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

• DOI: 10.1124/jpet.102.037218
• Publication Date: 2003-01-10T18:46:56Z
• Authors: A. Banan; J.Z. Fields; A. Farhadi; D.A. Talmage; L. Zhang; A. Keshavarzian
• Keywords: 0303 health sciences; Cell Membrane Permeability; Hydrogen Peroxide; Tetracycline; Oxidants; Transfection; Microtubules; Recombinant Proteins; Enzyme Activation; Isoenzymes; Protein Kinase C-delta; 03 medical and health sciences; Aminoglycosides; Cinnamates; Humans; Caco-2 Cells; Hygromycin B; Intestinal Mucosa; Oxidation-Reduction; Protein Kinase C

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 our parental cells to oxidants causes protein kinase C (PKC)-delta to be translocated to particulate fractions, we hypothesized that PKC-delta activation is required for these oxidant effects. Monolayers of parental Caco-2 cells were incubated with oxidant (H(2)O(2)) +/- modulators. Other cells were transfected with an inducible plasmid to stably overexpress PKC-delta or with a dominant negative plasmid to stably inhibit the activity of native PKC-delta. In parental cells, oxidants caused translocation of PKC-delta to the particulate (membrane + cytoskeletal) fractions, activation of PKC-delta 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-delta overexpression by itself (3.5-fold over its basal level) led to oxidant-like disruptive effects. Disruption induced by PKC-delta overexpression was potentiated by oxidants. Overexpressed PKC-delta resided in particulate fractions, indicating its activation. Stable inhibition of native PKC-delta activity (98%) by dominant negative transfection substantially protected against 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-delta isoform; and 2) overexpression and activation of PKC-delta is by itself a sufficient condition for disruption of these cytoskeleton and permeation pathways. Thus, PKC-delta activation may play a key role in intestinal dysfunction in oxidant-induced diseases such as inflammatory bowel disease.