B-230. ZAK is a Signaling Intermediary for Shiga Toxin Stimulated Cytokine Expression in Macrophage-Like Cells in vitro

D. M. Jandhyala, K. Poutsiaka, C. M. Thorpe;
Tufts-New England Med. Ctr., Boston, MA.

Up to 15 % of people infected with Shiga toxin-producing Escherichia coli (STEC) will develop severe disease characterized by microangiopathy. Shiga toxins (Stxs) are considered the main virulence factor causing microangiopathy following STEC infection, but the exact mechanism is poorly understood. Stxs damage host cells by cleaving an adenine from host 28S rRNA, stopping translation, and thus inducing a response called “ribotoxic stress”. Stx-induced ribotoxic stress is characterized by host SAPKinase activation, and can result in proinflammatory cytokine expression. As STEC are non-invasive, Stxs may play a role in enhancing their own uptake from the gut by augmenting local inflammation, thus increasing intestinal permeability. The Stx-induced ribotoxic stress response may also stimulate host macrophages to make proinflammatory cytokines, encouraging microangiopathy. The upstream signaling molecule(s) involved in sensing and/or transducing the Stx-induced ribosomal insult to the MAPKinase cascade are unknown, but may be potential therapeutic target(s). We have previously implicated the mixed lineage kinase, ZAK, as the major MAP3Kinase that transduces both the Stx- and ricin-mediated ribotoxic stress response in intestinal epithelial cells, resulting in both SAPK activation and interleukin-8 (IL-8) expression. We hypothesized that ZAK may also function in this capacity in macrophage-like cells. We used a ZAK-specific inhibitor called DHP-2 (also known as 7-[3-fluoro-4-aminophenyl-(4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl))]-quinoline) to evaluate the involvement of ZAK in Stx-induced SAPK activation and proinflammatory cytokine expression in differentiated THP-1 cells. We observed inhibition of Stx2-mediated JNK and p38 activation (measured by immunoprecipitation kinase assay), as well as decreases in Stx2-mediated IL-8 and TNF-α protein expression (measured by ELISA). Thus, ZAK is a critical intermediate that initiates the MAPKinase cascade in macrophage-like cells undergoing ribotoxic stress. ZAK inhibition may prevent and/or treat Stx-induced microangiopathy.