SENSITIZATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS TO SHIGA TOXIN - INVOLVEMENT OF PROTEIN-KINASE-C AND NF-KAPPA-B

Infection of humans with Shiga toxin-producing Escherichia coli O157:H 7 and Shigella dysenteriae 1 is strongly associated with vascular endo thelial cell damage and the development of hemolytic-uremic syndrome. The cytotoxic effect of Shiga toxins on vascular endothelial cells in vitro is enhanced by prior exposure to bacterial lipopolysaccharide (L PS) or either of the host cytokines tumor necrosis factor alpha (TNF) and interleukin-1 beta (IL-1), The purpose of this study was to examin e individual signal transduction components involved in the sensitizat ion of human umbilical vein endothelial cells (HUVEC) to Shiga toxin 1 . The results demonstrate that class I and II protein kinase C (PKC) i sozymes are required for sensitization of HUVEC to Shiga toxin by phor bol myristate acetate (PMA) or LPS but not by TNF or IL-1. Thus, the s pecific competitive inhibitor of class I/II PKC, 1-O-hexadecyl-2-O-met hyl-rac-glycerol (AMG), prevented only the action of PMA and LPS on HU VEC. Additional data obtained with ATP binding site inhibitors which a ffect all PKCs (i.e., classes I, II, and III) suggest that TNF may uti lize class III PKC isozymes in the Shiga toxin sensitization of HUVEC. Transcriptional activator NF-KB did not appear to be involved in the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA. Thus, the specific serine protease inhibitor L-1-tosylamido-2-phenylethyl c hloromethyl ketone (TPCK) did not inhibit the sensitization of HUVEC t o Shiga toxin by LPS, TNF, IL-1, or PMA despite its ability to inhibit NF-kappa B activation and the induction of the NF-kappa B-dependent t issue factor gene by these agents. Finally, all-trans retinoic acid pa rtially inhibited the sensitization of HUVEC to Shiga toxin, by unknow n mechanisms which also appeared to be independent of NF-kappa B activ ation. These results indicate that PKC plays a role in the sensitizati on of HUVEC to Shiga toxin in response to some, but not ail, sensitizi ng agents. In contrast, NF-kappa B activation appears not to be involv ed in the sensitization of HUVEC to Shiga toxin by LPS, TNF, IL-1, or PMA.