Reduction of tumor necrosis factor-alpha (TNF-α) related nuclear factor-kappaB (NF-κB) translocation but not inhibitor kappa-B (Iκ-B)-degradation by Rho protein inhibition in human endothelial cells

Abstract

Degradation of inhibitor kappa-B (Iκ-B) followed by translocation of nuclear factor-kappaB (NF-κB) into the nucleus and activation of gene expression is essential in tumor necrosis factor-alpha (TNF-α)-signaling. In order to analyze the role of Rho proteins in TNF-α-induced NF-κB-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 10ng/mL TcdB-10463 or 2.5μg/mL C3-toxin inhibited TNF-α (100ng/mL)-induced expression of a NF-κB-dependent reporter gene. Moreover, preincubation of HUVEC with 10ng/mL TcdB-10463 reduced TNF-α-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-κB DNA-binding as shown by electrophoretic mobility shift assays. TcdB-10463 and C3-toxin blocked TNF-α-related nuclear translocation of NF-κB although Iκ-Bα/β was still degraded. In contrast, TcdB-10463 had no effect on IL-1β-related NF-κB-translocation and activation in HUVEC. Neither 1μM Rho kinase inhibitor Y-27632 nor microfilament depolymerization by 50ng/mL C. botulinum C2-toxin blocked TNF-α-induced degradation of Iκ-B, nuclear NF-κB translocation or expression of a NF-κB-dependent reporter gene. Therefore, TNF-α-related Iκ-B-degradation is Rho-independent in HUVEC, whereas a Rho protein-dependent signal is necessary to induce nuclear transport of NF-κB in these cells pointing to a novel and unique role of Rho in NF-κB-translocation.