Interleukin-11 dependent NFkB activation in cultured intestinal epithelial cells
Leung, Jeffrey Daniel Hawk-Ling
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Interleukin-11 (IL-11) is a cytokine that promotes intestinal epithelial resistance to injury, however the mechanisms remain incompletely understood. Evidence from the Ropeleski Lab supports IL-11 dependent activation of the transcription factor Nuclear Factor кB (NFкB), without the degradation of the inhibitor кB (IкB), which deviates from the classical mechanism involving proteolytic processing of IкB. Also, IL-11 mediates the modulation of genes associated with healing, such as cyclooxygenase-2 (COX-2). It was hypothesized that IL-11 activates NFкB in intestinal epithelial cells by direct modulation of NFкB which, in a physiological setting, stimulates restitution during the healing response in the gut. Both cultured rat IEC-18 and human HIEC epithelial crypt cells were used as models to investigate whether the effect of IL-11 was species-specific. Activated NFкB is targeted to the nucleus therefore immunoblotting of nuclear extracts for expression of NFкB protein subunits including p65, activated p65 (phospho-p65Ser536), p50, and RelB, as well as by immunofluorescent detection of p65 were used. Inhibition of IL-11 signaling was carried out using various pharmacological inhibitors in order to determine their effect on p65 phosphorylation. Mechanically wounded cells were used as a model of gut injury and restitution where immunoblotting was used to examine IL-11 dependent effects on phospho-p65Ser536 and COX-2 expression. The binding of p65 to the кB binding site on DNA was detected with an ELISA-based system. IL-11 treatment was associated with the nuclear accumulation of phospho-p65ser536 in epithelial cell lines. Inhibition of PI3K/Akt signaling with LY294002 and AktiVIII suggested a partial reduction in phospho-p65Ser536 while inhibition of MEK1,2 signaling with U0126 indicated almost a complete abrogation of phospho-p65Ser536 accumulation in the nucleus. Inhibition of inhibitor of кB kinase-β (IKKβ) with SC-514 also revealed a strong attenuation of IL-11 induced phospho-p65Ser536. Inhibition of p90RSK1 with SL0101 was inconsistent but suggested a partial blockage of phospho-p65Ser536 whereas inhibition of Src kinase with PP2, did not affect phospho-p65Ser536 in IL-11 treated IEC-18 cells. There was no increased binding of p65 to the кB binding motif on DNA after IL-11 treatment. In mechanically wounded cells treated with IL-11, nuclear phospho-p65Ser536 was unaffected; however there was an evident potentiation of wound-induced COX-2 expression compared to untreated cells. In conclusion, IL-11 activates NFкB signaling in a non-classical manner through the phosphorylation of the p65 subunit. The predominant pathway appears to involve IKK and MEK signaling. Also, IL-11 modulates COX-2 expression in response to wounding in intestinal epithelial cells.