Abstract
Using monolayers of intestinal Caco-2 cells, we discovered that the θ isoform of protein kinase C (PKC), a member of the “novel” subfamily of PKC isoforms, is required for monolayer barrier function. However, the mechanisms underlying this novel effect remain largely unknown. Here, we sought to determine whether the mechanism by which PKC-θ disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC-θ-induced alterations in claudin isotypes. We used cell clones that we recently developed, clones that were transfected with varying levels of plasmid to either stably suppress endogenous PKC-θ activity (antisense, dominant-negative constructs) or to ectopically express PKC-θ activity (sense constructs). We then determined barrier function, claudin isotype integrity, PKC-θ subcellular activity, claudin isotype subcellular pools, and claudin phosphorylation. Antisense transfection to underexpress the PKC-θ led to monolayer instability as shown by reduced 1) endogenous PKC-θ activity, 2) claudin isotypes in the membrane and cytoskeletal pools (↓claud-1, ↓claud-4 assembly), 3) claudin isotype phosphorylation (↓ phospho-serine, ↓ phospho-threonine), 4) architectural stability of the claudin-1 and claudin-4 rings, and 5) monolayer barrier function. In these antisense clones, PKC-θ activity was also substantially reduced in the membrane and cytoskeletal cell fractions. In wild-type (WT) cells, PKC-θ (82 kDa) was both constitutively active and coassociated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-θ/claudin complexes. In a second series of studies, dominant-negative inhibition of the endogenous PKC-θ caused similar destabilizing effects on monolayer barrier dynamics, including claudin-1 and -4 hypophosphorylation, disassembly, and architectural instability as well as monolayer disruption. In a third series of studies, sense overexpression of the PKC-θ caused not only a mostly cytosolic distribution of this isoform (i.e., <12% in the membrane + cytoskeletal fractions, indicating PKC-θ inactivity) but also led to disruption of claudin assembly and barrier function of the monolayer. The conclusions of this study are that PKC-θ activity is required for normal claudin assembly and the integrity of the intestinal epithelial barrier. These effects of PKC-θ 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-θ to alter the dynamics of permeability protein claudins is a new function not previously ascribed to the novel subfamily of PKC isoforms.
Footnotes
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This work was supported in part by a grant from Rush University Medical Center, Department of Internal Medicine, and by National Institutes of Health Grants NIDDK 60511 and NCCAM 01581 (to A.B.) and National Institute on Alcohol Abuse and Alcoholism Grant 13745 (to A.K.). Portions of this work were presented in the abstract form during the annual meeting of the American Gastroenterological Association (Digestive Disease Week), May 2005.
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doi:10.1124/jpet.105.083428.
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ABBREVIATIONS: IBD, inflammatory bowel disease; PKC, protein kinase C; TRE, tetracycline-responsive expression; DMEM, Dulbecco's modified Eagle's medium; tTA, tetracycline-responsive transactivator; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid; LSCM, laser scanning confocal microscopy; ECL, enhanced chemiluminescence; TBST, Tris-buffered saline/Tween 20; AS, antisense; FD, fluorescein dextran; TTX, tetracycline.
- Received January 12, 2005.
- Accepted March 2, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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