Abstract
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.
Highlights
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
Confluent cell sheets to TPA causes a rapid decrease in the transepithelial resistance (TER) to less than 15% of its initial value [2] because of its effect on one type of cell junction known as tight junctions
As it relates to PKCmediated cell junction regulation in LLC-PK1 epithelial cells, we have begun to modulate the activities of individual isoforms by exogenously expressing wild type and dominant negative versions of the genes in an inducible expression system
Summary
Vol 272, No 23, Issue of June 6, pp. 14950 –14953, 1997 Printed in U.S.A. Protein Kinase C-␣ Activity Modulates Transepithelial Permeability and Cell Junctions in the LLC-PK1 Epithelial Cell Line*. 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. 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. The complexity of the PKC family and the differential expression of the isoforms suggest that the members serve distinct roles in signal transduction processes To examine this issue, as it relates to PKCmediated cell junction regulation in LLC-PK1 epithelial cells, we have begun to modulate the activities of individual isoforms by exogenously expressing wild type and dominant negative versions of the genes in an inducible expression system. LLCPK1 cells express the ␣, ␦, ⑀, and isoforms of PKC, and, in this study, we report that alteration of the activity of the ␣ isoform of PKC modulates the integrity of cell-cell junctions as measured by sensitivity to TPA
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