title: theta Isoform of protein kinase C 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 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- disrupts monolayer permeability and dynamics in intestinal epithelium involves PKC--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- activity (antisense, 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. Antisense transfection to underexpress the PKC- led to monolayer instability as shown by reduced 1) endogenous PKC- activity, 2) claudin isotypes in the membrane and cytoskeletal pools ( downward arrowclaud-1, downward arrowclaud-4 assembly), 3) claudin isotype phosphorylation ( downward arrow phospho-serine, downward arrow phospho-threonine), 4) architectural stability of the claudin-1 and claudin-4 rings, and 5) monolayer barrier function. In these antisense clones, PKC- activity was also substantially reduced in the membrane and cytoskeletal cell fractions. In wild-type (WT) cells, PKC- (82 kDa) was both constitutively active and coassociated with claudin-1 (22 kDa) and claudin-4 (25 kDa), forming endogenous PKC-/claudin complexes. In a second series of studies, dominant-negative inhibition of the endogenous PKC- 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- 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 claudin assembly and barrier function of the monolayer. The conclusions of this study are that 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.
authors.affiliation: Department of Internal Medicine, Rush University Medical Center, Chicago, IL 60612, USA. ali_banan@rush.edu
authors.forename: A
authors.initials: A
authors.lastname: Banan
authors.forename: L J
authors.initials: LJ
authors.lastname: Zhang
authors.forename: M
authors.initials: M
authors.lastname: Shaikh
authors.forename: J Z
authors.initials: JZ
authors.lastname: Fields
authors.forename: S
authors.initials: S
authors.lastname: Choudhary
authors.forename: C B
authors.initials: CB
authors.lastname: Forsyth
authors.forename: A
authors.initials: A
authors.lastname: Farhadi
authors.forename: A
authors.initials: A
authors.lastname: Keshavarzian
mesh_terms: D018938:Caco-2 Cells; D062445:Claudin-1; D062506:Claudin-4; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D007527:Isoenzymes / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology*; D010539:Permeability; D010766:Phosphorylation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000502:physiology*; D000074121:Protein Kinase C-theta; D011956:Receptors, Cell Surface / Q000502:physiology
chemical_list: C568108:CLDN1 protein, human; C568157:CLDN4 protein, human; D062445:Claudin-1; D062506:Claudin-4; D007527:Isoenzymes; D008565:Membrane Proteins; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; C076233:PRKCQ protein, human; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta