Inhibition of Oxidant-Induced Nuclear Factor-κB Activation and Inhibitory-κBα Degradation and Instability of F-Actin Cytoskeletal Dynamics and Barrier Function by Epidermal Growth Factor: Key Role of Phospholipase-γ Isoform

Abstract

Using monolayers of intestinal (Caco-2) cells as a model for studying inflammatory bowel disease (IBD), we previously showed that nuclear factor-κB (NF-κB) activation is required for oxidant-induced disruption of cytoskeletal and barrier integrity. Epidermal growth factor (EGF) stabilizes the F-actin cytoskeleton and protects against oxidant damage, but the mechanism remains unclear. We hypothesized that the mechanism involves activation of phospholipase C-γ (PLC-γ), which prevents NF-κB activation and the consequences of this activation, namely, cytoskeletal and barrier disruption. We studied wild-type and transfected cells. The latter were transfected with varying levels (1-5 μg) of cDNA to either stably overexpress PLC-γ or to inhibit its activation. Cells were pretreated with EGF before exposure to oxidant (H₂O₂). Stably overexpressing PLC-γ (+2.0-fold) or preincubating with EGF protected against oxidant injury as indicated by 1) decreases in several NF-κB-related variables [NF-κB (p50/p65 subunit) nuclear translocation, NF-κB subunit activity, inhibitory-κBα (I-κBα) phosphorylation and degradation]; 2) increases in F-actin and decreases in G-actin; 3) stabilization of the actin cytoskeletal architecture; and 4) enhancement of barrier function. Overexpression induced inactivation of NF-κB was potentiated by EGF. PLC-γ was found mostly in membrane and cytoskeletal fractions (<9% in the cytosolic fractions), indicating its activation. Dominant negative inhibition of endogenous PLC-γ (-99%) substantially prevented all measures of EGF protection against NF-κB activation. We concluded 1) EGF protects against oxidant-induced barrier disruption through PLC-γ activation, which inactivates NF-κB; 2) Activation of PLC-γ by itself is protective against NF-κB activation; 3) the ability to modulate the dynamics of NF-κB/I-κ Bα is a novel mechanism not previously attributed to the PLC family of isoforms in cells; and 4) development of PLC-γ mimetics represents a possible new therapeutic strategy for IBD.