Key role of PLC-gamma in EGF protection of epithelial barrier against iNOS upregulation and F-actin nitration and disassembly.
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.
Banan
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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
Zhang
L J
LJ
Shaikh
M
M
Fields
J Z
JZ
Farhadi
A
A
Keshavarzian
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A
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
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