OVEREXPRESSION OF PROTEIN-KINASE C-DELTA INCREASES TIGHT JUNCTION PERMEABILITY IN LLC-PK1 EPITHELIA

The Ca2+-independent delta-isoform of protein kinase C (PKC-delta) was overexpressed in LLC-PK1 epithelia and placed under control of a tetr acycline-responsive expression system. In the absence of tetracycline, the exogenous PKC-delta is expressed. Western immunoblots show that t he overexpressed PKC-delta is found in the cytosolic, membrane-associa ted, and Triton-insoluble fractions. Overexpression of PKC-delta produ ced subconfluent and confluent epithelial morphologies similar to that observed on exposure of wild-type cells to the phorbol ester 12-O-tet radecanoylphorbol-13-acetate. Transepithelial electrical resistance (R -T) in cell sheets overexpressing PKC-delta was only 20% of that in ce ll sheets incubated in the presence of tetracycline, in which the amou nt of PKC-delta and R-T were similar to those in LLC-PK1 parental cell sheets. Overexpression of PKC-delta also elicited a significant incre ase in transepithelial Aux of D-[C-14]mannitol and a radiolabeled 2 x 10(6)-molecular-weight dextran, suggesting with the R-T decrease that overexpression increased paracellular, tight junctional permeability. Electron microscopy showed that PKC-delta overexpression results in a multilayered cell sheet, the tight junctions of which are almost unifo rmly permeable to ruthenium red. Freeze-fracture electron microscopy i ndicates that overexpression of PKC-delta results in a more disorganiz ed arrangement of tight junctional strands. As with LLC-PK1 cell sheet s treated with 12-O-tetradecanoylphorbol-13-acetate, the reduced R-T, increased D-mannitol flux, and tight junctional leakiness to ruthenium red that are seen with PKC-delta overexpression suggest the involveme nt of PKC-delta id regulation of tight junctional permeability.