Verotoxin-1 Induces Tissue Factor Expression in Human Umbilical Vein Endothelial Cells through Activation of NF-κB/Rel and AP-1

Summary

This study examined the effect of verotoxin-1 (VT-1), which is released from Escherichia coli O157:H7, on endothelial expression of tissue factor (TF), a cofactor required to initiate blood coagulation. In order to elucidate the molecular basis for development of hemolytic uremic syndrome (HUS) in patients infected with E. coli O157:H7, human umbilical vein endothelial cells (HUVECs) were exposed to purified VT-1. VT-1 increased both TF activity and TF mRNA in HUVECs without loss of cell viability in a time-and dose-dependent manner from 0.1 to 10 ng/ml VT-1. Nuclear proteins extracted from VT-1-stimulated HUVECs bound to the consensus NF-κB/Rel and AP-1 binding oligonucleotides in a dose-dependent manner within 2 h after the stimulation in electrophoretic mobility shift assays (EMSA). Nuclear proteins from VT-1-stimulated HUVECs formed two complexes with the NF-κB/Rel binding motif in the human TF promoter (TF-κB motif). The supershift assays, using antibodies for human p65, p50 or c-Rel, indicated that the lower complex was composed of p65/p50 and the higher complex was a p65 homo-or hetero-dimer with the Rel family, except c-Rel. The human TF promoter contains two AP-1 binding sites, the proximal and distal AP-1 binding sites. The supershift assays indicated that AP-1 containing mainly c-Jun and JunD, positively bound to the proximal AP-1 motif of TF (TF-AP-1). The distal TF-AP-1 motif did not show positive binding with nuclear proteins from VT-1-stimulated HUVECs. Pretreatment of HUVECs with curcumin, an inhibitor of NF-κB/Rel activation, synthesis of c-Jun mRNA and binding of activated AP-1 with AP-binding oligonucleotide, prevented the VT-1 induced increase in TF mRNA and activity in VT-1-stimulated HUVECs. Curcumin also inhibited NF-κB and AP-1 binding to TF-κB and proximal TF-AP-1 oligonucleotides, respectively, in a dose-dependent manner. The present work suggests that both the NF-κB/Rel and AP-1 activated in endothelial cells by stimulation with VT-1 binds to the TF-κB and proximal AP-1 binding sites, respectively, of the TF promoter.

Key words

Endothelial cells - tissue factor - Escherichia coli O157 - verotoxin

• References

• 1 Griffin PM, Tauxe RV. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol Rev 1991; 13: 60-98.
• 2 Rose PE, Clark AJ. Haematology of the haemolytic uremic syndrome. Blood Rev 1989; 03: 136-40.
• 3 Zoja C, Remuzzi G. The Pivotal Role of the Endothelial Cell in the Pathogenesis of HUS. In: The Hemolytic Syndromes/Thrombotic Thrombocytopenic Purpura. Kaplan B, Trompeter R, Moake J. eds. New York: Dekker; 1992: 89-404.
• 4 Richardson SE, Rotman TA, Jay V, Smith CR, Becker LE, Petric M, Olivieri NF, Karmali MA. Experimental verocytotoxemia in rabbits. Infect Immun 1992; 60: 4154-67.
• 5 Richardson SE, Karmali MA, Becker LE, Smith CR. The histopathology of the hemolytic uremic syndrome (HUS) associated with verocytotoxinproducing Escherichia coli (VTEC) infections. Hum Pathol 1988; 19: 1102-8.
• 6 Moake JL. Haemolytic-uremic syndrome: basic-science. Lancet 1994; 343: 393-7.
• 7 Monteagudo J, Pereira A, Reverter JC, Pijoan J, Tusell J, Puig L, Ordians A, Castillo R. Thrombin generation and fibrinolysis in the thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome. Thromb Haemost 1991; 66: 515-9.
• 8 Nevard CH, Jurd KM, Lane DA, Philippou H, Haycock GB, Hunt BJ. Activation of coagulation and fibrinolysis in childhood diarrhoea-associated haemolytic uraemic syndrome. Thromb Haemost 1997; 78: 1450-5.
• 9 Moake JL, Byrnes JJ, Troll JH, Rudy CK, Weinstein MJ, Colannino NM, Hong SL. Abnormal VIII: von Willebrand factor patterns in the plasma of patients with the hemolytic-uremic syndrome. Blood 1984; 64: 592-8.
• 10 van de Kar NC, van Hinsbergh VW, Brommer EJ, Monmens LA. The fibrinolytic system in the hemolytic uremic syndrome: in vivo and in vitro studies. Pediatr Res 1994; 36: 257-64.
• 11 Nemerson T. Tissue factor and hemostasis. Blood 1988; 71: 1-8.
• 12 Mann KG, Nesheim ME, Church WR, Haley P, Krishnaswamy S. Surfacedependent reaction of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16.
• 13 Camerer E, Kolsto AB, Prydz H. Cell biology of tissue factor, the principal initiator of blood coagulation. Thromb Res 1996; 81: 1-41.
• 14 Colucci M, Balconi G, Lorenzet R, Pietra A, Locati D, Donati MB, Semeraro N. Cultured human endothelial cells generate tissue factor in response to endotoxin. J Clin Invest 1983; 71: 1893-6.
• 15 Nawroth PP, Handley DA, Esmon CT, Stern DM. Interleukin 1 induces endothelial cell procoagulant while suppressing cell-surface anticoagulant activity. Proc Natl Acad Sci USA 1986; 83: 3460-4.
• 16 Scarpati EM, Sadler JE. Regulation of endothelial cell coagulant properties. Modulation of tissue factor, plasminogen activator inhibitors, and thrombomodulin by phorbol 12-myristate 13-acetate and tumor necrosis factor. J Biol Chem 1989; 264: 20705-13.
• 17 Nawroth PP, Stern DM. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp Med 1986; 163: 740-5.
• 18 Bevilacqua MP, Pober JS, Majeau GR, Cotran RS, Gimbrone MR. Interleukin 1 (IL-1) induces biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells. J Exp Med 1984; 160: 618-23.
• 19 Ishii H, Horie S, Kizaki K, Kazama M. Retinoic acid counteracts both the downregulation of thrombomodulin and the induction of tissue factor in cultured human endothelial cells exposed to tumor necrosis factor. Blood 1992; 80: 2556-62.
• 20 Tannenbaum SH, Finko R, Cines DB. Antibody and immune complexes induce tissue factor production by human endothelial cells. J Immunol 1986; 137: 1532-7.
• 21 Louise CB, Obrig TG. Specific interaction of Escherichia coli O157:H7-derived Shiga-like toxin II with human renal endothelial cells. J Infect Dis 1995; 172: 1397-401.
• 22 O’Brien AD, LaVeck GD. Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli . Infect Immun 1983; 40: 675-83.
• 23 Olsnes S, Reisbig R, Eiklid K. Subunit structure of Shigella cytotoxin. J Biol Chem 1981; 256: 8732-8.
• 24 Lingwood CA, Law H, Richardson S, Petric M, Brunton JL, DeGrandis S, Karmali M. Glycolipid binding of purified and recombinant Escherichia coli produced verotoxin in vitro. J Biol Chem 1987; 262: 8834-9.
• 25 Lindberg AA, Brown JE, Stromberg N, Westling-Ryd M, Schultz JE, Karlsson KA. Identification of the carbohydrate receptor for Shiga toxin produced by Shigella dysenteriae type 1. J Biol Chem 1987; 262: 1779-85.
• 26 Obrig TG, Moran TP, Brown JE. The mode of action of Shiga toxin on peptide elongation of eukaryotic protein synthesis. Biochem J 1987; 244: 287-94.
• 27 Morigi M, Micheletti G, Figliuzzi M, Imberti B, Karmali MA, Remuzzi A, Remuzzi G, Zoja C. Verotoxin-1 promotes leukocyte adhesion to cultured endothelial cells under physiologic flow conditions. Blood 1995; 86: 4553-8.
• 28 Louise CB, Tran MC, Obrig TG. Sensitization of human umbilical vein endothelial cells to Shiga toxin: involvement of protein kinase C and NF-κB. Infect Immun 1997; 65: 3337-44.
• 29 Noda M, Yutsudo T, Nakabayashi N, Hirayama T, Kakeda Y. Purification and some properties of Shiga-like toxin from Escherichia coli O157:H7 that is immunologically identical to Shiga toxin. Microb Pathol 1987; 02: 339-49.
• 30 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: 2745-56.
• 31 Ishii H, Kizaki K, Horie S, Kazama M. Oxidized low density lipoprotein reduces thrombomodulin transcription in cultured human endothelial cells through degradation of the lipoprotein in lysosomes. J Biol Chem 1996; 271: 8458-65.
• 32 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55-63.
• 33 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-9.
• 34 Schreiber E, Matthias P, Muller MM, Schaffner W. Rapid detection of octamer binding proteins with “mini-extracts”, prepared from a small number of cells. Nucleic Acids Res 1989; 17: 6419.
• 35 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-54.
• 36 Conway EM, Bach R, Rosenberg RD, Konigsberg WH. Tumor necrosis factor enhances expression of tissue factor mRNA in endothelial cells. Thromb Res 1989; 53: 231-41.
• 37 Crossman DC, Carr DP, Tuddenham EG, Pearson JD, McVey JH. The regulation of tissue factor mRNA in human endothelial cells in response to endotoxin or phorbol ester. J Biol Chem 1990; 265: 9782-7.
• 38 Mackman N. Regulation of the tissue factor gene. FASEB J 1995; 09: 883-9.
• 39 Mackman N, Morrissey JH, Fowler B, Edgington TS. Complete sequence of the human tissue factor gene, a highly regulated cellular receptor that initiates the coagulation protease cascade. Biochemistry 1989; 28: 1755-62.
• 40 Parry GC, Mackman N. A set of inducible genes expressed by activated human monocytic and endothelial cells contain κB-like sites that specifically bind c-Rel-p65 heterodimers. J Biol Chem 1994; 269: 20823-5.
• 41 Parry GC, Mackman N. Transcriptional regulation of tissue factor expression in human endothelial cells. Arterioscler Thromb Vasc Biol 1995; 15: 612-21.
• 42 Pendurthi UR, Williams JT, Rao LV. Inhibition of tissue factor gene activation in cultured endothelial cells by curcumin. Suppression of activation of transcription factors Egr-1, AP-1, and NF-κB. Arterioscler Thromb Vasc Biol 1997; 17: 3406-13.
• 43 Bierhaus A, Zhang Y, Quehenberger P, Luther T, Haase M, Müller M, Mackman N, Ziegler R, Nawroth PP. The dietary pigment curcumin reduces endothelial tissue factor gene expression by inhibiting binding of AP-1 to the DNA and activation of NF-κB. Thromb Haemost 1997; 77: 772-82.
• 44 Thanos D, Maniatis T. NF-κB: a lesson in family values. Cell 1995; 80: 529-32.
• 45 Beg AA, Ruben SM, Scheinman RI, Haskill S, Rosen CA, Baldwin AS. IκB interacts with the nuclear localization sequences of the subunits of NF-κB: a mechanism for cytoplasmic retention. Genes Dev 1992; 06: 1899-913.
• 46 Moll T, Czyz M, Holzmuller H, Hofer-Warbinek R, Wagner E, Winkler H, Bach FH, Hofer E. Regulation of the tissue factor promoter in endothelial cells. Binding of NF-κB-, AP-1-, and Sp1-like transcription factors. J Biol Chem 1995; 270: 3849-57.
• 47 Sakiri R, Ramegowda B, Tesh VL. Shiga toxin type 1 activates tumor necrosis factor-α gene transcription and nuclear translocation of the transcriptional activators nuclear factor-κB and activator protein-1. Blood 1998; 92: 558-66.
• 48 Ledebur HC, Parks TP. Transcriptional regulation of the intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells. Essential roles of a variant NF-kB site and p65 homodimers. J Biol Chem 1995; 270: 933-43.
• 49 Bierhaus A, Zhang Y, Deng Y, Mackman N, Quehenberger P, Haase M, Luther T, Muller M, Bohrer H, Greten J, Martin E, Baeuerle PA, Waldherr R, Kisiel W, Ziegler R, Stern DM, Nawroth PP. Mechanism of the tumor necrosis factor α-mediated induction of endothelial tissue factor. J Biol Chem 1995; 270: 26419-32.
• 50 Zhu Y, Lin JH, Liao HL, Friedli OJ, Verna L, Marten NW, Straus DS, Stemerman MB. LDL induces transcription factor activator protein-1 in human endothelial cells. Arterioscler Thromb Vasc Biol 1998; 18: 473-80.
• 51 Singh S, Aggarwal BB. Activation of transcription factor NF-κB is suppressed by curcumin. J Biol Chem 1995; 270: 24995-5000.
• 52 Huang TS, Lee SC, Lin JK. Suppression of c-Jun/AP-1 activation by an inhibitor of tumor promotion in mouse fibroblast cells. Proc Natl Acad Sci USA 1991; 88: 5292-6.
• 53 Oeth P, Parry GC, Mackman N. Regulation of the tissue factor gene in human monocytic cells. Arterioscler Thromb Vasc Biol 1997; 17: 365-74.
• 54 Böhrer H, Qiu F, Zimmermann T, Zhang Y, Jllmer T, Männel D, Böttiger BW, Stern DM, Waldherr R, Saeger HD, Ziegler R, Bierhaus A, Martin E, Nawroth PP. Role of NF-κB in the mortality of sepsis. J Clin Invest 1997; 100: 972-85.