Fli-1 Inhibits Collagen Type I Production in Dermal Fibroblasts via an Sp1-dependent Pathway

Abstract

Fibrosis is characterized by the excessive deposition of extracellular matrix (ECM), especially collagen. Because Ets factors are implicated in physiological and pathological ECM remodeling, the aim of this study was to investigate the role of Ets factors in collagen production. We demonstrate that the expression of collagenous proteins and collagen α2(I) (COL1A2) mRNA was inhibited following stable transfection of Fli-1 in dermal fibroblasts. Subsequent analysis of the COL1A2 promoter identified a critical Ets binding site that mediates Fli-1 inhibition. In contrast, Ets-1 stimulates COL1A2 promoter activity. In vitro binding assays demonstrate that both Fli-1 and Ets-1 form DNA-protein complexes with sequences present in COL1A2 promoter. Furthermore, Fli-1 binding to the COL1A2 is enhanced via Sp1-dependent interaction. Studies using Fli-1 dominant interference and DNA binding mutants indicate that Fli-1 inhibition is mediated by both direct (DNA binding) and indirect (via protein-protein interaction) mechanisms and that Sp1 is an important mediator of the Fli-1 function. Furthermore, experiments using the Gal4 system in the context of different cell types as well as experiments with theCOL1A2 promoter in different cell lines demonstrate that the direction and magnitude of the effect of Fli-1 is promoter- and cell context-specific. We propose that Fli-1 inhibitsCOL1A2 promoter activity by competition with Ets-1. In addition, we postulate that another factor (co-repressor) may be required for maximal inhibition after recruitment to the Fli-1-Sp1 complex. We conclude that the ratio of Fli-1 to Ets-1 and the presence of co-regulatory proteins ultimately control ECM production in fibroblasts. Fibrosis is characterized by the excessive deposition of extracellular matrix (ECM), especially collagen. Because Ets factors are implicated in physiological and pathological ECM remodeling, the aim of this study was to investigate the role of Ets factors in collagen production. We demonstrate that the expression of collagenous proteins and collagen α2(I) (COL1A2) mRNA was inhibited following stable transfection of Fli-1 in dermal fibroblasts. Subsequent analysis of the COL1A2 promoter identified a critical Ets binding site that mediates Fli-1 inhibition. In contrast, Ets-1 stimulates COL1A2 promoter activity. In vitro binding assays demonstrate that both Fli-1 and Ets-1 form DNA-protein complexes with sequences present in COL1A2 promoter. Furthermore, Fli-1 binding to the COL1A2 is enhanced via Sp1-dependent interaction. Studies using Fli-1 dominant interference and DNA binding mutants indicate that Fli-1 inhibition is mediated by both direct (DNA binding) and indirect (via protein-protein interaction) mechanisms and that Sp1 is an important mediator of the Fli-1 function. Furthermore, experiments using the Gal4 system in the context of different cell types as well as experiments with theCOL1A2 promoter in different cell lines demonstrate that the direction and magnitude of the effect of Fli-1 is promoter- and cell context-specific. We propose that Fli-1 inhibitsCOL1A2 promoter activity by competition with Ets-1. In addition, we postulate that another factor (co-repressor) may be required for maximal inhibition after recruitment to the Fli-1-Sp1 complex. We conclude that the ratio of Fli-1 to Ets-1 and the presence of co-regulatory proteins ultimately control ECM production in fibroblasts. extracellular matrix type I collagen α2(I) transforming growth factor matrix metalloproteinase Ets binding site electrophoretic mobility-shift assay DNA binding domain base pair GA-binding protein ECM1 remodeling is a complex and tightly regulated process that occurs during embryogenesis, the female reproductive cycle, angiogenesis, and wound repair. However, in the majority of normal adult tissues only limited turnover of the ECM takes place. In contrast, in many pathological conditions the balance between ECM synthesis and degradation is disrupted, leading to abnormal ECM remodeling. Excessive ECM breakdown is associated with rheumatoid arthritis, osteoarthritis and periodontitis as well as tumor invasion and metastasis (1Vincenti M.P. Clark I.M. Brinckerhoff C.E. Arthritis Rheum. 1994; 37: 1115-1126Crossref PubMed Scopus (190) Google Scholar, 2Malemud C.J. Goldberg V.M. Front. Biosci. 1999; 4: D762-D771Crossref PubMed Google Scholar, 3Forget M.A. Desrosiers R.R. Beliveau R. Can. J. Physiol. Pharmacol. 1999; 77: 465-480Crossref PubMed Scopus (142) Google Scholar, 4Curran S. Murray G.I. J. Pathol. 1999; 189: 300-308Crossref PubMed Scopus (611) Google Scholar), whereas excessive ECM deposition occurs in fibrotic diseases such as scleroderma, liver cirrhosis, and glomerulosclerosis (5Trojanowska M. LeRoy E.C. Eckes B. Krieg T. J. Mol. Med. 1998; 76: 266-274Crossref PubMed Scopus (150) Google Scholar, 6Friedman S.L. N. Engl. J. Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar, 7Border W.A. Yamamoto T. Noble N.A. Diabetes Metab. Rev. 1996; 12: 309-339Crossref PubMed Scopus (97) Google Scholar). The type I collagen, a product of two coordinately regulated genes, α1(I) and α2(I) (COL1A2), is a predominant ECM component of the fibrotic lesion. Studies using diverse experimental systems have indicated that transcriptional regulation of the type I collagen genes is species- and tissue-specific (5Trojanowska M. LeRoy E.C. Eckes B. Krieg T. J. Mol. Med. 1998; 76: 266-274Crossref PubMed Scopus (150) Google Scholar). TheCOL1A2 promoter has been extensively used as an experimental model system to delineate transcriptional regulation of the collagen gene in human tissues. This promoter constitutively exhibits high levels of expression in activated dermal fibroblasts, which are “turned on” by routine in vitro tissue culture conditions such as growth on plastic in the presence of serum. Previous studies have identified the ubiquitous factors, Sp1 and Sp3, and CAAT binding factor as primary activators of this promoter (9Ihn H. Trojanowska M. Nucleic Acids Res. 1997; 25: 3712-3717Crossref PubMed Scopus (98) Google Scholar, 10Ihn H. Ohnishi K. Tamaki T. LeRoy E.C. Trojanowska M. J. Biol. Chem. 1996; 271: 26717-26723Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). In contrast, very little is currently known about the nature of the transcriptional repressors that may counteract or modulate the potent stimulatory effects of Sp1 and Sp3 on this promoter. The activity of the COL1A2 promoter can be modulated further by transcriptional factors mediating cytokine responses. Thus, Smad3 in cooperation with p300/CREB-binding protein mediates activation of this promoter by TGF-β (8Zhang W. Ou J. Inagaki Y. Greenwel P. Ramirez F. J. Biol. Chem. 2000; 275: 39237-39245Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 11Ghosh A.K. Yuan W. Mori Y. Varga J. Oncogene. 2000; 19: 3546-3555Crossref PubMed Scopus (201) Google Scholar, 12Chen S.J. Yuan W. Mori Y. Levenson A. Trojanowska M. Varga J. J. Invest. Dermatol. 1999; 112: 49-57Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 13Chen S.J. Yuan W. Lo S. Trojanowska M. Varga J. J. Cell. Physiol. 2000; 183: 381-392Crossref PubMed Scopus (171) Google Scholar), whereas CAAT/enhancer-binding protein as well as nuclear factor-κB seem to be involved in tumor necrosis factor-α inhibition of the COL1A2 activity (14Kouba D.J. Chung K.Y. Nishiyama T. Vindevoghel L. Kon A. Klement J.F. Uitto J. Mauviel A. J. Immunol. 1999; 162: 4226-4234PubMed Google Scholar, 15Greenwel P. Tanaka S. Penkov D. Zhang W. Olive M. Moll J. Vinson C. Di Liberto M. Ramirez F. Mol. Cell. Biol. 2000; 20: 912-918Crossref PubMed Scopus (144) Google Scholar). Moreover, recent studies provide evidence for Sp1 as a critical mediator of TGF-β induction of this promoter via interaction with Smad3 (8Zhang W. Ou J. Inagaki Y. Greenwel P. Ramirez F. J. Biol. Chem. 2000; 275: 39237-39245Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 16Poncelet A.C. Schnaper H.W. J. Biol. Chem. 2000; 276: 6983-6992Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Ets proteins comprise a family of transcription factors with over 30 members identified to date that are characterized by the presence of a highly conserved DNA binding domain termed the Ets domain (17Graves B.J. Petersen J.M. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar). Ets proteins are grouped into subfamilies based on the sequence similarity of the Ets domain and its location within the protein as well as additional sequence similarities (17Graves B.J. Petersen J.M. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar). Fli-1 and the closely related Erg are the members of the ERG subfamily. Extensive in vitrostudies as well as the data obtained from various Fli-1 null mice support a crucial role for Fli-1 in hematopoiesis (18Spyropoulos D.D. Pharr P.N. Lavenburg K.R. Jackers P. Papas T.S. Ogawa M. Watson D.K. Mol. Cell. Biol. 2000; 20: 5643-5652Crossref PubMed Scopus (255) Google Scholar). Fli-1 is also expressed in embryonic tissues at the sites of cell migration and epithelial-mesenchymal transition, suggesting additional functions during development (19Mager A.M. Grapin-Botton A. Ladjali K. Meyer D. Wolff C.M. Stiegler P. Bonnin M.A. Remy P. Int. J. Dev. Biol. 1998; 42: 561-572PubMed Google Scholar). It may be relevant that Fli-1 is an inducer of tenascin-C, an ECM molecule, which is also expressed at the sites of cell migration (20Shirasaki F. Makhluf H.A. LeRoy C. Watson D.K. Trojanowska M. Oncogene. 1999; 18: 7755-7764Crossref PubMed Scopus (85) Google Scholar). However, the specific role of Fli-1 in these processes is presently not well understood. Targeted disruption of the Fli-1 gene resulted in hemorrhage into the neural tube and the ventricles of the brain at E11, resulting in embryonic death shortly thereafter. In addition to hematopoietic defects, these mutant embryos demonstrated a disruption of basement membrane tissues (18Spyropoulos D.D. Pharr P.N. Lavenburg K.R. Jackers P. Papas T.S. Ogawa M. Watson D.K. Mol. Cell. Biol. 2000; 20: 5643-5652Crossref PubMed Scopus (255) Google Scholar). There is increasing evidence that some Ets proteins, particularly the members of Ets-1 and E1AF/PEA3 subfamilies, play an important role in regulating transcriptional activation of ECM-degrading enzymes including serine proteases and matrix metalloproteinases (MMPs) (21Calmels T.P. Mattot V. Wernert N. Vandenbunder B. Stehelin D. Biol. Cell. 1995; 84: 53-61Crossref PubMed Scopus (28) Google Scholar,22Bolon I. Gouyer V. Devouassoux M. Vandenbunder B. Wernert N. Moro D. C. J. Pathol. 1995; Google Scholar). Extensive studies of the as well as and have a for the role of Ets in the regulation of this of genes C. A. R. B. J. PubMed Scopus Google Scholar). Ets binding and The members of the Ets and that to the are also of the protein B. J. A. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the transcriptional activation of this of genes on the nature of proteins Ets and are as well as the the L. D. A. D. P. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). on recent that a matrix tenascin-C, is also regulated by Ets factors (20Shirasaki F. Makhluf H.A. LeRoy C. Watson D.K. Trojanowska M. Oncogene. 1999; 18: 7755-7764Crossref PubMed Scopus (85) Google Scholar), we that the role of Ets in ECM remodeling may not be limited to regulation of the may also regulation of synthesis of the ECM proteins M. Oncogene. 2000; 19: PubMed Scopus Google Scholar). this we a well characterized system with physiological and pathological the In the present study we demonstrate that the collagen gene is a for Ets we provide evidence that Fli-1 as a transcriptional of this whereas Ets-1 as an Furthermore, we that context an important role in both the magnitude and direction of to dermal from obtained from the of tissue was by type I and in with from and from the Fli-1 and control stable in human by the with Fli-1 or control and in the presence of of and for the expression of Fli-1 The analysis of into protein was as Y. LeRoy E.C. Trojanowska M. J. 1997; Google Scholar). in and to The was to with and for the of the a in activity was for The was from and the and The was in a and in and to The of for was to the cell of enhanced by in and by the levels of expression of Ets-1 and Fli-1 proteins, cell nuclear in and membrane with the with primary by with and with complexes using enhanced The was obtained from the was obtained from and the was (18Spyropoulos D.D. Pharr P.N. Lavenburg K.R. Jackers P. Papas T.S. Ogawa M. Watson D.K. Mol. Cell. Biol. 2000; 20: 5643-5652Crossref PubMed Scopus (255) Google Scholar). was from and by as Y. LeRoy E.C. Trojanowska M. J. 1997; Google Scholar). with for α2(I) and The with a The COL1A2 sequences between and to the gene was H. Ohnishi K. Tamaki T. LeRoy E.C. Trojanowska M. J. Biol. Chem. 1996; 271: 26717-26723Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The was by the to promoter into The promoter mutants by in the sequence with using the promoter as a with the of the to the used in electrophoretic mobility-shift assays The COL1A2 promoter binding site was as K.Y. A. Uitto J. Mauviel A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). a that DNA DNA binding domain and (20Shirasaki F. Makhluf H.A. LeRoy C. Watson D.K. Trojanowska M. Oncogene. 1999; 18: 7755-7764Crossref PubMed Scopus (85) Google Scholar). and a of The was from of the mutants in the in a with the indicated and control in in using to the at using two different and in of protein to and Nucleic Acids Res. 19: PubMed Scopus Google with vitro and human and proteins using the system as T. Ohnishi K. C. LeRoy E.C. Trojanowska M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). of nuclear for 30 on in of the binding and and of In some or with nuclear 30 to the addition of a and from and was from was characterized (18Spyropoulos D.D. Pharr P.N. Lavenburg K.R. Jackers P. Papas T.S. Ogawa M. Watson D.K. Mol. Cell. Biol. 2000; 20: 5643-5652Crossref PubMed Scopus (255) Google Scholar). The of DNA from DNA-protein complexes was on a was in at at The and to at The or was used to In the of experiments we Fli-1 ECM production by stable Fli-1 in human dermal fibroblasts. that levels of Fli-1 protein for further of Fli-1 in dermal not the effect of Fli-1 on the production of collagenous proteins, from with was by in expression of Fli-1 protein with the production of collagenous data indicate that expression of Fli-1 has a potent effect on the collagenous levels of the collagenous proteins in may from the activation of the collagen or Fli-1 collagen gene COL1A2 mRNA levels in Fli-1 stable and control in and Fli-1 COL1A2 mRNA levels by at suggesting that its effects occurs at via regulating collagen mRNA expression The effect of Fli-1 on the activity of the COL1A2 promoter was with the effect of Fli-1 on the COL1A2 mRNA Fli-1 inhibited COL1A2 promoter activity in a and the Fli-1 in the COL1A2 we promoter H. Ohnishi K. Tamaki T. LeRoy E.C. Trojanowska M. J. Biol. Chem. 1996; 271: 26717-26723Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The inhibition was with the whereas the was inhibited about The the and by Fli-1 Thus, the effect of Fli-1 was mediated by at two COL1A2 promoter between and and the between and Because the of the COL1A2 promoter is well we on this to further delineate the of theCOL1A2 by of the sequence of this promoter the presence of Fli-1 at and are within the identified to that Sp1 binding sites T. Ohnishi K. C. LeRoy E.C. Trojanowska M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). which of the for mediating the Fli-1 the and in and The in transfection in the Fli-1 inhibition of theCOL1A2 whereas the in Because Ets factors are known to with in the regulation of and 1996; PubMed Scopus Google Scholar), we the characterized site K.Y. A. Uitto J. Mauviel A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google in the be involved in the Fli-1 inhibition. The effect of Fli-1 on the COL1A2 promoter was not by of the characterized However, the COL1A2 promoter in the binding sites to the effects of Fli-1 This is Sp1 and Sp3 have been to be potent activators of this promoter and have resulted in a further of the promoter activity. In promoter in or binding sites in a of the promoter only in the binding sites the to This that Sp1 and Sp3, in this promoter may be for further the between Fli-1 and we Fli-1 with DNA this we binding of the Sp1 and Sp3 to the to COL1A2 promoter Sp1 binding sites and or using nuclear from human and a In with data (9Ihn H. Trojanowska M. Nucleic Acids Res. 1997; 25: 3712-3717Crossref PubMed Scopus (98) Google Scholar, T. Ohnishi K. C. LeRoy E.C. Trojanowska M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), specific DNA-protein complexes that be by an of of the complexes was not by an of suggesting that the binding in this complex is related to Ets Furthermore, of the complex was was used as a The presence of Sp1 and Sp3 in the complexes with the and by with Sp1 and Sp3 in the not Sp1 or Sp3 suggesting that competition for DNA binding not to the effect of interaction of Fli-1 with the collagen we used the to of theCOL1A2 promoter and nuclear from human dermal fibroblasts. This promoter was in to a of a complex with this promoter Sp1 and Sp3 have been identified as the of this complex (9Ihn H. Trojanowska M. Nucleic Acids Res. 1997; 25: 3712-3717Crossref PubMed Scopus (98) Google Scholar). we interaction of Sp1 and Sp3 with the to promoter Fli-1 also to this promoter as indicated by a with the The of the to Sp1 and Sp3 complexes in addition to the Fli-1 complex the of protein-protein interaction between these the effects of in on binding of we used the to in assays indicated that mediates the Fli-1 we to to the of binding to Fli-1 Sp1 binding to this promoter was by of the Fli-1 from experiments between Fli-1 binding to the and the the role of in Fli-1 with theCOL1A2 we used as a the promoter in which Sp1 binding sites these binding Fli-1 binding was also with this as indicated by with the analysis of experiments a in Fli-1 binding between the and the in these in data indicate that Fli-1 with the in the of the the binding of to this promoter to Fli-1 this we two Fli-1 an mutant a in the Ets domain that its to DNA K. R. J. F. M. J. Mol. Cell. Biol. 1994; PubMed Scopus Google and a mutant only the Ets domain The effects of of these two Fli-1 mutants on the In to the both mutants in the COL1A2 promoter suggesting that direct (via DNA binding) and indirect (via protein-protein interaction) mechanisms to the effects of Fli-1 on the COL1A2 promoter. is the for the dermal Ets factors including and In to Ets-1 and the COL1A2 promoter in dermal fibroblasts. Ets-1 the COL1A2 promoter with not and was for further we have that and are expressed in dermal and that and are activators of the promoter in (20Shirasaki F. Makhluf H.A. LeRoy C. Watson D.K. Trojanowska M. Oncogene. 1999; 18: 7755-7764Crossref PubMed Scopus (85) Google Scholar), is that is involved in the and which to DNA as a two of the which are not present in the of the COL1A2 the of competition between Ets-1 and Fli-1 for the COL1A2 a of Fli-1 and was with increasing of Ets-1 into dermal Ets-1 was to the effects of Fli-1 in a suggesting that these two factors may for binding to the COL1A2 promoter. with this Ets-1 binding to the COL1A2 promoter was by Ets-1 binding to be that with Fli-1 the binding of Fli-1 and Ets-1 to theCOL1A2 the in vitro Ets-1 and Fli-1 with the promoter in the presence or of the The Fli-1 protein not with of the promoter not whereas the Ets-1 only to the promoter not data that Ets-1 or Fli-1 have binding for theCOL1A2 promoter. Thus, presence in the DNA-protein complexes on the presence of transcription factors with this promoter. the may be Sp1 and the interaction between and Ets factors we the Gal4 Sp1 or Sp3 to the Gal4 promoter in human Sp3 was a potent Sp1 in the context of the Gal4 of Ets-1 with or further enhanced promoter activity This a model for interaction between and Ets-1 in dermal fibroblasts. Fli-1 was also to with the and proteins these experimental with data using the promoter (20Shirasaki F. Makhluf H.A. LeRoy C. Watson D.K. Trojanowska M. Oncogene. 1999; 18: 7755-7764Crossref PubMed Scopus (85) Google Scholar), that Fli-1 can as a or an on the promoter In to fibroblasts, in Fli-1 not have a effect on or activation whereas Ets-1 with both Sp1 and Sp3 in the Gal4 promoter data that cell may be required to Fli-1 with with this studies have demonstrated that the of a complex and the interaction between Sp1 and Ets-1 required presence of the protein J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The between Fli-1-Sp1 interaction further that Ets-1 and Fli-1 interaction with these data that the of Fli-1 on cell and promoter further the of different cell types to we cell lines of different in the of Ets-1 and Fli-1 regulation of theCOL1A2 promoter Ets-1 theCOL1A2 promoter activity in cell lines In contrast, Fli-1 cell In human fibroblasts, Fli-1 inhibited the In human and in Fli-1 a whereas in Fli-1 was stimulatory for the COL1A2 promoter. We have also the expression of Ets-1 and Fli-1 proteins in the used in this Ets-1 and Fli-1 expressed in cell types and some in expression the ratio of Ets-1 to Fli-1 protein to be in The present study for the that Ets factors play a critical role in regulating the expression of the collagen type I gene in dermal fibroblasts. The following support this the expression of Fli-1 in dermal to a of the collagenous proteins and the COL1A2 transfection experiments have a in theCOL1A2 promoter. binding assays have demonstrated that Fli-1 and Ets-1 form DNA-protein complexes with sequences present in the COL1A2 promoter. Furthermore, this study has also to specific mechanisms involved in the transcriptional control of the COL1A2 gene by Ets using Fli-1 dominant interference and DNA binding mutants indicate that Fli-1 inhibition is mediated by both direct (DNA binding) and indirect interaction) on the studies with the promoter mutants as well as the we postulate that interaction of Fli-1 with Sp1 and Sp3 is for the of It also that additional tissue-specific factors are involved in these The nature of these is presently Furthermore, the competition experiments that Fli-1 the COL1A2 promoter by Ets-1. these that the ratio of Fli-1 to Ets-1 and the presence of co-regulatory proteins may to the control of collagen production in fibroblasts. that in the context of the Sp1 may play a role of an and a of this promoter Moreover, Fli-1 and a tissue-specific may to of this complex. direct interaction between Sp1 and further support this A. H. M. V. C. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). such a role has been for a tumor M. J. Dev. 1999; PubMed Scopus Google Scholar, W. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). It has been demonstrated that of specific genes on of with and M. J. Dev. 1999; PubMed Scopus Google Scholar), whereas the of can be enhanced by W. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). It is that in Sp1 can as an and a in the context of the promoter. the between these two to be However, has been that Sp1 is involved in mediating TGF-β activation of this promoter (8Zhang W. Ou J. Inagaki Y. Greenwel P. Ramirez F. J. Biol. Chem. 2000; 275: 39237-39245Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), suggesting a role of in this Studies using dermal indicate that Fli-1 can as a in the context of the collagen and is a potent for the promoter (20Shirasaki F. Makhluf H.A. LeRoy C. Watson D.K. Trojanowska M. 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