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 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 x 10(6)-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.
Mullin
J M
JM
Lankenau Medical Research Center, Wynnewood, Pennsylvania 19096-3411, USA.
Kampherstein
J A
JA
Laughlin
K V
KV
Clarkin
C E
CE
Miller
R D
RD
Szallasi
Z
Z
Kachar
B
B
Soler
A P
AP
Rosson
D
D
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002463:Cell Membrane Permeability; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D005614:Freeze Fracturing; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D007672:Kidney Cortex; D018374:LLC-PK1 Cells; D008854:Microscopy, Electron; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000235:genetics; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D011994:Recombinant Proteins / Q000096:biosynthesis; D013552:Swine; D013752:Tetracycline / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019108:Tight Junctions / Q000502:physiology* / Q000648:ultrastructure; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D013752:Tetracycline; D013755:Tetradecanoylphorbol Acetate