Key role of PKC and Ca2+ in EGF protection of microtubules and intestinal barrier against oxidants.
Using monolayers of human intestinal (Caco-2) cells, we showed that growth factors (GFs) protect microtubules and barrier integrity against oxidative injury. Studies in nongastrointestinal cell models suggest that protein kinase C (PKC) signaling is key in GF-induced effects and that cytosolic calcium concentration ([Ca2+](i)) is essential in cell integrity. We hypothesized that GF protection involves activating PKC and maintaining normal ([Ca2+](i)) Monolayers were pretreated with epidermal growth factor (EGF) or PKC or Ca2+ modulators before exposure to oxidants (H2O2 or HOCl). Oxidants disrupted microtubules and barrier integrity, and EGF protected from this damage. EGF caused rapid distribution of PKC-alpha, PKC-betaI, and PKC-zeta isoforms to cell membranes, enhancing PKC activity of membrane fractions while reducing PKC activity of cytosolic fractions. EGF enhanced (45)Ca2+ efflux and prevented oxidant-induced (sustained) rises in ([Ca2+](i)). PKC inhibitors abolished and PKC activators mimicked EGF protection. Oxidant damage was mimicked by and potentiated by a Ca2+ ionophore (A-23187), exacerbated by high-Ca2+ media, and prevented by calcium removal or chelation or by Ca2+ channel antagonists. PKC activators mimicked EGF on both (45)Ca2+ efflux and ([Ca2+](i)). Membrane Ca2+-ATPase pump inhibitors prevented protection by EGF or PKC activators. In conclusion, EGF protection of microtubules and the intestinal epithelial barrier requires activation of PKC signal transduction and normalization of ([Ca2+](i)).
Banan
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Department of Internal Medicine (Division of Digestive Diseases), Rush University Medical Center, 1725 W. Harrison, Suite 206, Chicago, IL 60612, USA. ali_banan@rush.edu
Fields
J Z
JZ
Zhang
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Keshavarzian
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D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D006997:Hypochlorous Acid / Q000633:toxicity; D007211:Indoles / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007527:Isoenzymes / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D008954:Models, Biological; D016877:Oxidants / Q000633:toxicity*; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D000099221:Protein Kinase C zeta
D000001:Calcimycin; D002118:Calcium; D000252:Calcium-Transporting ATPases; D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor; D006861:Hydrogen Peroxide; D006997:Hypochlorous Acid; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D016877:Oxidants; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate; D000099221:Protein Kinase C zeta; C496628:PRKCA protein, human; C070515:bisindolylmaleimide I