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Protein and Vesicle Trafficking, Cytoskeleton

PKC-1 isoform activation is required for EGF-induced NF-B inactivation and IB stabilization and protection of F-actin assembly and barrier function in enterocyte monolayers

Division of Digestive Diseases, Department of Internal Medicine, Department of Pharmacology,and Department of Molecular Physiology, Rush University Medical Center, Chicago, Illinois 60612

Submitted 31 July 2003 ; accepted in final form 2 November 2003

Using monolayers of intestinal Caco-2 cells, we reported that activation of NF-B is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-1 prevents oxidant-induced activation of NF-B and the consequences of NF-B activation, F-actin, and barrier dysfunction. We used wild-type (WT) and transfected cells. The latter were transfected with varying levels of cDNA to overexpress or underexpress PKC-1. Cells were pretreated with EGF or PKC modulators ± oxidant. Pretreatment with EGF protected monolayers by increasing native PKC-1 activity, decreasing IB phosphorylation/degradation, suppressing NF-B activation (p50/p65 subunit nuclear translocation/activity), enhancing stable actin (increased F-actin-to-G-actin ratio), increasing stability of actin cytoskeleton, and reducing barrier hyperpermeability. Cells stably overexpressing PKC-1 were protected by low, previously nonprotective doses of EGF or modulators. In these clones, we found enhanced IB stabilization, NF-B inactivation, actin stability, and barrier function. Low doses of the modulators led to increases in PKC-1 in the particulate fractions, indicating activation. Stably inhibiting endogenous PKC-1 substantially prevented all measures of EGF's protection against NF-B activation. We conclude that EGF-mediated protection against oxidant disruption of the intestinal barrier function requires PKC-1 activation and NF-B suppression. The molecular event underlying this unique effect of PKC-1 involves inhibition of phosphorylation and increases in stabilization of IB. The ability to inhibit the dynamics of NF-B/IB and F-actin disassembly is a novel mechanism not previously attributed to the classic subfamily of PKC isoforms.

F-actin; cytoskeleton; growth factors; Caco-2 cells; gut barrier; protection; protein kinase C isoforms

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