PKC-β1 isoform activation is required for EGF-induced NF-κB inactivation and IκBα stabilization and protection of F-actin assembly and barrier function in enterocyte monolayers

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Abstract
Using monolayers of intestinal Caco-2 cells, we reported that activation of NF-κB is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-β1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-β1 prevents oxidant-induced activation of NF-κB and the consequences of NF-κB 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-β1. Cells were pretreated with EGF or PKC modulators ± oxidant. Pretreatment with EGF protected monolayers by increasing native PKC-β1 activity, decreasing IκBα phosphorylation/degradation, suppressing NF-κB 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-β1 were protected by low, previously nonprotective doses of EGF or modulators. In these clones, we found enhanced IκBα stabilization, NF-κB inactivation, actin stability, and barrier function. Low doses of the modulators led to increases in PKC-β1 in the particulate fractions, indicating activation. Stably inhibiting endogenous PKC-β1 substantially prevented all measures of EGF's protection against NF-κB activation. We conclude that EGF-mediated protection against oxidant disruption of the intestinal barrier function requires PKC-β1 activation and NF-κB suppression. The molecular event underlying this unique effect of PKC-β1 involves inhibition of phosphorylation and increases in stabilization of IκBα. The ability to inhibit the dynamics of NF-κB/IκBα and F-actin disassembly is a novel mechanism not previously attributed to the classic subfamily of PKC isoforms.

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