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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Key role of PLC- in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly

Departments of Internal Medicine (Section of Gastroenterology and Nutrition), Pharmacology, and Molecular Physiology, Rush University School of Medicine, Chicago, Illinois 60612

Submitted 1 April 2003 ; accepted in final form 29 May 2003

Upregulation of inducible nitric oxide synthase (iNOS) is key to oxidant-induced disruption of intestinal (Caco-2) monolayer barrier, and EGF protects against this disruption by stabilizing the cytoskeleton. PLC- appears to be essential for monolayer integrity. We thus hypothesized that PLC- activation is essential in EGF protection against iNOS upregulation and the consequent cytoskeletal oxidation and disarray and monolayer disruption. Intestinal cells were transfected to stably overexpress PLC- or to inhibit its activation and were then pretreated with EGF ± oxidant (H₂O₂). Wild-type (WT) intestinal cells were treated similarly. Relative to WT monolayers exposed to oxidant, pretreatment with EGF protected monolayers by: increasing native PLC- activity; decreasing six iNOS-related variables (iNOS activity/protein, NO levels, oxidative stress, actin oxidation/nitration); increasing stable F-actin; maintaining actin stability; and enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected monolayers overexpressing PLC- (+2.3-fold) were protected, as indicated by decreases in all measures of iNOS-driven pathway and enhanced actin and barrier integrity. Overexpression-induced inhibition of iNOS was potentiated by low doses of EGF. Stable inhibition of PLC- prevented all measures of EGF protection against iNOS upregulation. We conclude that 1) EGF protects against oxidative stress disruption of intestinal barrier by stabilizing F-Actin, largely through the activation of PLC- and downregulation of iNOS pathway; 2) activation of PLC- is by itself essential for cellular protection against oxidative stress of iNOS; and 3) the ability to suppress iNOS-driven reactions and cytoskeletal oxidation and disassembly is a novel mechanism not previously attributed to the PLC family of isoforms.

actin cytoskeleton; gut barrier; growth factors; oxidative stress; nitration and carbonylation; reactive nitrogen metabolites; phospholipase C isoform; inflammatory bowel disease; Caco-2 cells

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