Search

The Ca2+-independent δ-isoform of protein kinase C (PKC-δ) was overexpressed in LLC-PK1epithelia 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 ( RT) 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 RTwere 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 RTdecrease 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-PK1cell sheets treated with 12- O-tetradecanoylphorbol-13-acetate, the reduced RT, 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.

For rat distal colon, the transepithelialelectrical parameters, short circuit current(Iscc) and transepithelial electricalresistance (TER), respectively, measure nettransepithelial electrolyte transport activity and the barrier function of theepithelium. Studies with a variety of epithelial cellcultures have shown greater than 90% decreases of TERwithin minutes of exposure of in vitro cell sheets to phorbol esters. The phorbol ester and proteinkinase C (PKC) activator, phorbol dibutyrate (PDBU), wasobserved to produce an over 100% elevation ofIscc but only a small yet significant 20-30%decrease of TER across rat distal colon. Inhibition ofthe above effects of PDBU by the PKC inhibitorbisindolylmaleimide (GFX) is further evidence that inrat distal colon, Iscc and TER are underregulatory control by PKC. When animals received anesthesia withintraperitoneal pentobarbital prior to removal of thecolon, the effect of PDBU on Iscc wassignificantly reduced, and the effect of PDBU on TER wasalmost completely inhibited. This effect ofpentobarbital on PKC-mediated transepithelialpermeability parameters is consistent with the knownability of anesthetics to alter protein kinase Cactivity. Exposure of rat colon to pentobarbital produced as muchas a 90% inhibition of calcium-dependent PKC activity,whereas calcium-independent activity was stimulated byas much as 35%. Prior anesthetic use may be therefore a complicating factor in observingPKC-mediated effects on epithelial barrier functionusing epithelial tissue models.

Modulation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) disrupts the cell-cell junctions of the epithelial cell line LLC-PK1. To examine the role of specific PKC isoforms in this process we have created modified LLC-PK1 subclones that express wild-type and dominant negative versions of PKC-alpha under control of the tetracycline-responsive expression system. Overexpression of wild-type PKC-alpha 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-alpha 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 alpha isoform of PKC is at least one of the isoforms that regulate tight junctions and other cell-cell junctions of LLC-PK1 epithelia.

Acute exposure (up to 4 h) of LLC-PK1 epithelial cell sheets to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a rapid decrease of transepithelial electrical resistance (TER) to less than 15% of initial value. As the TPA exposure period is continued by chronically passaging cells in the presence of TPA, TER begins to recover. By 6 weeks of exposure, TER recovers to almost 50% of its initial value, suggesting that tight junctions (TJs) are recovering barrier function even in the continued presence of TPA. Between 6 and 8 weeks, TER then decreases a second time to approximately 20 to 40% of initial values, and TER values remain at this level for at least 18 weeks of exposure. Transepithelial (paracellular) fluxes of D-mannitol inversely correspond with TER changes. Across chronically treated cell sheets, rates are higher than those across control cell sheets, but lower than those across acutely treated cell sheets. The decrease of TER at 6-8 weeks coincides with the appearance of multilayered, polyp-like foci (PLFs) on the otherwise one cell layer thick epithelium. Electron microscopy shows that the electron-dense dye ruthenium red cannot penetrate across TJs of control cells but passes across all of the TJs of a cell sheet treated acutely with TPA. In chronically treated cultures, ruthenium red penetrates TJs between most cells of PLFs, but not TJs of adjacent morphologically normal epithelium. A clonal subline derived from cells of a PLF (clone PLF-A) is multilayered almost throughout and exhibits ruthenium red penetration across nearly all of its tight junctions, monolayer or multilayer. Acute exposure of control cell sheets to TPA induces activation, translocation, and down-regulation of protein kinase C-alpha (PKC-alpha). In chronically TPA-treated and clone PLF-A cells, total PKC-alpha levels are reduced even further and almost all remaining PKC-alpha is found in the membrane-associated and Triton-insoluble fractions. Immunofluorescence shows that PKC-alpha expression is restricted to the PLFs in chronically TPA-treated cells and is more homogeneously distributed in clone PLF-A cultures. In summary, the data show that chronic treatment of epithelial cells with a tumor promoter induces the formation of abnormal cell architecture (PLFs) associated with increased leakiness of TJs and membrane translocation of PKC-alpha. Recovery of barrier function in portions of chronically TPA-treated cultures does not correlate with up-regulation of PKC-alpha nor translocation back to the cytosolic compartment.

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.