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Molecular Damage and Induction of Proinflammatory Cytokines in Human Endothelial Cells Exposed to Shiga Toxin 1, Shiga Toxin 2, and -Sarcin

Dipartimento di Patologia Sperimentale, Università di Bologna,¹ Dipartimento di Chimica Industriale e dei Materiali dell'Università degli Studi di Bologna,² Bologna Istituto di Farmacologia e Farmacognosia Università degli Studi di Urbino Carlo Bo, Urbino,³ Dipartimento di Medicina Sperimentale, Sezione di Patologia Molecolare e Immunologia, Università di Parma, Parma,⁴ Istituto di Ricerca sulle Attività Motorie, Università degli Studi di Urbino Carlo Bo, Urbino, Italy⁵

Received 25 October 2006/ Returned for modification 28 November 2006/ Accepted 24 January 2007

Treatment of human endothelial cells with Shiga toxin 1 and 2 leads to the upregulation of genes encoding proinflammatory molecules involved in the pathogenesis of hemolytic-uremic syndrome. The paradoxical effect of inhibitors of mRNA translation, such as Shiga toxins, that at the same time induce protein expression was investigated by studying the relationship between their enzymatic activity (abstraction of adenine from nucleic acids) and the induction of interleukin-8 and granulocyte-macrophage colony-stimulating factor in human endothelial cells. As a positive control, the fungal toxin -sarcin, acting on the same rRNA sequence targeted by Shiga toxins with a different mechanism (RNase activity), was used. The three toxins caused ribosomal lesions that, in turn, induced the activation of p38 stress kinase with kinetics that paralleled the inhibition of translation. -Sarcin was devoid of activity on DNA. Shiga toxin 2 targeted nuclear DNA with more rapid kinetics than did Shiga toxin 1. Since the fungal ribotoxin was fully effective in the induction of proinflammatory proteins, we conclude that damage to ribosomes is indispensable and sufficient to activate protein expression via induction of the stress-kinase cascade. However, gene upregulation events induced by Shiga toxin 2 were much more efficient than those triggered by Shiga toxin 1, although the two toxins impaired translation to the same extent and had overlapping time courses of stress kinase activation. Regulations independent of the ribotoxic stress were assumed to operate in intoxicated cells. We hypothesized that the two bacterial toxins recognize different DNA sequences inducing different regulating effects on gene expression.

Published ahead of print on 12 February 2007.

Editor: A. D. O'Brien