θ 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 (2claud-1, 2claud-4 assembly), 3) claudin isotype phosphorylation (2 phospho-serine, 2 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., Ͻ12% in the membrane ϩ 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.The epithelium of the intestinal mucosa is the largest interface between the body and the external environment. An important characteristic of this interface is its ability to maintain a highly selective permeability barrier that protects the internal milieu from hostile factors in the lumenal environment. Barrier permeability, which in general is maintained by epithelial tight-junctional proteins, pe...

Protein Kinase C Activation Modulates Reversible Increase in Cortical Blood–Brain Barrier Permeability and Tight Junction Protein Expression during Hypoxia and Posthypoxic Reoxygenation

“…In the current study, we have assessed and determined changes in cortical endothelial cell distribution and expression of nPKC-y, and aPKC-z isozymes in isolated microvessels and membrane-and cytosolic-enriched fractions, induced by Hx and HR. The selection of these PKC isozymes was based on reports that these two isozymes are associated with TJ proteins (GonzalezMariscal et al, 2008), previous studies in our laboratory (Fleegal et al, 2005) and a recent report by Banan et al (2007) showing an association between PKC-y and claudin-5 in the intestine. We have shown increased expression and activation of nPKC-y and aPKC-z after Hx.…”

“…Immunofluorescence studies have shown PKC isozymes to colocalize with ZO-1 in Madin-Darby canine kidney and human colon adenocarcinoma cells (Dodane and Kachar, 1996). In other studies, activation of nPKC-y was reported to affect intestinal epithelial barrier function through modulation of claudin isotypes (Banan et al, 2004(Banan et al, , 2007.…”

“…The protein kinase C (PKC) family of serine/ threonine protein kinases includes at least 12 known isozymes divided into three groups depending on the enzymes' cofactor requirements: (1) conventional (cPKC; PKC-a, PKC-bI, PKC-bII, and PKC-g), (2) novel (nPKC; PKC-d, PKC-e, PKC-Z, and PKC-y), and (3) atypical (aPKC; PKC-l and PKC-z) (Steinberg, 2008). These isozymes differ in their mechanism of activation, subcellular distribution, substrate type, and expression, suggesting that each of these PKC isozymes can perform unique biological tasks (Banan et al, 2003(Banan et al, , 2004(Banan et al, , 2007. Although several studies have shown the importance of PKC in regulating BBB function (Andreeva et al, 2001;Rao et al, 2002), the role of specific PKC isozymes in barrier regulation and their mechanisms after ischemia remain largely unknown.…”

Phosphorylation of claudin-4 by PKCε regulates tight junction barrier function in ovarian cancer cells

“…Some investigations have shown modulation of claudin-4 by aPKC [30] and PKCθ [34]. In Caco-2 cells, constitutively active PKCθ is necessary to maintain barrier function and this process involves the phosphorylation of claudin-1 and -4 [34].…”

“…Interestingly, several studies have demonstrated the involvement of various kinases in the phosphorylation and regulation of claudin proteins [29][30][31][32][33][34][35][36][37], and we have recently shown that phosphorylation of claudin-3 by PKA can affect TJ properties in ovarian cancer cells [38]. Protein kinase C (PKC) isoforms are present in ovarian cancer and are known to modulate TJ function by phosphorylation of the proteins in the complex [24,34,[39][40][41][42][43], but it is unclear whether PKC can directly phosphorylate and regulate claudin proteins. We have previously shown that claudin-4 can be phosphorylated in ovarian cancer cells upon 12-OTetradecanoylophorbol-13-acetate (TPA) stimulation [38], but the exact PKC isoforms involved, the phosphorylation sites on claudin-4, and the consequences of this phosphorylation have remained unknown.…”