Abstract
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.
Footnotes
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This work was supported in part by a grant from Rush University Medical Center, Department of Internal Medicine, and by a grant from the American College of Gastroenterology. Portions of this work were presented in abstract form as an oral presentation: “Research Forum”, during the annual meeting of the American Gastroenterological Association (Digestive Disease World), May 2002.
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DOI: 10.1124/jpet.102.037218
- Abbreviations:
- GI
- gastrointestinal
- IBD
- inflammatory bowel disease
- PKC
- protein kinase C
- DMEM
- Dulbecco's modified Eagle's medium
- TRE
- tetracycline-responsive expression
- tTA
- tetracycline-responsive transactivator
- LSCM
- laser scanning confocal microscopy
- PAGE
- polyacrylamide gel electrophoresis
- O.D.
- optical density
- Received April 10, 2002.
- Accepted May 23, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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