in osteoblasts of transgenic mice, whereas the segment of COL1a1 located between -3.2 and -2.3 kb drives high level expression of the lacZ reporter gene specifically in tendon, ligament, and fascia fibroblasts (4Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (212) Google Scholar, 6Rossert J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (93) Google Scholar, 7Terraz C. Toman D. Delauche M. Ronco P. Rossert J. J. Biol. Chem. 2001; 276: 37011-37019Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 8Terraz C. Brideau G. Ronco P. Rossert J. J. Biol. Chem. 2002; 277: 19019-19026Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Analysis of this latter segment has shown that two short cis-acting elements, named TSE1 4The abbreviations used are: TSE1, tendon-specific element 1; SCX, scleraxis; AgE, E-box-containing sequence of the aggrecan promoter; bHLH, basic helix-loop-helix; CsA, cyclosporin A; LacZ, E. coli β-galactosidase; TSE2, tendon-specific element 2; RT, reverse transcription; ANOVA, analysis of variance; DTT, dithiothreitol; E15.5, embryonic day 15.5. and TSE2, are necessary for activation of the COL1a1 promoter in tendon fibroblasts (8Terraz C. Brideau G. Ronco P. Rossert J. J. Biol. Chem. 2002; 277: 19019-19026Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). TSE2 is located between -2,363 and -2,316 bp. It contains a consensus E-box (CACGTG) that is located at -2,325 bp, and it binds a tendon-specific nuclear protein (8Terraz C. Brideau G. Ronco P. Rossert J. J. Biol. Chem. 2002; 277: 19019-19026Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). TSE1 is located between -2,845 and -2,741 bp, and its 3′-half is necessary for expression of the lacZ reporter gene in tendons of transgenic mice (8Terraz C. Brideau G. Ronco P. Rossert J. J. Biol. Chem. 2002; 277: 19019-19026Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Molecular mechanisms responsible for the differentiation of mesenchymal cells into tendon fibroblasts are still largely unknown (9Edom-Vovard F. D. PubMed Scopus Google Scholar). proteins have a of expression to developing tendons, which that tendon cell The and genes are expressed in tendons, and largely to the tendons and to the tendons G. P. B. A. B. 1995; PubMed Google Scholar, J. J.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). (SCX), which is a of the basic helix-loop-helix of transcription is specifically expressed in tendons and ligaments, it can from cells to fibroblasts P. D. D.J. 1995; PubMed Google Scholar, A. 2001; PubMed Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar). most transcription factors that have a of SCX the gene and binds as or SCX/E47 P. D. D.J. 1995; PubMed Google Scholar, D.J. Biol. PubMed Scopus Google Scholar, C. Biol. PubMed Scopus Google Scholar). it has shown that overexpression of SCX in tendon fibroblasts the expression of the gene C. A. M. Biol. PubMed Scopus Google Scholar). This latter gene a protein that is expressed selectively in tendons, ligaments, and of and can as a of tendon fibroblasts C. PubMed Scopus Google Scholar). a of It has also shown that a of the differentiation of mesenchymal cells into tendon fibroblasts in the of protein that this is in the differentiation of mesenchymal cells into tendon fibroblasts A. G. G. P. H. K. A. S. G. G. D. J. 116: PubMed Scopus Google Scholar). In particular, it the expression of the SCX, and COL1a1 genes. the genes that are of have The NFATc of transcription factors is composed of and the is a of the of these proteins the action of a NFATc proteins are in the in a in to and NFATc and translocation into the to cis-acting elements A. C. PubMed Scopus Google Scholar). Although in cells, of the NFATc are expressed in cell of the and are in such as and 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Cell Biol. 2002; PubMed Scopus Google Scholar). In this study, we that SCX binds TSE2, preferentially as a SCX/E47 and the COL1a1 proximal promoter in transfection also show that proteins of the NFATc are present in tendon fibroblasts, TSE1, and the COL1a1 proximal promoter in transfection inhibition of the nuclear translocation of these proteins strongly the expression of COL1a1 in tendon cells. Taken together, these suggest that SCX and NFATc activate the COL1a1 gene in tendon fibroblasts, and are of a in differentiation of mesenchymal cells into tendon fibroblasts. transfection experiments, various segments of the COL1a1 proximal promoter cloned upstream of the lacZ gene in the reporter (4Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (212) Google Scholar). contains a segment of the COL1a1 promoter from to bp. contains four copies of TSE1 the COL1a1 promoter sequence from to -2,741 cloned upstream of the COL1a1 minimal promoter that from to bp. contains four copies of TSE2 the sequence of the COL1a1 promoter from -2,363 to -2,316 cloned upstream of the COL1a1 minimal promoter. is to that the E-box in TSE2 by using the and the The mouse encoding SCX from E. cloned in the expression which contains an upstream of the to The mouse encoding and E47 from C. of at cloned into the expression and The expression and by C. of the cloned upstream of the reporter used as for transfection Cell fibroblasts and cells from and in at in a cells, a of H. in and at in a TT-D6 tendon fibroblasts in and at in a H. K. M. A. M. Cell PubMed Scopus Google Scholar). Gel gel shift assays, we used nuclear extracts from TT-D6 cells or from cells or and The nuclear extracts as but PubMed Scopus Google Scholar). The by in using the of the coli I The used as or TSE1 TSE2 and the binding of SCX to TSE2, the TSE2 of nuclear extracts from cells for at in a DTT, in the of of The on in SCX binds the sequence of the aggrecan we used it as a H. A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). experiments, a of to the In nuclear extracts from cells and of for at of the binding of NFATc proteins to TSE1, the TSE1 of nuclear extracts from TT-D6 cells, for at in a DTT, of and in the of of NFATc proteins the sequence K. 1995; Full Text PDF PubMed Scopus Google we used it as a The sequence but a used as experiments, of to the The by on in using and the transfection experiments, fibroblasts in and the gene of by transfection experiments, cells various of expression of and of a lacZ reporter for the cells that a reporter cells in DTT, and the and using the reporter gene and the cells a lacZ reporter to to for in transfection the cells, to the of transfection experiments in and at cells in whereas in the of then the and I It using and reverse a an performed the reverse for for each NFATc for using the in the of the to the expressed gene and of the that used in in a experiments, performed in a using the I of and of COL1a1 or of used for are expressed as a of COL1a1 or expression in cells. In experiments, TT-D6 or cells of either cyclosporin or S. J. G. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google the by cell cells or and used for experiments in and at we used nuclear extracts from TT-D6 cells and fibroblasts, which as and cell from cells an expression encoding or NFATc4 as cells or as the cells and in of nuclear proteins or of cell on a a a the mouse or NFATc4 protein and then a by using the In mouse in and for in to as F. B. D. Biol. 2002; PubMed Scopus Google Scholar, C. S. F. P. D. PubMed Scopus (51) Google Scholar). The SCX used as C. S. F. P. D. PubMed Scopus (51) Google Scholar). The for and NFATc4 from the in the of to The by using F. B. D. Biol. 2002; PubMed Scopus Google Scholar). cells in using the chain a and as F. B. D. Biol. 2002; PubMed Scopus Google Scholar). are as performed by by or by as using as TSE2 an E-box the shift have shown that TSE2 binds a tendon-specific nuclear protein and that the binding site for this protein a consensus E-box (CACGTG) located at -2,325 (8Terraz C. Brideau G. Ronco P. Rossert J. J. Biol. Chem. 2002; 277: 19019-19026Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). are to transcription we that TSE2 this we performed gel shift using an TSE2 SCX can as an or as a or SCX/E47 P. D. D.J. 1995; PubMed Google Scholar, D.J. Biol. PubMed Scopus Google this SCX SCX and or SCX and TSE2 SCX E47 but it SCX or SCX, or results gel shift using TSE2 and nuclear extracts from cells or of TSE2 nuclear extracts from cells the of a that nuclear extracts from cells or This that TSE2 binds the The of an at the of SCX us to experiments in the of The of an to the largely the of the In as shown in the by of TSE2 nuclear extracts from cells by of TSE2 but also of is a sequence of the aggrecan promoter that has shown to Taken together, these results that TSE2 can SCX it is The of SCX/E47 to TSE2 the of the COL1a1 of SCX/E47 to TSE2 and the activity of the COL1a1 minimal promoter in transfection expression encoding SCX and E47 a reporter four copies of TSE2 cloned upstream of of the mouse COL1a1 proximal promoter and of the lacZ reporter gene in cells. This segment of the COL1a1 proximal promoter has shown to have characteristics of a minimal promoter J.A. Chen S.S. Eberspaecher H. Smith C.N. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (93) Google Scholar). The cells are embryonic cells that type I collagen but SCX of a and in the of expression of of each of these two expression the of expression of increased of but of E47 experiments then using of contains a 3.2-kb segment of the mouse COL1a1 proximal promoter cloned upstream of the lacZ reporter and it is expressed at high in tendons and of transgenic mice (4Rossert J. Eberspaecher H. de Crombrugghe B. J. Cell Biol. 1995; 129: 1421-1432Crossref PubMed Scopus (212) Google Scholar, 8Terraz C. Brideau G. Ronco P. Rossert J. J. Biol. Chem. 2002; 277: 19019-19026Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). of SCX E47 increased the activity of to and this By contrast, transfection of on and transfection of of SCX also on the activity of in a mesenchymal cell line that type I collagen, of enhanced the expression of This that SCX/E47 to cooperate other nuclear proteins that are present in cells but in cells. the 3.2-kb segment of the COL1a1 proximal promoter contains consensus transfection experiments using a reporter construct in which the E-box of TSE2 The of this E-box by the activation by transfection of of of Taken together, these results that SCX can reporter constructs TSE2, and these to by the binding of SCX/E47 to the E-box of TSE2. SCX/E47 to to the COL1a1 experiments have shown that TSE2 to cooperate tendon-specific cis-acting named TSE1, to reporter gene expression in tendon fibroblasts (8Terraz C. Brideau G. Ronco P. Rossert J. J. Biol. Chem. 2002; 277: 19019-19026Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This led us to the of overexpression of SCX and E47 on in transfection and expression in of cells that in transfection experiments, overexpression of SCX E47 the activity of the lacZ reporter gene as shown in transgenic experiments, SCX to cooperate other elements to its on the COL1a1 it is into the NFATc in analysis of the 3′-half of TSE1 the of binding for transcription factors but a at bp, which to the sequence of a site Chen J. A. PubMed Scopus Google this is between NFATc proteins have shown to a in the of which tendons such as the expression of L.A. H. H. E. J. A. PubMed Scopus Google Scholar, M.J. de C. PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). have also shown to cooperate to the expression of COL1a1 in osteoblasts M. de Crombrugghe B. K. H. Med. PubMed Scopus Google Scholar). to the expression of the NFATc genes in tendon fibroblasts and the of the proteins to TSE1 and the COL1a1 promoter. performed experiments to the of encoding the NFATc proteins in TT-D6 cells. This cell line has from tendons of transgenic mice harboring a of and it has characteristics of tendon fibroblasts H. K. M. A. M. Cell PubMed Scopus Google Scholar). used as and to a NFATc4 are present in this shown in to and but in TT-D6 cells. experiments performed using cell from TT-D6 cells showed that the and NFATc4 proteins expressed in these cells but at The activity of the NFATc proteins is by and the NFATc proteins present in the can and activate the NFATc proteins present in TT-D6 cells present in the we performed experiments using nuclear extracts from TT-D6 cells. extracts from cells expression encoding or NFATc4 used as NFATc4 in nuclear extracts from TT-D6 cells, whereas and proteins and suggest that the NFATc4 protein is and into the in TT-D6 fibroblasts. cells NFATc4 the in of the of NFATc proteins in TT-D6 cells, we performed in experiments to NFATc expression in tendons of mouse The tendons using a for the tendon SCX and shown by the of SCX and NFATc4 expressed in and tendons and and these type I collagen genes expressed in developing tendons NFATc4 also around elements and In these NFATc4 and expression in developing tendons, whereas genes expressed in results show that mouse tendon fibroblasts and a tendon cell line the NFATc4 gene. NFATc TSE1 and the COL1a1 the of NFATc proteins present in TT-D6 cells to TSE1, we performed gel shift of nuclear extracts from TT-D6 cells an TSE1 the of a This by an of TSE1 but by a of the it by a to a site in the mouse promoter but by an in which this binding site Taken together, these results that TSE1 binds NFATc proteins that are present in tendon fibroblasts. the binding of NFATc to TSE1 can the COL1a1 proximal cells an expression encoding or NFATc4 and a reporter construct harboring four copies of TSE1 cloned upstream of the mouse COL1a1 minimal promoter and of the lacZ gene of and NFATc4 increased the activity of Taken together, these that NFATc proteins can TSE1 and the COL1a1 proximal promoter. NFATc the of Type I in the of NFATc proteins to the transcription of we used the By the activity of the of NFATc and its nuclear translocation A. C. PubMed Scopus Google Scholar). The expression of COL1a1 in TT-D6 cells various of analyzed by shown in a dramatic and in the of COL1a1 this TT-D6 cells of is a of COL1a1 and we analyzed the of NFATc proteins to the expression of of TT-D6 cells a and inhibition of the expression of the collagen gene that the inhibition of the expression of COL1a1 by of of NFATc proteins and to of that are of TT-D6 fibroblasts This specifically the between NFATc and S. J. G. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). CsA, we a and in the of COL1a1 of TT-D6 cells also strongly inhibited the expression of the collagen gene NFATc transcription factors to of the expression of COL1a1 and in tendon cells. NFATc Type I in from NFATc proteins are expressed in a of cell we to the expression of the NFATc genes in a cell line from TT-D6 cells and the of and on the expression of COL1a1 in this cell to cells, which is a cell line J. PubMed Google Scholar). experiments showed the of and NFATc4 in fibroblasts the of and NFATc4 proteins in cell and nuclear extracts from cells shown and experiments showed that of cells or an and inhibition of the expression of the COL1a1 and and and NFATc proteins are expressed in fibroblasts from tendon fibroblasts, and also to the expression of type I collagen genes in these cells. Molecular mechanisms the expression of type I collagen genes are still In particular, transcription factors that expression in fibroblasts have By transgenic mice harboring various segments of the COL1a1 proximal promoter cloned upstream of the lacZ reporter we have shown that two short cis-acting elements, named TSE1 and TSE2, are necessary for activation of this promoter specifically in tendon fibroblasts (8Terraz C. Brideau G. Ronco P. Rossert J. J. Biol. Chem. 2002; 277: 19019-19026Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). we have used gel shift to show that SCX can the E-box of TSE2, preferentially as an This is results that this E-box binds a protein expressed selectively in tendon fibroblasts (8Terraz C. Brideau G. Ronco P. Rossert J. J. Biol. Chem. 2002; 277: 19019-19026Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). It is also experiments that SCX can E47 to an E-box sequence of the which is a of a of proteins D.J. Biol. PubMed Scopus Google Scholar). other have shown that SCX can also as a or as an P. D. D.J. 1995; PubMed Google Scholar, H. A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). for other the sequence of the the E-box are to an in the binding of these C. H. M. H. D. Full Text PDF PubMed Scopus Google Scholar). that the binding of SCX to TSE2 the COL1a1 promoter. overexpression of SCX and E47 strongly enhanced the activity of a reporter construct four copies of TSE2 cloned upstream of the COL1a1 minimal promoter in transfection overexpression of these two proteins strongly a reporter construct harboring a 3.2-kb segment of the COL1a1 proximal promoter the E-box of TSE2 in it the of SCX/E47 The that a of this E-box the of SCX is of the of downstream of TSE2 in the COL1a1 proximal promoter. overexpression of SCX or of SCX on the COL1a1 proximal which is in the results of the gel shift experiments Although overexpression of SCX has shown to the expression of the gene in tendon fibroblasts C. A. M. Biol. PubMed Scopus Google gene shown to by SCX in tendon cells. COL1a1 is the of SCX to in tendon fibroblasts. It in transfection experiments performed using cells, overexpression of SCX E47 which that SCX nuclear proteins that are present in cells but in cells. this overexpression of SCX enhanced the expression of the gene in tendon fibroblasts but in cells such as or C. A. M. Biol. PubMed Scopus Google Scholar). the of the NFATc it has that of this of transcription factors have in the and of the F. PubMed Scopus Google Scholar). of mice have shown that are also in other such as and or 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, M. de Crombrugghe B. K. H. Med. PubMed Scopus Google Scholar). TSE1 contains a consensus binding site for NFATc proteins Chen J. A. PubMed Scopus Google we on a for these proteins in COL1a1 in tendon fibroblasts. Analysis of the expression of the NFATc genes showed that NFATc4 is expressed and in tendon fibroblasts. experiments performed using from TT-D6 cells the of NFATc4 and showed that NFATc4 present in nuclear extracts from these cells. In in experiments showed that NFATc4 present in tendons of developing mouse it SCX By contrast, we the of or proteins in nuclear extracts from TT-D6 cells, and in experiments also the in tendons of developing mouse experiments the of and in TT-D6 cells, and of cell showed of the and it that and are present in the of these cells but are and into the activation of NFATc proteins has J. Cell Biol. 2002; PubMed Scopus Google Scholar). tendon fibroblasts to we the of NFATc proteins to TSE1 and the COL1a1 gene. suggest that NFATc proteins the activity of COL1a1 in tendon fibroblasts binding to gel shift showed that TSE1 can NFATc proteins that are present in nuclear extracts from TT-D6 tendon cells. of a TSE1 nuclear extracts from TT-D6 cells the of a that by a to a consensus site but by a This also by a TSE1 in which the consensus binding site for NFATc in transfection experiments, overexpression of NFATc on a reporter construct four copies of TSE1 cloned upstream of the COL1a1 minimal promoter. inhibition of the nuclear translocation of NFATc proteins by two and a dramatic and inhibition of the expression of COL1a1 in TT-D6 tendon cells. is a that has widely used to the nuclear translocation of NFATc transcription factors and it the activity of its and can downstream of J. J. Full Text PDF PubMed Scopus Google Scholar). By contrast, is a that specifically of to NFATc by the between these two proteins and that the of other S. J. G. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). M. de Crombrugghe B. K. H. Med. PubMed Scopus Google have that NFATc proteins the activity of the COL1a1 gene in In particular, have shown the that overexpression of proteins but the activity of the COL1a1 proximal promoter in cells, and that of osteoblasts a that is to and also the of NFATc strongly the expression of COL1a1 M. de Crombrugghe B. K. H. Med. PubMed Scopus Google Scholar). NFATc have on COL1a1 in osteoblasts and tendon fibroblasts. In M. de Crombrugghe B. K. H. Med. PubMed Scopus Google have shown that a transcription factor that a in and differentiation K. G. de Crombrugghe B. 2002; Full Text Full Text PDF PubMed Scopus Google to activate the COL1a1 promoter. NFATc proteins to cooperate to activate the COL1a1 gene in osteoblasts and SCX to activate this gene in tendon fibroblasts. of mice have also shown that is for of L.A. H. H. E. J. A. PubMed Scopus Google Scholar, M.J. de C. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google but its in the differentiation of fibroblasts that also transcription such as H. A. J.A. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google to the differentiation of fibroblasts and expression of suggest that NFATc proteins are also in activation of the COL1a1 gene in cells, have a in activation of the collagen genes in cells. In show that NFATc proteins and SCX activate the COL1a1 gene in tendon fibroblasts binding to TSE1 and TSE2, two transcription factors are of a that induces the differentiation of mesenchymal cells into tendon fibroblasts. are to E. C. and C. for the of and to H. for the of the cell