θ-Isoform of PKC is required for alterations in cytoskeletal dynamics and barrier permeability in intestinal epithelium: a novel function for PKC-θ

Authors:
  • LZL.J. Zhang
    L.J. Zhang
  • MSM. Shaikh
    M. Shaikh
  • JFJ.Z. Fields
    J.Z. Fields
  • AFA. Farhadi
    A. Farhadi
  • AKA. Keshavarzian
    A. Keshavarzian
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Abstract
Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the θ-isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC-θ is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC-θ (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC-θ showed monolayer injury as indicated by decreased native PKC-θ activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC-θ was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC-θ was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-θ/tubulin complex. Second, DN transfection to inhibit the endogenous PKC-θ led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC-θ led to a mostly cytosolic distribution of θ-isoform (

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