title: Key role of PLC-gamma in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly.
abstract: 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-gamma appears to be essential for monolayer integrity. We thus hypothesized that PLC-gamma 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-gamma 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-gamma 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-gamma (+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-gamma 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-gamma and downregulation of iNOS pathway; 2) activation of PLC-gamma 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.
authors.affiliation: Department of Internal Medicine, Section of Gastroenterology and Nutrition, Rush University School of Medicine, 1725 W. Harrison, Suite 206, Chicago, IL 60612, USA. ali_banan@rush.edu
authors.forename: A
authors.initials: A
authors.lastname: Banan
authors.forename: L J
authors.initials: LJ
authors.lastname: Zhang
authors.forename: M
authors.initials: M
authors.lastname: Shaikh
authors.forename: J Z
authors.initials: JZ
authors.lastname: Fields
authors.forename: A
authors.initials: A
authors.lastname: Farhadi
authors.forename: A
authors.initials: A
authors.lastname: Keshavarzian
mesh_terms: D008841:Actin Cytoskeleton / Q000502:physiology; D000199:Actins / Q000378:metabolism*; D018938:Caco-2 Cells; D003599:Cytoskeleton / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D010446:Peptide Fragments / Q000494:pharmacology; D010539:Permeability; D054801:Phosphoinositide Phospholipase C; D051966:Phospholipase C gamma; D011499:Protein Processing, Post-Translational; D014018:Tissue Distribution; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000199:Actins; D009566:Nitrates; D016877:Oxidants; D010446:Peptide Fragments; D004815:Epidermal Growth Factor; D006861:Hydrogen Peroxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D010738:Type C Phospholipases; C499235:PLCZ1 protein, human; D054801:Phosphoinositide Phospholipase C; D051966:Phospholipase C gamma