The beta 1 isoform of protein kinase C mediates the protective effects of epidermal growth factor on the dynamic assembly of F-actin cytoskeleton and normalization of calcium homeostasis in human colonic cells.
Using intestinal monolayers, we showed that F-actin cytoskeletal stabilization and Ca(2+) normalization contribute to epidermal growth factor (EGF)-mediated protection against oxidant injury. However, the intracellular mediator responsible for these protective effects remains unknown. Since the protein kinase C-beta1 (PKC-beta1) isoform is abundant in our naive (N) cells, we hypothesized that PKC-beta1 is essential to EGF protection. Monolayers of N Caco-2 cells were exposed to H(2)O(2) +/- EGF, PKC, or Ca(2+) modulators. Other cells were transfected to over-express PKC-beta1 or to inhibit its expression and then pretreated with low or high doses of EGF or a PKC activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), before H(2)O(2). In N monolayers exposed to oxidant, pretreatment with EGF or PKC activators activated PKC-beta1, enhanced (45)Ca(2+) efflux, normalized Ca(2+), decreased monomeric G-actin, increased stable F-actin, and protected the cytoarchitecture of the actin. PKC inhibitors prevented these protective effects. Transfected cells stably over-expressing PKC-beta1 (+3.1-fold) but not N cell monolayers were protected from injury by even lower doses of EGF or OAG. EGF or OAG rapidly activated the over-expressed PKC-beta1. Antisense inhibition of PKC-beta1 expression (-90%) prevented all measures of EGF protection. Inhibitors of Ca(2+)-ATPase prevented EGF protection in N cells as well as protective synergism in transfected cells. EGF protects the assembly of the F-actin cytoskeleton in intestinal monolayers against oxidants in large part through the activation of PKC-beta1. EGF normalizes Ca(2+) by enhancing Ca(2+) efflux through PKC-beta1. We have identified novel biologic functions, protection of actin and Ca(2+) homeostasis, among the classical isoforms of PKC.
Banan
Ali
A
Department of Internal Medicine, Section of Gastroenterology and Nutrition, Rush University Medical Center, 1725 West Harrison, Chicago, IL 60612, USA. ali_banan@rush.edu
Fields
J Z
JZ
Farhadi
A
A
Talmage
D A
DA
Zhang
L
L
Keshavarzian
A
A
D000199:Actins / Q000378:metabolism*; D018938:Caco-2 Cells / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D006706:Homeostasis / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D016877:Oxidants / Q000037:antagonists & inhibitors / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D064546:Protein Kinase C beta; D014162:Transfection
D000199:Actins; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D016877:Oxidants; D004815:Epidermal Growth Factor; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D002118:Calcium