Am J Physiol Cell Physiol Journal of Applied Physiology
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Am J Physiol Cell Physiol 275: C544-C554, 1998;
0363-6143/98 $5.00
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Vol. 275, Issue 2, C544-C554, August 1998

Overexpression of protein kinase C-delta increases tight junction permeability in LLC-PK1 epithelia

James M. Mullin1, Jennifer A. Kampherstein1, Kathleen V. Laughlin1, Cheryl E. K. Clarkin1, R. Daniel Miller1, Zoltan Szallasi2, Bechara Kachar3, Alejandro Peralta Soler1, and Dan Rosson1

1 Lankenau Medical Research Center, Wynnewood, Pennsylvania 19096-3411; 2 Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda 20814; and 3 Laboratory of Cell Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20850

The Ca2+-independent delta -isoform of protein kinase C (PKC-delta ) was overexpressed in LLC-PK1 epithelia and placed under control of a tetracycline-responsive expression system. In the absence of tetracycline, the exogenous PKC-delta is expressed. Western immunoblots show that the overexpressed PKC-delta is found in the cytosolic, membrane-associated, and Triton-insoluble fractions. Overexpression of PKC-delta 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 (RT) in cell sheets overexpressing PKC-delta was only 20% of that in cell sheets incubated in the presence of tetracycline, in which the amount of PKC-delta and RT were similar to those in LLC-PK1 parental cell sheets. Overexpression of PKC-delta also elicited a significant increase in transepithelial flux of D-[14C]mannitol and a radiolabeled 2 × 106-molecular-weight dextran, suggesting with the RT 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 uniformly permeable to ruthenium red. Freeze-fracture electron microscopy indicates that overexpression of PKC-delta 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 RT, increased D-mannitol flux, and tight junctional leakiness to ruthenium red that are seen with PKC-delta overexpression suggest the involvement of PKC-delta in regulation of tight junctional permeability.

phorbol ester; paracellular; transepithelial; resistance; cytoskeleton; mannitol; freeze fracture; dextran


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