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Effect of ouabain on CFTR gene expression in human Calu-3 cells
Maryvonne Baudouin-Legros1, Franck Brouillard, Danielle Tondelier
1Institut National de la Santé et de la Recherche Médicale U. 467, Faculté de Médecine Necker, 75015 Paris, France. legros@necker.fr
Abstract:
We have previously shown that ouabain, which changes the electrochemical properties of cell membranes by inhibiting Na(+),K(+)-ATPase, induces the expression of multidrug resistance (MDR-1) gene in several human cell lines. Because the expressions of the MDR-1 and CFTR (which encodes the cAMP-activated Cl(-) channel associated with cystic fibrosis) genes are physiologically regulated in opposing directions, we wanted to determine whether ouabain also decreases CFTR transcripts and subsequently to analyze its mechanism of action. We found that the submicromolar concentrations of ouabain that increase MDR-1 mRNAs decrease the CFTR transcripts with analogous time-dependency in human pulmonary Calu-3 cells. By altering or reproducing the ouabain-induced changes in intracellular ionic activities (decreasing in external Na(+) or K(+) or using Na(+) ionophore), we show that the ouabain-induced regulations of both CFTR and MDR-1 transcripts depend on the Na(+)/K(+) pump inhibition but that the decrease in CFTR mRNAs also proceeds from cytoplasm reactions simultaneously activated by ouabain. These data, which emphasize the complex mechanism of action of ouabain, suggest that changes in intracellular ionic activities modulate CFTR/MDR-1 gene expressions.
Insights
Ouabain, a Na(+),K(+)-ATPase inhibitor, decreases cystic fibrosis transmembrane conductance regulator (CFTR) gene expression in lung cells. This effect, alongside increased multidrug resistance (MDR-1) gene expression, is linked to altered intracellular ion levels.
Area of Science:
- Cellular Biology
- Molecular Biology
- Physiology
Background:
- Ouabain (Na(+),K(+)-ATPase inhibitor) previously shown to induce multidrug resistance (MDR-1) gene expression.
- MDR-1 and cystic fibrosis transmembrane conductance regulator (CFTR) genes are physiologically regulated in opposing directions.
- CFTR encodes a cAMP-activated Cl(-) channel crucial in cystic fibrosis.
Purpose of the Study:
- To investigate if ouabain decreases CFTR transcripts.
- To analyze the mechanism of ouabain's action on CFTR gene expression.
- To understand the interplay between CFTR and MDR-1 gene regulation by ouabain.
Main Methods:
- Treatment of human pulmonary Calu-3 cells with submicromolar ouabain concentrations.
- Analysis of MDR-1 and CFTR mRNA levels over time.
- Manipulation of intracellular ionic activities (Na(+), K(+), ionophores) to probe ouabain's mechanism.
Main Results:
- Submicromolar ouabain decreased CFTR transcripts in Calu-3 cells, mirroring MDR-1 mRNA induction.
- Ouabain-induced regulation of both CFTR and MDR-1 transcripts depends on Na(+)/K(+) pump inhibition.
- CFTR mRNA decrease is also mediated by cytoplasm reactions activated by ouabain.
Conclusions:
- Ouabain modulates CFTR and MDR-1 gene expression in opposite directions.
- Intracellular ionic activity changes are key mediators of ouabain's complex regulatory effects on these genes.
- Findings highlight the intricate mechanisms by which ion homeostasis influences gene expression.