Reevaluation of the role of the multidrug-resistant P-glycoprotein in cellular cholesterol homeostasis

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-b-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 Pgp 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.-Le Goff, W., M. Settle, D. J. Greene, R. E. Morton, and J. D. Smith. Reevaluation of the role of the multidrug-resistant P-glycoprotein in cellular cholesterol homeostasis. J. Lipid Res. 2006. 47: 51-58. Supplementary key words cholesterol efflux . ABCA1 . cyclodextrin . apolipoprotein A-I P-glycoprotein (P-gp) is a member of the ATP binding cassette transporter family responsible for the multidrug resistance (MDR) phenotype. P-gp is the protein product of the human MDR1 gene (ABCB1 is the official gene symbol), and P-gp is highly expressed in the intestine, liver, kidney, placenta, and the blood-brain barrier. Plasma membrane P-gp mediates the efflux of numerous neutral and cationic organic compounds and drugs, among them chemotherapeutic drugs, thereby contributing to the MDR phenotype in many cancers. However, information concerning endogenous substrates for P-gp as well as the physiological role of P-gp is still lacking (1).P-gp has been reported to modulate cellular cholesterol homeostasis via several mechanisms. Stable transfection of a rat intestinal cell line with a P-gp expression vector led to a modest increase in the uptake of cholesterol-containing micelles (2). Nonspecific P-gp inhibitors have been reported to inhibit cholesterol biosynthesis in CHO-7 cells (3) and to inhibit cellular cholesterol esterification (4). Transfection of NIH 3T3 cells with a P-gp expression vector followed by selection for drug resistance was associated with increased esterification of plasma membrane cholesterol (5). Mice, unlike humans, have two copies of the gene for P-gp, abcb1a and abcb1b (previously mdr1a and mdr1b). Mice deficient in both of these genes have been constructed, and althou...

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“…RNA was quantified using RiboGreen by Life Technologies and all samples were diluted to 0.2mg/mL in Tris-EDTA buffer prepared in DEPC-treated water. The reverse transcription reaction was completed using 1.0μg of RNA and a combination of OligodT 20 and random hexamers in the SuperScript III First-Strand SuperMix from Life Technologies. The cDNA products were characterized using the OliGreen Assay by Life Technologies and all samples diluted to 2ng/ μL.…”

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