The effect of vero toxin produced by enterohemorrhagic Escherichia coli on ion transporter

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Background. Although shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli (Stx-producing E. coli) has been shown to induce cell injury and play a central role in the pathogenesis of hemolytic uremic syndrome (HUS), the intracellular mechanism by which Stx causes damage to cells remains unclear. We examined whether the mitogen-activated protein kinase (MAPK) pathway is involved in Stx-induced Vero cell injury.Methods. We used Stx1, Stx2, and a non-toxic mutant Stx1, E167Q. The activation of MAPK pathways by Stx was assessed mainly by immunoblotting with anti-phospho-extracellular signal-regulated kinase 1/2 (ERK1/2) or -phospho-p38 MAPK antibodies. Cell viability was determined by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay. The role of MAPK and CaィイD12+ィエD1 was assessed using specific biochemical inhibitors.Results. Stx1 and Stx2 but not E167Q induced cell death in a dose-dependent and time-dependent manner. Consonant with cell injury, Stx1 and Stx2 caused a transient phosphorylation of ERK1/2 and a sustained phosphorylation of p38 MAPK. SB 203580 and PD 169316, both inhibitors of p38 MAPK, but not PD 98059, an inhibitor ERK kinase (MEK 1/2), partially inhibited the Stx1-induced cell death. BAPTA-AM, an intracellu1ar CaィイD12+ィエD1 chelator, and verapamil and nicardipine which are CaィイD12+ィエD1 channel blockers, reduced both cell injury and phosphorylation of p38 MAPK.Conclusion. These data indicate that MAPK pathways can be activated by Stx in Vero cells and that the activation of p38 MAPK, which is triggered by intracellular CaィイD12+ィエD1 elevation, plays a role in Stx-induced cell death.