RT Journal Article
SR Electronic
T1 Activation of δ-Isoform of Protein Kinase C Is Required for Oxidant-Induced Disruption of Both the Microtubule Cytoskeleton and Permeability Barrier of Intestinal Epithelia
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 17
OP 28
DO 10.1124/jpet.102.037218
VO 303
IS 1
A1 A. Banan
A1 J. Z. Fields
A1 A. Farhadi
A1 D. A. Talmage
A1 L. Zhang
A1 A. Keshavarzian
YR 2002
UL http://jpet.aspetjournals.org/content/303/1/17.abstract
AB Using monolayers of intestinal (Caco-2) cells, we showed that oxidants disassemble the microtubule cytoskeleton and disrupt barrier integrity (permeability) (Banan et al., 2000a). Because exposure of ourparental cells to oxidants causes protein kinase C (PKC)-δ to be translocated to particulate fractions, we hypothesized that PKC-δ activation is required for these oxidant effects. Monolayers of parental Caco-2 cells were incubated with oxidant (H2O2) ± modulators. Other cells were transfected with an inducible plasmid to stably overexpress PKC-δ or with a dominant negative plasmid to stably inhibit the activity of native PKC-δ. In parentalcells, oxidants caused translocation of PKC-δ to the particulate (membrane + cytoskeletal) fractions, activation of PKC-δ isoform, increases in monomeric (S1) tubulin and decreases in polymerized (S2) tubulin, disruption of the microtubule cytoarchitecture, and loss of barrier integrity (hyperpermeability). In transfected cells, induction of PKC-δ overexpression by itself (3.5-fold over its basal level) led to oxidant-like disruptive effects. Disruption induced by PKC-δ overexpression was potentiated by oxidants. Overexpressed PKC-δ resided in particulate fractions, indicating its activation. Stable inhibition of native PKC-δ activity (98%) by dominant negative transfection substantially protectedagainst all measures of oxidative disruption. We conclude that 1) oxidants induce loss of intestinal epithelial barrier integrity by disassembling the microtubules in large part through the activation of the PKC-δ isoform; and 2) overexpression and activation of PKC-δ is by itself a sufficient condition for disruption of these cytoskeleton and permeation pathways. Thus, PKC-δ activation may play a key role in intestinal dysfunction in oxidant-induced diseases such as inflammatory bowel disease. The American Society for Pharmacology and Experimental Therapeutics