PKC-zeta prevents oxidant-induced iNOS upregulation and protects the microtubules and gut barrier integrity.
Using intestinal (Caco-2) monolayers, we reported that inducible nitric oxide synthase (iNOS) activation is key to oxidant-induced barrier disruption and that EGF protects against this injury. PKC-zeta was required for protection. We thus hypothesized that PKC-zeta activation and iNOS inactivation are key in EGF protection. Wild-type (WT) Caco-2 cells were exposed to H(2)O(2) (0.5 mM) +/- EGF or PKC modulators. Other cells were transfected to overexpress PKC-zeta or to inhibit it and then pretreated with EGF or a PKC activator (OAG) before oxidant. Relative to WT cells exposed to oxidant, pretreatment with EGF protected monolayers by 1) increasing PKC-zeta activity; 2) decreasing iNOS activity and protein, NO levels, oxidative stress, tubulin oxidation, and nitration); 3) increasing polymerized tubulin; 4) maintaining the cytoarchitecture of microtubules; and 5) enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected cells overexpressing PKC-zeta (+2.9-fold) were protected as indicated by decreases in all measures of iNOS-driven pathways and enhanced stability of microtubules and barrier function. Overexpression-induced inhibition of iNOS was OAG independent, but EGF potentiated this protection. Antisense inhibition of PKC-zeta (-95%) prevented all measures of EGF protection against iNOS upregulation. Thus EGF protects against oxidative disruption of the intestinal barrier by stabilizing the cytoskeleton in large part through the activation of PKC-zeta and downregulation of iNOS. Activation of PKC-zeta is by itself required for cellular protection against oxidative stress of iNOS. We have thus discovered novel biologic functions, suppression of the iNOS-driven reactions and cytoskeletal oxidation, among the atypical PKC isoforms.
Banan
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Department of Internal Medicine, Section of Gastroenterology and Nutrition, Rush University Medical Center, Chicago, Illinois 60612, USA. ali_banan@rush.edu
Zhang
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L
Fields
J Z
JZ
Farhadi
A
A
Talmage
D A
DA
Keshavarzian
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A
D018938:Caco-2 Cells; D004357:Drug Synergism; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007422:Intestines / Q000502:physiology*; D008870:Microtubules / Q000502:physiology* / Q000648:ultrastructure; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D000099221:Protein Kinase C zeta
D004815:Epidermal Growth Factor; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D016877:Oxidants; D011493:Protein Kinase C; D014404:Tubulin; D000099221:Protein Kinase C zeta; C496319:NOS2 protein, human