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Cellular variability in the development of tight junctions after activation of protein kinase C
B Ellis1, E E Schneeberger, C A Rabito
1Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
Abstract:
Phorbol 12-myristate 13-acetate (PMA) decreases the tight junction conductance (TJC) during the reorganization of LLC-PK1A monolayers, but has the opposite effect in LLC-PK1B4, MDCK, and MDCK4 cells. Because no protein synthesis was required for the effects of PMA on the TJC of LLC-PK1A monolayers, we conclude that the regulation of the tight junction by protein kinase C (PKC) is a posttranslational event. In LLC-PK1A monolayers with existing tight junctions, PMA produced an initial increase in the TJC that reverted later to control values despite the continuous presence of PMA and cycloheximide. The inhibitory effect of PMA on the other cell lines was not revertible. A downregulation of total PKC activity and phorbol ester receptors was only observed during the reorganization of LLC-PK1A monolayers. PMA further increases this downregulation. This indicates that the peculiar response to PMA observed in LLC-PK1A monolayers is the result of two concurrent events: 1) the early activation of the enzyme just before the reorganization of the tight junctions begin, and 2) its late downregulation induced after prolonged exposure to phorbol esters. We conclude that PKC regulates the development of the occluding junctions, but through different mechanisms dependent on the characteristics of the cells.
Insights
Phorbol 12-myristate 13-acetate (PMA) differentially affects tight junction conductance (TJC) in kidney cell lines. Protein kinase C (PKC) regulation of TJC is a posttranslational event, with cell-specific mechanisms influencing junction development.
Area of Science:
- Cell Biology
- Molecular Biology
- Physiology
Background:
- Tight junctions (TJs) are crucial for epithelial barrier function.
- Phorbol 12-myristate 13-acetate (PMA) is a potent activator of protein kinase C (PKC).
- PKC's role in regulating TJ permeability is cell-type dependent.
Purpose of the Study:
- To investigate the differential effects of PMA on tight junction conductance (TJC) in various kidney cell lines.
- To elucidate the posttranslational mechanisms by which PKC regulates TJs.
- To understand the cell-specific regulation of TJ development by PKC.
Main Methods:
- Treatment of LLC-PK1A, LLC-PK1B4, MDCK, and MDCK4 cell monolayers with PMA.
- Measurement of TJC.
- Assessment of protein synthesis requirements.
- Evaluation of PKC activity and phorbol ester receptor levels.
Main Results:
- PMA decreased TJC in LLC-PK1A cells but increased it in LLC-PK1B4, MDCK, and MDCK4 cells.
- PKC regulation of TJC in LLC-PK1A cells was a posttranslational event, independent of protein synthesis.
- LLC-PK1A cells showed a transient increase in TJC with PMA, followed by a return to baseline, unlike other cell lines.
- Downregulation of PKC activity and receptors occurred during LLC-PK1A cell reorganization, exacerbated by PMA.
Conclusions:
- PKC regulates TJ development through distinct, cell-specific posttranslational mechanisms.
- The unique response of LLC-PK1A cells to PMA involves early PKC activation and subsequent downregulation.
- PKC's influence on TJ formation and maintenance is context-dependent on cellular characteristics.