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

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 theta-isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC-theta 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-theta (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 under-expressing PKC-theta showed monolayer injury as indicated by decreased native PKC-theta 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-theta was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC-theta was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-theta/tubulin complex. Second, DN transfection to inhibit the endogenous PKC-theta led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC-theta led to a mostly cytosolic distribution of theta-isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1) PKC-theta isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2) the molecular event underlying this novel biological effect of PKC-theta involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-theta.