The Transcription Factor EGR-1 Suppresses Transformation of Human Fibrosarcoma HT1080 Cells by Coordinated Induction of Transforming Growth Factor-β1, Fibronectin, and Plasminogen Activator Inhibitor-1

Abstract

Re-expression of EGR-1 in fibrosarcoma HT1080 suppresses transformation including tumorigenicity (Huang, R.-P., Liu, C., Fan, Y., Mercola, D., and Adamson, E. (1995) Cancer Res. 55, 5054–5062) owing in part to up-regulation of the transforming growth factor (TGF)-β1 promoter by EGR-1 which suppresses growth by an autocrine mechanism (Liu, C., Adamson, E., and Mercola, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11831–11836). Here we show that enhanced cell attachment contributes to the suppression via increased secretion of fibronectin (FN) and also of plasminogen activator inhibitor-1 (PAI-1). The secretion of FN and PAI-1 is strongly correlated with EGR-1 expression (R PEARSON = 0.971 and 0.985, respectively). Addition of authentic TGF-β1 to parental cells greatly stimulated secretion of PAI-1 but not FN, whereas addition of TGF-β antibody or lipofection with specific antisense TGF-β1oligonucleotides to EGR-1-regulated cells completely inhibits the secretion of PAI-1 but not FN. However, in gel mobility shift assays pure EGR-1 or nuclear extracts of EGR-1-regulated cells specifically bind to two GC-rich elements of the human FN promoter at positions −75/−52 and −4/+18, indicating that the increased secretion of FN is likely due to direct up-regulation by EGR-1. Moreover, adhesion was greatly enhanced in EGR-1-regulated cells and was reversed by treatment with Arg-Gly-Asp (RGD) or PAI-1 antibody indicating that the secreted proteins are functional. We conclude that EGR-1 regulates the coordinated expression of gene products important for cell attachment (“oikis” factor) and normal growth control. Re-expression of EGR-1 in fibrosarcoma HT1080 suppresses transformation including tumorigenicity (Huang, R.-P., Liu, C., Fan, Y., Mercola, D., and Adamson, E. (1995) Cancer Res. 55, 5054–5062) owing in part to up-regulation of the transforming growth factor (TGF)-β1 promoter by EGR-1 which suppresses growth by an autocrine mechanism (Liu, C., Adamson, E., and Mercola, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11831–11836). Here we show that enhanced cell attachment contributes to the suppression via increased secretion of fibronectin (FN) and also of plasminogen activator inhibitor-1 (PAI-1). The secretion of FN and PAI-1 is strongly correlated with EGR-1 expression (R PEARSON = 0.971 and 0.985, respectively). Addition of authentic TGF-β1 to parental cells greatly stimulated secretion of PAI-1 but not FN, whereas addition of TGF-β antibody or lipofection with specific antisense TGF-β1oligonucleotides to EGR-1-regulated cells completely inhibits the secretion of PAI-1 but not FN. However, in gel mobility shift assays pure EGR-1 or nuclear extracts of EGR-1-regulated cells specifically bind to two GC-rich elements of the human FN promoter at positions −75/−52 and −4/+18, indicating that the increased secretion of FN is likely due to direct up-regulation by EGR-1. Moreover, adhesion was greatly enhanced in EGR-1-regulated cells and was reversed by treatment with Arg-Gly-Asp (RGD) or PAI-1 antibody indicating that the secreted proteins are functional. We conclude that EGR-1 regulates the coordinated expression of gene products important for cell attachment (“oikis” factor) and normal growth control. egr-1 was originally identified as an immediate early growth response gene that exhibits a rapid and transient burst of transcription without de novo protein synthesis, following stimulation by a variety of mitogens (1Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Crossref PubMed Scopus (996) Google Scholar). EGR-1 is a nuclear phosphoprotein that contains three zinc finger motifs in the C-terminal portion of the molecule, which confer specific DNA-binding properties (2Christy B. Nathans D. Mol. Cell. Biol. 1989; 9: 4889-4895Crossref PubMed Scopus (172) Google Scholar, 3Gashler A.L. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (565) Google Scholar, 4Lemaire P. Vesque C. Schmitt J. Stunnenberg H. Frank R. Charnay P. Mol. Cell. Biol. 1990; 10: 3456-3467Crossref PubMed Google Scholar). EGR-1 preferentially but not exclusively binds GC-rich regulatory elements (GCE) 1The abbreviations used are: GCE, GC-rich regulatory elements; TGF-β1, transforming growth factor-β1; FN, fibronectin; PAI-1, plasminogen activator inhibitor-1; RGD, Arg-Gly-Asp; WT-1, Wilm's tumor-1; rh, recombinant human; ECM, extracellular matrix; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; uPA, urokinase plasminogen; uPAR, urokinase plasminogen receptor; GST, glutathioneS-transferase. with a consensus sequence of 5′-GCG(T/G)GGGCG-3′ (5Nakagama H. Heinrich G. Pelletier J. Housman D.E. Mol. Cell. Biol. 1995; 15: 1489-1498Crossref PubMed Scopus (166) Google Scholar). For some GCEs in certain genes, other transcription factors also bind and influence transcription. Examples to the GC-rich of gene promoter C. Proc. Natl. Acad. Sci. U. S. 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J. Biol. 1995; PubMed Scopus Google Addition of recombinant FN or PAI-1 proteins to parental HT1080 cells or addition of specific of FN or PAI-1 to HT1080 cells that proteins are in growth with cell that EGR-1 to a coordinated of gene expression to increased extracellular with enhanced HT1080 cells and cells by of cells with expression for egr-1 as C. D. E. Cancer Res. 1995; Google and in with and in the of and and for by direct cell to C. D. E. Cancer Res. 1995; Google Scholar). at the of with phosphate-buffered and by the with The a to the for and at for The protein protein of protein by gel and with by enhanced The of was by a For the plasminogen activator cells in in with and cells to in the or of of recombinant human TGF-β1 in the to or for at which was to for an was as J. Sci. Google Scholar). cell with PBS, and the and nuclear proteins by with and The extracellular proteins by addition of to the following by The to and the with for by and For the FN cells in The cells with or without TGF-β1 or in The was to for The and to in the of as J. R. J. Mol. Cell. Biol. PubMed Google Scholar). The by and the with for by and to the human and the sequence by and of the antisense sequence of and sequence of to completely and specifically TGF-β1 expression C. B. J. 1995; PubMed Scopus Google Scholar). the fibrosarcoma HT1080 cells or the a was used R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). in of with in of and for at the with of and to cells that to and with The of and in the was and normal growth was the cells for the expression of PAI-1 or FN. extracts the of EGR-1 expression and cells as J. Scopus Google Scholar). The protein in the nuclear extracts by protein to to or to of the and S. Proc. Natl. Acad. Sci. U. S. A. 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