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.

Modulation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) disrupts the cell-cell junctions of the epithelial cell line LLC-PK 1 . To examine the role of specific PKC isoforms in this process we have created modified LLC-PK 1 subclones that express wildtype and dominant negative versions of PKC-␣ under control of the tetracycline-responsive expression system. Overexpression of wild-type PKC-␣ rendered the cells more sensitive to the effects of TPA on transepithelial permeability as measured by loss of transepithelial resistance across the cell sheet. Conversely, expression of a dominant negative PKC-␣ rendered the cells more resistant to the effects of TPA as measured both by loss of transepithelial resistance as well as cell scattering. The properties of both subclones could be modulated by the addition of tetracycline, which suppressed the effect of the exogenous genes. These results indicate that the ␣ isoform of PKC is at least one of the isoforms that regulate tight junctions and other cell-cell junctions of LLC-PK 1 epithelia.

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Although exposure of LLC-PK1 epithelial cell sheets to phorbol esters (TPA) causes a near immediate and total decrease of transepithelial electrical resistance (TER), continuation of exposure for 3 to 4 days results in a tachyphylactic response as TER begins to return to control levels. Recovery of TER is maximal by 5 to 6 days, but reaches only 70 to 80% of control level. A reciprocal change in the transepithelial flux of D-mannitol indicates that the TER decrease is indicative of an increase in tight junction permeability. Exposure of cell sheets to TPA for several days also results in the appearance of multilayered polyp-like foci (PLFs) across the otherwise one cell layer thick cell sheets. The pattern of penetration of the electron dense dye, ruthenium red, from the apical surface, across the tight junction and into the lateral intercellular space indicates that the tight junctions of the cell sheet become uniformly leaky after acute exposure to TPA. However, when exposure is continued for several days, only the junctions of cells in the PLFs manifest leakiness. The decrease in TER following acute TPA exposure correlates with the translocation of protein kinase C-alpha (PKC alpha) into a membrane-associated compartment. With exposure of several days, only a trace of PKC alpha is visible by Western immunoblot, and this is in the membrane-associated compartment. Immunofluorescent microscopy indicates that the trace of PKC alpha seen in the Western immunoblots is ascribable distinctly to cells of the PLFs. Monolayer areas between PLFs show no discernible immunofluorescent signal. The data therefore indicate that tight junction barrier function may be restored in certain areas by the down regulation of PKC alpha from the membrane-associated compartment. Failure to down regulate may result in the paracellular leakiness and abnormal cell architecture of the PLFs. Possible implications of this model for in vivo epithelial tumor promotion are discussed.