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

Using monolayers of intestinal (Caco-2) cells as a model for studying inflammatory bowel disease (IBD), we previously showed that nuclear factor-kappaB (NF-kappaB) 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-gamma (PLC-gamma), which prevents NF-kappaB activation and the consequences of this activation, namely, cytoskeletal and barrier disruption. We studied wildtype and transfected cells. The latter were transfected with varying levels (1 - 5 mug) of cDNA to either stably overexpress PLC-gamma or to inhibit its activation. Cells were pretreated with EGF before exposure to oxidant (H2O2). Stably overexpressing PLC-gamma ( + 2.0-fold) or preincubating with EGF protected against oxidant injury as indicated by 1) decreases in several NF-kappaB-related variables [NF-kappaB (p50/p65 subunit) nuclear translocation, NF-kappaB subunit activity, inhibitory-kappaBalpha (I-kappaBalpha) 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-kappaB was potentiated by EGF. PLC-gamma was found mostly in membrane and cytoskeletal fractions ( < 9% in the cytosolic fractions), indicating its activation. Dominant negative inhibition of endogenous PLC-gamma ( - 99%) substantially prevented all measures of EGF protection against NF-kappa B activation. We concluded 1) EGF protects against oxidant-induced barrier disruption through PLC-gamma activation, which inactivates NF-kappa B; 2) Activation of PLC-gamma by itself is protective against NF-kappa B activation; 3) the ability to modulate the dynamics of NF-kappa B/I-kappa B alpha is a novel mechanism not previously attributed to the PLC family of isoforms in cells; and 4) development of PLC-gamma mimetics represents a possible new therapeutic strategy for IBD.