Activation of the Classical Mitogen-Activated Protein Kinases Is Part of the Shiga Toxin-Induced Ribotoxic Stress Response and May Contribute to Shiga Toxin-Induced Inflammation

Abstract

Infection with enterohemorrhagic Escherichia coli (EHEC) can result in severe disease, including hemorrhagic colitis and the hemolytic uremic syndrome. Shiga toxins (Stx) are the key EHEC virulence determinant contributing to severe disease. Despite inhibiting protein synthesis, Shiga toxins paradoxically induce the expression of proinflammatory cytokines from various cell types in vitro, including intestinal epithelial cells (IECs). This effect is mediated in large part by the ribotoxic stress response (RSR). The Shiga toxin-induced RSR is known to involve the activation of the stress-activated protein kinases (SAPKs) p38 and JNK. In some cell types, Stx also can induce the classical mitogen-activated protein kinases (MAPKs) or ERK1/2, but the mechanism(s) by which this activation occurs is unknown. In this study, we investigated the mechanism by which Stx activates ERK1/2s in IECs and the contribution of ERK1/2 activation to interleukin-8 (IL-8) expression. We demonstrate that Stx1 activates ERK1/2 in a biphasic manner: the first phase occurs in response to StxB1 subunit, while the second phase requires StxA1 subunit activity. We show that the A subunit-dependent ERK1/2 activation is mediated through ZAK-dependent signaling, and inhibition of ERK1/2 activation via the MEK1/2 inhibitors U0126 and PD98059 results in decreased Stx1-mediated IL-8 mRNA. Finally, we demonstrate that ERK1/2 are activated in vivo in the colon of Stx2-intoxicated infant rabbits, a model in which Stx2 induces a primarily neutrophilic inflammatory response. Together, our data support a role for ERK1/2 activation in the development of Stx-mediated intestinal inflammation.