title: Zeta isoform of protein kinase C prevents oxidant-induced nuclear factor-kappaB activation and I-kappaBalpha degradation: a fundamental mechanism for epidermal growth factor protection of the microtubule cytoskeleton and intestinal barrier integrity.
abstract: Oxidant damage and gut barrier disruption contribute to the pathogenesis of a variety of inflammatory gastrointestinal disorders, including inflammatory bowel disease (IBD). In our studies using a model of the gastrointestinal (GI) epithelial barrier, monolayers of intestinal (Caco-2) cells, we investigated damage to and protection of the monolayer barrier. We reported that activation of nuclear factor-kappaB (NF-kappaB) via degradation of its endogenous inhibitor I-kappaBalpha is key to oxidant-induced disruption of barrier integrity and that growth factor (epidermal growth factor, EGF) protects against this injury by stabilizing the cytoskeletal filaments. Protein kinase C (PKC) activation seems to be required for monolayer maintenance, especially activation of the atypical zeta isoform of PKC. In an attempt to investigate, at the molecular level, the fundamental events underlying EGF protection against oxidant disruption, we tested the intriguing hypothesis that EGF-induced activation of PKC-zeta prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB activation, namely, cytoskeletal and barrier disruption. Monolayers of wild-type (WT) Caco-2 cells were incubated with oxidant (H2O2) with or without EGF or modulators. In other studies, we used the first gastrointestinal cell clones created by stable transfection of varying levels (1-5 microg) of cDNA to either overexpress PKC-zeta or to inhibit its expression. Transfected cell clones were then pretreated with EGF or a PKC activator (diacylglycerol analog 1-oleoyl-2-acetyl-glycerol, OAG) before oxidant. We monitored the following endpoints: monolayer barrier integrity, stability of the microtubule cytoskeleton, subcellular distribution and activity of the PKC-zeta isoform, intracellular levels and phosphorylation of the NF-kappaB inhibitor I-kappaBalpha, and nuclear translocation and activity of NF-kappaB subunits p65 and p50. Monolayers were also fractionated and processed to assess alterations in the structural protein of the microtubules, polymerized tubulin (S2), and monomeric tubulin (S1). Our data indicated that relative to WT monolayers exposed only to oxidant, pretreatment with EGF protected cell monolayers by 1) increasing native PKC-zeta activity; 2) decreasing several variables related to NF-kappaB activation [NF-kappaB (both p50 and p65 subunits) nuclear translocation, NF-kappaB subunits activity, I-kappaBalpha degradation, and phosphorylation]; 3) increasing stable tubulin (increased polymerized S2 tubulin and decreased monomeric S1 tubulin); 4) maintaining the cytoarchitectural integrity of microtubules; and 5) preventing hyperpermeability (barrier disruption). In addition, relative to WT cells exposed to oxidant, monolayers of transfected cells stably overexpressing PKC-zeta (approximately 3.0-fold increase) were protected as indicated by decreases in all measures of NF-kappaB activation as well as enhanced stability of microtubule cytoarchitecture and barrier function. Overexpression induced stabilization of I-kappaBalpha and inactivation of NF-kappaB was OAG-independent, although EGF potentiated this protection. Approximately 90% of the overexpressed PKC-zeta resided in particulate (membrane + cytoskeletal) fractions (with less than 10% in cytosolic fractions), indicating constitutive activation of the zeta isoform of PKC. Furthermore, antisense transfection to stably inhibit native PKC-zeta expression (-95%) and activation (-99%) prevented all measures of EGF-induced protection against NF-kappaB activation and monolayer disruption. We conclude the following: 1) EGF protects against oxidant disruption of the intestinal barrier integrity, in large part, through the activation of PKC-zeta and inactivation of NF-kappaB (an inflammatory mediator); 2) activation of PKC-zeta is by itself required for monolayer protection against oxidant stress of NF-kappaB activation; 3) the mechanism underlying this novel biological effect of the atypical PKC isoform zeta seems to involve suppression of phosphorylation and enhancement of stabilization of I-kappaBalpha; and 4) development of agents that can mimic or enhance PKC-zeta-induced suppression of NF-kappaB activation may be a useful therapeutic strategy for preventing oxidant damage to GI mucosal epithelium in disorders such as IBD. To our knowledge, this is the first report that PKC-zeta can inhibit the dynamics of NF-kappaB and cytoskeletal disassembly in cells.
authors.affiliation: Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois, USA. ali_banan@rush.edu
authors.forename: A
authors.initials: A
authors.lastname: Banan
authors.forename: J Z
authors.initials: JZ
authors.lastname: Fields
authors.forename: L J
authors.initials: LJ
authors.lastname: Zhang
authors.forename: M
authors.initials: M
authors.lastname: Shaikh
authors.forename: A
authors.initials: A
authors.lastname: Farhadi
authors.forename: A
authors.initials: A
authors.lastname: Keshavarzian
mesh_terms: D021581:Active Transport, Cell Nucleus; D018938:Caco-2 Cells; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D006861:Hydrogen Peroxide; D020672:I-kappa B Proteins / Q000378:metabolism*; D007422:Intestines / Q000502:physiology; D008870:Microtubules / Q000187:drug effects / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D000099221:Protein Kinase C zeta
chemical_list: D004815:Epidermal Growth Factor; D006861:Hydrogen Peroxide; D020672:I-kappa B Proteins; D016328:NF-kappa B; D020319:Oligodeoxyribonucleotides, Antisense; D016877:Oxidants; D011493:Protein Kinase C; D012694:Serine; D014404:Tubulin; D000099221:Protein Kinase C zeta