Received for publication January 13, 2005.
Revised March 2, 2005.
Accepted for publication March 2, 2005.

Theta () Isoform of PKC Alters Barrier Function in Intestinal Epithelium through Modulation of Distinct Claudin Isotypes: A Novel Mechanism for Regulation of Permeability

Abstract

Using monolayers of intestinal Caco-2 cells, we discovered that the theta isoform of PKC, a member of the "novel" subfamily of PKC isoforms, is required for monolayer barrier function. However, the mechanisms underlying this novel effect remains largely unknown. Here, we sought to determine whether the mechanism by which PKC- disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC- induced alterations in claudin isotypes. We utilized cell clones that we recently developed, clones which were transfected with varying levels of plasmid to either stably suppress endogenous PKC- activity (anti-sense, dominant negative constructs) or to ectopically express PKC- activity (sense constructs). We then determined barrier function; claudin isotype integrity; PKC- subcellular activity; claudin isotype subcellular pools; and claudin phosphorylation. Anti-sense transfection to under-express the PKC- led to monolayer instability as shown by reduced: [1] endogenous PKC- activity, [2] claudin isotypes in the membrane & cytoskeletal pools (decreased claud-1, claud-4 assembly), [3] claudin isotypes phosphorylation (decreased phospho-serine, phospho-threonine), [4] architectural stability of the claudin-1 & claudin-4 rings, and [5] monolayer barrier function. In these anti-sense clones PKC- activity was also substantially reduced in the membrane & cytoskeletal cell fractions. In wild type (WT) cells, PKC- (82 kDa) was both constitutively active and co-associated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-/claudin complexes. In a 2nd series of studies, dominant negative inhibition of the endogenous PKC- caused similar destabilizing effects on monolayer barrier dynamics, including claudin-1 & -4 hypo-phosphorylation, disassembly, and architectural instability as well as monolayer disruption. In a 3rd series of studies, sense over-expression of the PKC- caused not only a mostly cytosolic distribution of this isoform (i.e., <12% in the membrane + cytoskeletal fractions, indicating PKC- inactivity), but also led to disruption of claudins assembly and barrier function of the monolayer. Conclusions: PKC- activity is required for normal claudin assembly and the integrity of the intestinal epithelial barrier. These effects of PKC- are mediated at the molecular level by changes in phosphorylation, membrane assembly, and/or organization of the subunit components of two barrier function proteins: claudin-1 and claudin-4 isotypes. The ability of PKC- to alter the dynamics of permeability protein claudins is a new function not previously ascribed to the "novel" subfamily of PKC isoforms.

Key words: Cytoskeleton, Epithelial Barrier Function, GI, IBD, Inflammation, PKC isoform

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