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Am J Physiol Cell Physiol 287: C218-C234, 2004. First published February 25, 2004; doi:10.1152/ajpcell.00575.2003
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Protein and Vesicle Trafficking, Cytoskeleton

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

A. Banan, L.J. Zhang, M. Shaikh, J.Z. Fields, A. Farhadi, and A. Keshavarzian

Division of Digestive Diseases, Department of Internal Medicine, Department of Pharmacology, and Department of Molecular Physiology, Rush University Medical Center, Chicago, Illinois 60612

Submitted 19 December 2003 ; accepted in final form 17 February 2004

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 underexpressing 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}.

microtubules; tubulin; occludin; epithelial barrier permeability; protein kinase C isoform


Address for reprint requests and other correspondence: A. Banan, Director of Research, Section of Gastroenterology and Nutrition, Rush Univ. of Chicago School of Medicine, 1725 W. Harrison, Suite 206, Chicago, IL 60612 (E-mail: ali_banan{at}rush.edu).




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