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The role of phosphorylation in development of tight junctions in cultured renal epithelial (MDCK) cells
S K Nigam1, N Denisenko, E Rodriguez-Boulan
1Dept. of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, MA.
Abstract:
We have explored the effect of the protein kinase inhibitor H7 on tight junction formation in a MDCK cell model for the development of cell-cell contact, tight junctions and epithelial polarity: the "Ca++ switch" model. In this developmental model, which is thought to mimic processes during the early morphogenesis of epithelial tissues, the protein kinase inhibitor H7 markedly inhibits the development of transepithelial resistance of confluent MDCK cells during the "switch" from low (1-5 microM) to normal (1.8 mM) Ca++ media compared with control MDCK cells. Moreover, indirect immunofluorescence using specific antisera against two tight junctional proteins, ZO1 and cingulin, revealed that H7 inhibits the sorting of these proteins from an intracellular site to the lateral surfaces of MDCK cells when the Ca++ in the medium is raised. These data suggest protein kinase mediation in sorting events that lead to the assembly of tight junctions.
Insights
The protein kinase inhibitor H7 blocks tight junction formation in Madin-Darby canine kidney (MDCK) cells. H7 disrupts the sorting of key junctional proteins, indicating protein kinases are crucial for epithelial development.
Area of Science:
- Cell Biology
- Epithelial Biology
- Molecular Biology
Background:
- Tight junctions are crucial for epithelial barrier function and polarity.
- The "Ca++ switch" model mimics early epithelial morphogenesis.
- Protein kinases are implicated in cellular signaling pathways.
Purpose of the Study:
- To investigate the role of protein kinase inhibitor H7 in tight junction formation.
- To elucidate the effect of H7 on cell-cell contact and epithelial polarity in MDCK cells.
- To understand the mechanism of tight junction assembly during epithelial development.
Main Methods:
- Utilized the Madin-Darby canine kidney (MDCK) cell "Ca++ switch" model.
- Measured transepithelial electrical resistance to assess barrier function.
- Employed indirect immunofluorescence to visualize tight junctional proteins ZO-1 and cingulin.
Main Results:
- H7 significantly inhibited the development of transepithelial resistance in MDCK cells during the Ca++ switch.
- H7 treatment prevented the proper localization of ZO-1 and cingulin to the lateral cell surfaces.
- These effects were observed when calcium levels were increased in the culture medium.
Conclusions:
- Protein kinase activity is essential for the proper assembly of tight junctions.
- H7 interferes with the sorting and trafficking of tight junctional proteins.
- These findings highlight the role of protein kinases in regulating epithelial morphogenesis and barrier formation.