PKC-beta1 mediates EGF protection of microtubules and barrier of intestinal monolayers against oxidants.
Using monolayers of human intestinal (Caco-2) cells, we found that oxidants and ethanol damage the cytoskeleton and disrupt barrier integrity; epidermal growth factor (EGF) prevents damage by enhancement of protein kinase C (PKC) activity and translocation of the PKC-beta1 isoform. To see if PKC-beta1 mediates EGF protection, cells were transfected to stably over- or underexpress PKC-beta1. Transfected monolayers were preincubated with low or high doses of EGF (1 or 10 ng/ml) or 1-oleoyl-2-acetyl-sn-glycerol [OAG; a PKC activator (0.01 or 50 microM)] before treatment with oxidant (0.5 mM H(2)O(2)). Only in monolayers overexpressing PKC-beta1 (3.1-fold) did low doses of EGF or OAG initiate protection, increase tubulin polymerization (assessed by quantitative immunoblotting) and microtubule architectural integrity (laser scanning confocal microscopy), maintain normal barrier permeability (fluorescein sulfonic acid clearance), and cause redistribution of PKC-beta1 from cytosolic pools into membrane and/or cytoskeletal fractions (assessed by immunoblotting), thus indicating PKC-beta1 activation. Antisense inhibition of PKC-beta1 expression (-90%) prevented these changes and abolished EGF protection. We conclude that EGF protection against oxidants requires PKC-beta1 isoform activation. This mechanism may be useful for development of novel therapies for the treatment of inflammatory gastrointestinal disorders including inflammatory bowel disease.
Banan
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A
Division of Digestive Diseases, Department of Internal Medicine, Rush University Medical Center, Chicago, Illinois 60612, USA. ali_banan@rush.edu
Fields
J Z
JZ
Talmage
D A
DA
Zhang
Y
Y
Keshavarzian
A
A
D018938:Caco-2 Cells; D002463:Cell Membrane Permeability / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology / Q000187:drug effects; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D014162:Transfection
D020536:Enzyme Activators; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D016877:Oxidants; D004815:Epidermal Growth Factor; D006861:Hydrogen Peroxide; D011493:Protein Kinase C; D064546:Protein Kinase C beta