title: Reduction of tumor necrosis factor-alpha (TNF-alpha) related nuclear factor-kappaB (NF-kappaB) translocation but not inhibitor kappa-B (Ikappa-B)-degradation by Rho protein inhibition in human endothelial cells.
abstract: Degradation of inhibitor kappa-B (Ikappa-B) followed by translocation of nuclear factor-kappaB (NF-kappaB) into the nucleus and activation of gene expression is essential in tumor necrosis factor-alpha (TNF-alpha)-signaling. In order to analyze the role of Rho proteins in TNF-alpha-induced NF-kappaB-activation in human umbilical cord vein endothelial cells (HUVEC) we used Clostridium difficile toxin B-10463 (TcdB-10463) which inactivates RhoA/Rac1/Cdc42 by glucosylation and Clostridium botulinum C3-toxin which inhibits RhoA/B/C by ADP-ribosylation. Exposure of HUVEC to 10 ng/mL TcdB-10463 or 2.5 microg/mL C3-toxin inhibited TNF-alpha (100 ng/mL)-induced expression of a NF-kappaB-dependent reporter gene. Moreover, preincubation of HUVEC with 10 ng/mL TcdB-10463 reduced TNF-alpha-related expression of interleukin-8 (IL-8), TNF-receptor associated factor-2 (TRAF2), and human inhibitor of apoptosis protein 1 (hIAP1)-mRNA. Blocking of Rho reduced NF-kappaB DNA-binding as shown by electrophoretic mobility shift assays. TcdB-10463 and C3-toxin blocked TNF-alpha-related nuclear translocation of NF-kappaB although Ikappa-Balpha/beta was still degraded. In contrast, TcdB-10463 had no effect on IL-1beta-related NF-kappaB-translocation and activation in HUVEC. Neither 1 microM Rho kinase inhibitor Y-27632 nor microfilament depolymerization by 50 ng/mL C. botulinum C2-toxin blocked TNF-alpha-induced degradation of Ikappa-B, nuclear NF-kappaB translocation or expression of a NF-kappaB-dependent reporter gene. Therefore, TNF-alpha-related Ikappa-B-degradation is Rho-independent in HUVEC, whereas a Rho protein-dependent signal is necessary to induce nuclear transport of NF-kappaB in these cells pointing to a novel and unique role of Rho in NF-kappaB-translocation.
authors.affiliation: Charité, Department of Internal Medicine, Humboldt-University, Augustenburger Platz 1, 13353 Berlin, Germany. stefan.hippenstiel@charite.de
authors.forename: Stefan
authors.initials: S
authors.lastname: Hippenstiel
authors.forename: Bernd
authors.initials: B
authors.lastname: Schmeck
authors.forename: Joachim
authors.initials: J
authors.lastname: Seybold
authors.forename: Matthias
authors.initials: M
authors.lastname: Krüll
authors.forename: Christoph
authors.initials: C
authors.lastname: Eichel-Streiber
authors.forename: Norbert
authors.initials: N
authors.lastname: Suttorp
mesh_terms: D001692:Biological Transport; D002478:Cells, Cultured; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D020672:I-kappa B Proteins; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D020742:rhoA GTP-Binding Protein