Abstract
The multidrug resistance P-glycoprotein (P-gp) was recently proposed to redistribute cholesterol in the plasma membrane, suggesting that P-gp could modulate cholesterol efflux to cholesterol acceptors. To address this hypothesis and to reevaluate the role of P-gp in cholesterol homeostasis, we first analyzed the role of P-gp expression on cholesterol efflux in P-gp stably transfected drug-selected LLC-MDR1 cells. Cholesterol efflux to methyl-beta-cyclodextrin (CD) was 4-fold higher in LLC-MDR1 cells compared with control LLC-PK1 cells, indicating that the accessible pool of plasma membrane cholesterol was increased by P-gp expression. However, using the P-gp-inducible cells lines HeLa MDR-Tet and 77.1 MDR-Tet, cholesterol efflux to CD, apolipoprotein A-I, or HDL was not associated with P-gp expression. In addition, we did not observe any effect of P-gp expression on cellular free and esterified cholesterol content, cholesteryl ester uptake from LDL and HDL particles, or acyl-CoA:cholesterol acyltransferase activity. Therefore, we conclude that P-gp expression does not play a major role in cholesterol homeostasis in P-gp-inducible cells and that the effects of P-gp on cholesterol homeostasis previously described in drug-selected cells might result from non-P-gp pathways that were also induced by selection for drug resistance.
Highlights
The multidrug resistance P-glycoprotein (P-gp) was recently proposed to redistribute cholesterol in the plasma membrane, suggesting that P-gp could modulate cholesterol efflux to cholesterol acceptors
P-gp activity was measured in the pig kidney polarized epithelial cell line LLC-PK1 and in the LLC-MDR1 cell line, which was derived from LLC-PK1 cells by stable transfection with the human MDR1 gene followed by selection for vincristine resistance [7]
Verapamil (100 mM), a competitive inhibitor of P-gp activity, increased rhodamine 123 (Rho-123) accumulation in LLC-MDR1 cells by 93% compared with nontreated cells (P, 0.01), and similar results were found with 10 mM verapamil
Summary
The multidrug resistance P-glycoprotein (P-gp) was recently proposed to redistribute cholesterol in the plasma membrane, suggesting that P-gp could modulate cholesterol efflux to cholesterol acceptors. We found that drug-selected LLC-MDR1 cells that overexpress human P-pg, versus control cells, had increased FC efflux to methyl-b-cyclodextrin (CD), suggesting that P-gp increased FC cholesterol content in the plasma membrane. Our experiments in drug-selected LLC-MDR1 cells suggest that expression of human P-gp increases the FC content accessible to CD in plasma membrane, supporting a role of P-gp in cellular cholesterol homeostasis.
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