Overexpression of protein kinase C-δ increases tight junction permeability in LLC-PK₁epithelia

Abstract

The Ca2+-independent δ-isoform of protein kinase C (PKC-δ) was overexpressed in LLC-PK1 epithelia and placed under control of a tetracycline-responsive expression system. In the absence of tetracycline, the exogenous PKC-δ is expressed. Western immunoblots show that the overexpressed PKC-δ is found in the cytosolic, membrane-associated, and Triton-insoluble fractions. Overexpression of PKC-δ produced subconfluent and confluent epithelial morphologies similar to that observed on exposure of wild-type cells to the phorbol ester 12- O-tetradecanoylphorbol-13-acetate. Transepithelial electrical resistance ( R T) in cell sheets overexpressing PKC-δ was only 20% of that in cell sheets incubated in the presence of tetracycline, in which the amount of PKC-δ and R Twere similar to those in LLC-PK1parental cell sheets. Overexpression of PKC-δ also elicited a significant increase in transepithelial flux ofd-[14C]mannitol and a radiolabeled 2 × 106-molecular-weight dextran, suggesting with the R T decrease that overexpression increased paracellular, tight junctional permeability. Electron microscopy showed that PKC-δ overexpression results in a multilayered cell sheet, the tight junctions of which are almost uniformly permeable to ruthenium red. Freeze-fracture electron microscopy indicates that overexpression of PKC-δ results in a more disorganized arrangement of tight junctional strands. As with LLC-PK1 cell sheets treated with 12- O-tetradecanoylphorbol-13-acetate, the reduced R T, increasedd-mannitol flux, and tight junctional leakiness to ruthenium red that are seen with PKC-δ overexpression suggest the involvement of PKC-δ in regulation of tight junctional permeability.

Protein Kinase C Signaling Regulates ZO-1 Translocation and Increased Paracellular Flux of T84 Colonocytes Exposed toClostridium difficile Toxin A

“…In pulmonary epithelial monolayers, PKC␣ activation modulates TNF␣-induced increase in paracellular permeability (31). Similarly, it has been reported (17,18) that PKC␣ and PKC␦ expression were correlated with tight junctional leakiness in renal epithelial cells. Marano et al (33) reported that phorbol ester increased paracellular permeability in IEC-18 intestinal cells by a mechanism involving translocation of PKC␣ from the cytosolic to membrane compartment and phosphorylation of the tight junctional protein occludin.…”

“…A substantial body of experimental data indicates that PKC regulates paracellular permeability in epithelial (17,18) and endothelial (31) monolayers. The PKC activators diacylglycerol and phorbol esters increase paracellular permeability and diminish TER (15,32).…”

“…The PKC activators diacylglycerol and phorbol esters increase paracellular permeability and diminish TER (15,32). PKC regulates the sorting and assembly of the TJ proteins ZO-1 and the development of TER in confluent renal epithelial cells (17,18). In pulmonary epithelial monolayers, PKC␣ activation modulates TNF␣-induced increase in paracellular permeability (31).…”

“…For example, decreased transepithelial electrical resistance (TER) and increased paracellular flux are observed in cells exposed to the PKC agonist (12-O-tetradecanoylphorbol-13-acetate) (16). Both PKC␣ and -␦ are implicated in the PKC-dependent regulation of TJ assembly and barrier function following activation of a G-protein-coupled receptor (17)(18)(19)(20). Freeze fracture electron micrographs reveal abnormal TJ morphology in transfectants overexpressing mutant forms of the Rho family of GTPases, suggesting involvement of Rho proteins in TJ assembly (21).…”

Role of Protein Kinase C Zeta (PKCζ) in Regulation of Epithelial Tight Junctions

“…Protein kinase C activation has been shown to decrease 34,107,116 or increase 106,117,118 paracellular permeability. This suggests a complex signaling pathway at the junctional complex.…”