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INTERLEUKIN-11 DEPENDENT ACTIVATION OF MCP-1 IN INTESTINAL EPITHELIA INVOLVES PI3K, MEK AND PKA PATHWAYS CONVERGING ON p65 NFkappaB
JD Leung, K Parhar, KA Baer, Y Shu, MJ Ropeleski
Gastrointestinal Disease Research Unit (GIDRU), Depts of Medicine and Anatomy/Cell Biology, Queen’s University Kingston, Ontario
IL-11 promotes epithelial resistance to injury, yet its mechanisms of action are incompletely understood. We have previously shown that IL-11 induces genes associated with healing such as monocyte chemoattractant protein-1 (MCP-1) and that this was associated with non-classical activation of NF-kappaB. We hypothesized that IL-11 stimulates multi-site phosphorylation of NFkappaB in intestinal epithelial cells to confer its effect during the healing response.
Rat IEC-18 and human HIEC epithelial crypt cells were used. MCP-1 expression was monitored by ELISA. Stealth duplex siRNA to rat p65 was carried out using Silentfect. NFkappaB activation was determined by immunoblotting of total and nuclear extracts for phospho-p65Ser536, phospho-p65Ser276 and total p65 as well as by immunofluorescence. Phosphorylation of IKKalpha,betaSer180/181 and phospho-MSK-1Ser376 was monitored in parallel. Inhibition of p65 phosphorylation was carried out using LY294002, U0126, SC-514 and the PKA inhibitor peptide(14-22).
SiRNA knockdown achieved 90% knockdown of p65 by immunoblot and significant decreases in IL-11 inducible MCP-1 transcripts, an effect reversed by mutant siRNA. IL-11 induced accumulation of nuclear p65, phospho-p65Ser536 and phospho-p65Ser276 which was associated with increased phospho-IKKalpha,betaSer180/181 and phospho-MSK-1Ser376. Inhibition of IKKbeta with SC-514 led to a partial reduction in phospho-p65Ser536 and MCP-1 expression. Inhibition of PI3K with LY294002 led to a partial reduction in phospho-p65Ser536 while inhibition with U0126 led to complete inhibition of p65 phosphorylation and MCP-1 expression. PKA inhibition decreased phospho-p65Ser276 expression in IL-11 treated cells.
IL-11 is as a non-classical activator of NFkappaB signaling in intestinal epithelial cells. It’s activation of NFkappaB predominantly involves MEK pathways as well as novel involvement of PKA signaling.
Supported by the Crohn’s and Colitis Foundation of Canada