title: PKC-beta1 isoform activation is required for EGF-induced NF-kappaB inactivation and IkappaBalpha stabilization and protection of F-actin assembly and barrier function in enterocyte monolayers.
abstract: Using monolayers of intestinal Caco-2 cells, we reported that activation of NF-kappaB is required for oxidative disruption and that EGF protects against this injury but the mechanism remains unclear. Activation of the PKC-beta1 isoform is key to monolayer barrier integrity. We hypothesized that EGF-induced activation of PKC-beta1 prevents oxidant-induced activation of NF-kappaB and the consequences of NF-kappaB activation, F-actin, and barrier dysfunction. We used wild-type (WT) and transfected cells. The latter were transfected with varying levels of cDNA to overexpress or underexpress PKC-beta1. Cells were pretreated with EGF or PKC modulators +/- oxidant. Pretreatment with EGF protected monolayers by increasing native PKC-beta1 activity, decreasing IkappaBalpha phosphorylation/degradation, suppressing NF-kappaB activation (p50/p65 subunit nuclear translocation/activity), enhancing stable actin (increased F-actin-to-G-actin ratio), increasing stability of actin cytoskeleton, and reducing barrier hyperpermeability. Cells stably overexpressing PKC-beta1 were protected by low, previously nonprotective doses of EGF or modulators. In these clones, we found enhanced IkappaBalpha stabilization, NF-kappaB inactivation, actin stability, and barrier function. Low doses of the modulators led to increases in PKC-beta1 in the particulate fractions, indicating activation. Stably inhibiting endogenous PKC-beta1 substantially prevented all measures of EGF's protection against NF-kappaB activation. We conclude that EGF-mediated protection against oxidant disruption of the intestinal barrier function requires PKC-beta1 activation and NF-kappaB suppression. The molecular event underlying this unique effect of PKC-beta1 involves inhibition of phosphorylation and increases in stabilization of IkappaBalpha. The ability to inhibit the dynamics of NF-kappaB/IkappaBalpha and F-actin disassembly is a novel mechanism not previously attributed to the classic subfamily of PKC isoforms.
authors: Division of Digestive Diseases, Department of Internal Medicine, Department of Pharmacology, and Department of Molecular Physiology, Rush University Medical Center, Chicago, Illinois 60612, USA. ali_banan@rush.edu
authors: A
authors: A
authors: Banan
authors: L J
authors: LJ
authors: Zhang
authors: A
authors: A
authors: Farhadi
authors: J Z
authors: JZ
authors: Fields
authors: M
authors: M
authors: Shaikh
authors: A
authors: A
authors: Keshavarzian
mesh_terms: D000199:Actins / Q000378:metabolism*; D018938:Caco-2 Cells; D002467:Cell Nucleus / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D020895:Enterocytes / Q000187:drug effects / Q000201:enzymology*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D020319:Oligodeoxyribonucleotides, Antisense; D018384:Oxidative Stress / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D012694:Serine / Q000378:metabolism; D014162:Transfection
chemical_list: D000199:Actins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D020319:Oligodeoxyribonucleotides, Antisense; D000072000:NF-KappaB Inhibitor alpha; D012694:Serine; D004815:Epidermal Growth Factor; D011493:Protein Kinase C; D064546:Protein Kinase C beta