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Tumor necrosis factor α induces endothelial galactosyl transferase activity and verocytotoxin receptors. Role of specific tumor necrosis factor receptors and protein kinase C

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Author: Kar, N.C.A.J. van de · Kooistra, T. · Vermeer, M. · Lesslauer, W. · Monnens, L.A.H. · Hinsbergh, V.W.M. van
Institution: TNO Preventie en Gezondheid
Source:Blood, 3, 85, 734-743
Identifier: 232940
Keywords: Toxicology · 1 (5 isoquinolinesulfonyl) 2 methylpiperazine · 2 [1 (3 amidinothiopropyl) 1h indol 3 yl] 3 (1 methyl 1h indol 3 yl)maleimide · endothelial leukocyte adhesion molecule 1 · galactosyltransferase · globotriaosylceramide · interleukin 1 · n (2 guanidinoethyl) 5 isoquinolinesulfonamide · phorbol 13 acetate 12 myristate · phorbol ester · plasminogen activator inhibitor 1 · protein kinase C · protein kinase C inhibitor · recombinant interleukin 1beta · recombinant tumor necrosis factor alpha · tumor necrosis factor receptor · verotoxin · article · autoradiography · controlled study · endothelium cell · endothelium injury · gene expression · hemolytic uremic syndrome · human · human cell · priority journal · protein synthesis inhibition · signal transduction · umbilical vein · Autoradiography · Bacterial Toxins · Carbon Radioisotopes · Cell Adhesion Molecules · Cells, Cultured · Cytotoxins · E-Selectin · Endothelium, Vascular · Enzyme Induction · Galactose · Galactosyltransferases · Glucose · Glycolipids · Human · Kinetics · Protein Kinase C · Receptors, Cell Surface · Receptors, Tumor Necrosis Factor · Shiga-Like Toxin I · Support, Non-U.S. Gov't · Tumor Necrosis Factor · Umbilical Veins


Infections with verocytotoxin (VT) producing Escherichia coil have been strongly implicated in the epidemic form of hemolytic uremic syndrome (HUS). Endothelial damage plays a central role in the pathogenesis of HUS. In vitro studies have shown that VT can damage endothelial cells after interaction with its cellular receptor globotriaosylceramide (GbOse3cer). Cytokines, such as tumor necrosis factor α (TNFα) and interleukin-1 (IL-1) can potentiate the toxic effect of VT by inducing a protein-synthesis dependent increase in VT receptors on endothelial cells. In this study, the mechanisms underlying the increase in endothelial VT receptors induced by TNFα were studied in more detail. To investigate which proteins were involved in this induction, endothelial cells were incubated with and without TNFα in the presence of 14C-galactose or 14C-glucose. Thin-layer chromatography (TLC) analysis of the glycolipid extracts of these cells demonstrated a markedly enhanced incorporation of 14C-galactose in GbOse3cer and other galactose- containing glycolipids, suggesting that TNFα enhanced galactosyl-transferase activity. To examine the role of the two recently cloned TNF-receptors (TNFR- p75 and TNFR-p55) in the TNFα-induced increase in GbOse3cer in human endothelial cells, cells were incubated with TNFα, the TNFR-p55 selective R32W-S86T-TNFα-mutant, or the TNFR-p75 selective D143N-A145R-TNFα-mutant. The effect of TNFα activation,determined by binding-experiments with 125I-VT-1, could be largely, but not completely mimicked by R32W-S86T- TNFα. Although incubation of cells with D143N-A145R-TNFα did not show an increase in VT-1 binding, the monoclonal antibody utr-1, which prevents binding to TNFR-p75, decreased the TNFα-induced VT-1 binding. Activation of protein kinase C (PKC) by phorbol ester increases the expression of VT-1 receptors; this effect was prevented by the PKC inhibitor Ro31-8220 and by homologous desensitization by pretreatment with phorbol ester. In contrast, the presence of the protein kinase inhibitor Ro31-8220 or desensitization of PKC activity reduced the TNFα-induced increase in VT-1 receptors maximally by 50% end 24%, respectively. Comparable reductions in overall protein synthesis and the synthesis of E-selectin and plasminogen activator inhibitor-1 (PAI-1) were observed. This suggests an effect on general protein synthesis rather than a specific effect of PKC in the signal transduction pathway, by which TNFα induces VT-1 receptors. Our results indicate that TNFα can increase the VT-1 receptors on endothelial cells by inducing galactosyl-transferase activity, that this action of TNFα mainly occurs via the TNFR-p55; and that PKC activation increases expression of VT-1 receptors by a separate mechanism that acts additively to the TNFα-induced increase in VT-1 receptors.