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012 INTERLEUKIN-11 STIMULATES MCP-1 SECRETION IN AN AKT AND NFkappaB DEPENDANT MANNER K Parhar, KA Baer, and MJ Ropeleski IL-11 is an anti-inflammatory mesenchymal-derived cytokine that plays an important role in intestinal resistance to injury and healing. The mechanisms through which IL-11 acts are not well understood. The AKT pathway orchestrates anti-apoptotic signaling and also modulates NFkappaB activity. We hypothesized that IL-11 could stimulate NFkappaB in an AKT-dependent manner to induce genes important for the healing response such as monocyte chemoattractant protein-1 (MCP-1).
Gastrointestinal Disease Research Unit (GIDRU), Departments of Medicine and Anatomy/Cell Biology, Queen's University Kingston, Ontario
IEC-18 intestinal epithelial crypt cells were stimulated with varying doses of IL-11, using TNFalpha as a positive control. AKT activity was assessed using phospho-specific immunoblotting and kinase assays. NFkappaB activation was determined by immunoblotting for IkappaB, phospho-p65Ser536 and nuclear p65/p50, by EMSA to assess DNA binding on generic kappaB and MCP-1-specific promoter/enhancer NFkappaB oligos, and luciferase assays to assess NFkappaB transcriptional activity. IL-11 mediated MCP-1 gene activation was examined using semi-quantitative RT-PCR and luciferase assays while secreted protein was detected by ELISA.
IL-11 treatment resulted in a dose-dependant increase in both phosphorylated AKT and kinase activity. IL-11 treatment also resulted in the activation of NFkappaB-dependant luciferase activity. Examination of MCP-1, a target gene of NFkappaB implicated in healing, showed an increase in promoter activity with a corresponding increase in both MCP-1 mRNA and secreted protein. Inhibition of NFkappaB using specific IKKbeta inhibitors (SC-514 and Bay 11-7082) as well as a PI3K inhibitor (LY294002) blocked MCP-1 mRNA synthesis. Interestingly, NFkappaB activation did not involve degradation of IkappaB, increased p65 nuclear localization or increased DNA binding, but did involve increased phospho-p65Ser536 suggesting a non-classical mechanism of NFkappaB activation.
IL-11 may act as a non-classical activator of NFkappaB signaling in intestinal epithelial cells which may mediate the anti-apoptotic and healing characteristics of IL-11 in various models of intestinal injury.
Supported by the Crohn's and Colitis Foundation of Canada