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Relationship between Na(+),K(+)-ATPase and cell attachment
R G Contreras1, L Shoshani, C Flores-Maldonado
1Center for Research and Advanced Studies of México (CINVESTAV), Av. Instituto Politecnico Nacional 2508, Codigo Postal, 07660 Mexico.
Abstract:
A prolonged ouabain blockade of the Na(+),K(+)-ATPase detaches cells from each other and from the substrate. This suggests the existence of a link between pump (P) and attachment (A). In the present work, we report that MDCK-W cells treated with ouabain increase tyrosine phosphorylation and content of active MAP kinase, redistribute molecules involved in cell attachment (occludin, ZO-1, desmoplakin, cytokeratin, alpha-actinin, vinculin and actin), and detach. Genistein and UO126, inhibitors of protein tyrosine kinase and of MAP kinase kinase, respectively, block this detachment. The content of P190(Rho-GAP), a GTPase activating protein of the Rho small G-protein subfamily, is increased by ouabain, suggesting that both the Rho/Rac and MAPK pathways are involved. Another clone of MDCK cells whose Na(+),K(+)-ATPase has a negligible affinity for the drug, show none of the effects described for MDCK-W and remain attached. Ma104 cells, a line that has a high affinity for ouabain and stops pumping, fail to modify phosphorylation, as well as the pattern of distribution of attaching molecules, and remain in the monolayer. Taken together, these results suggest that there is a mechanism (P-->A) that transduces a blockade of the pump in a detachment of the cell from neighbors and substrate, in which Ma104 cells are faulty.
Insights
Ouabain blockade of the sodium-potassium pump (Na(+),K(+)-ATPase) triggers cell detachment by increasing tyrosine phosphorylation and activating MAP kinase signaling pathways. This pump-to-attachment (P-A) mechanism is crucial for cell adhesion.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- The Na(+),K(+)-ATPase (sodium-potassium pump) is vital for cellular homeostasis.
- Previous observations suggest a link between pump activity and cell attachment.
- The precise mechanism linking pump function to cell adhesion remains unclear.
Purpose of the Study:
- To investigate the molecular mechanisms by which ouabain blockade of the Na(+),K(+)-ATPase affects cell adhesion.
- To identify signaling pathways involved in ouabain-induced cell detachment.
- To explore the role of specific cell types in this pump-to-attachment (P-A) phenomenon.
Main Methods:
- Treatment of Madin-Darby Canine Kidney-W (MDCK-W) cells with ouabain.
- Analysis of tyrosine phosphorylation and MAP kinase activation.
- Assessment of cell adhesion molecule distribution (occludin, ZO-1, desmoplakin, etc.).
- Inhibition studies using genistein (tyrosine kinase inhibitor) and UO126 (MAP kinase kinase inhibitor).
- Comparison with MDCK cells having low ouabain affinity and Ma104 cells.
Main Results:
- Ouabain treatment induced cell detachment, increased tyrosine phosphorylation, and activated MAP kinase in MDCK-W cells.
- Key cell attachment molecules were redistributed upon ouabain exposure.
- Inhibitors of tyrosine kinase and MAP kinase kinase blocked ouabain-induced detachment.
- Ouabain increased P190(Rho-GAP) content, implicating Rho/Rac and MAPK pathways.
- MDCK cells with low ouabain affinity and Ma104 cells did not exhibit detachment or associated molecular changes.
Conclusions:
- A pump-to-attachment (P-A) mechanism exists, linking Na(+),K(+)-ATPase activity to cell detachment.
- MAPK and Rho/Rac signaling pathways are involved in mediating this effect.
- Cellular differences in Na(+),K(+)-ATPase ouabain affinity influence the response.
- Ma104 cells appear faulty in transducing the pump blockade signal for detachment.