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CELLULAR AND MOLECULAR

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

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

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

Address correspondence to: Dr. A. Banan, GI Physiology and Pharmacology, Section of Gastroenterology and Nutrition, Rush University of Chicago, School of Medicine, 1725 W. Harrison, Suite 206, Chicago, IL 60612. E-mail: ali_banan{at}rush.edu