Interferon-gamma Inhibits Enterocyte Migration by Reversibly Displacing Connexin43 from Lipid Rafts

Necrotizing enterocolitis (NEC) is associated with the release of interferon-gamma (IFN) by enterocytes and delayed intestinal restitution. Our laboratory has recently demonstrated that IFN inhibits enterocyte migration by impairing enterocyte gap junctions, intercellular channels that are composed of connexin43 (Cx43) monomers and that are required for enterocyte migration to occur.  We show that lipid rafts are required for enterocyte migration, that IFN displaces Cx43 from lipid rafts, and that the phorbol ester phorbol 12-myristate 13-acetate (PMA) restores Cx43 to lipid rafts after treatment with IFN in a protein kinase C-dependent manner. IFN also reversibly decreased the phosphorylation of Cx43 on lipid rafts, which was restored by PMA. Strikingly, restoration of Cx43 to lipid rafts by PMA or by transfection of enterocytes with adenoviruses expressing wild-type Cx43 but not mutant Cx43 is associated with the restoration of enterocyte migration after IFN treatment. Taken together, these findings suggest an important role for lipid raft-Cx43 interactions in the regulation of enterocyte migration during exposure to IFN, such as NEC.
As shown above, IFN reversibly displaces Cx43 from lipid rafts in IEC-6 cells and decreases the phosphorylation of Cx43 at lipid rafts. These events are reversed by PMA. In set A, IEC-6 cells were either untreated (top row), treated with IFN (middle) or treated with IFN in the presence of PMA (bottom). Cells were stained with antibodies against Cx43 (red) and Cav-1 (green) along with the nuclear marker Draq-5 (blue) and were examined by immunoconfocal microscopy. Arrows indicate sites of colocalization of Cx43 and Cav-1 on the merged images. In set B, confocal micrographs of IEC-6 cells that were either untreated (left) or were treated with IFN in the absence (center) or presence (right) of PMA, treated with cholera toxin and immunostained with antibodies against phosphorylated Cx43. The colocalization of phosphorylated Cx43 with lipid rafts is revealed by arrows.
Leaphart CL, Dai S, Gribar SC, Richardson W, Ozolek J, Shi XH, Bruns JR, Branca M, Li J, Weisz OA, Sodhi C, Hackam DJ
Am J Physiol Gastrointest Liver Physiol
2008 Sep.
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January 27, 2011
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Last Update
January 27, 2011