Biochem. J. (2007) 406
(273–283) (Printed in Great Britain)
NPC1L1 (Niemann–Pick C1-like 1) mediates sterol-specific unidirectional transport of non-esterified cholesterol in McArdle-RH7777 hepatoma cells
J. Mark BROWN, Lawrence L. RUDEL and Liqing YU1
Department of Pathology, Section on Lipid Sciences, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1040, U.S.A.
Recent evidence suggests that NPC1L1 (Niemann–Pick C1-like 1) is critical for intestinal sterol absorption in mice, yet mechanisms by which NPC1L1 regulates cellular sterol transport are lacking. In the study we used a McArdle-RH7777 rat hepatoma cell line stably expressing NPC1L1 to examine the sterol-specificity and directionality of NPC1L1-mediated sterol transport. As previously described, cholesterol-depletion-driven recycling of NPC1L1 to the cell surface facilitates cellular uptake of non-esterified (free) cholesterol. However, it has no impact on the uptake of esterified cholesterol, indicating free sterol specificity. Interestingly, the endocytic recycling of NPC1L1 was also without effect on b-sitosterol uptake, indicating that NPC1L1 can differentiate between free sterols of animal and plant origin in hepatoma cells. Furthermore, NPC1L1-driven free cholesterol transport was unidirectional, since cellular cholesterol efflux to apolipoprotein A-I, high-density lipoprotein or serum was unaffected by NPC1L1 expression or localization. Additionally, NPC1L1 facilitates mass non-esterified-cholesterol uptake only when it is located on the cell surface and not when it resides intracellularly. Finally, NPC1L1-dependent cholesterol uptake required adequate intracellular K+, yet did not rely on intracellular Ca2+, the cytoskeleton or signalling downstream of protein kinase A, protein kinase C or pertussis-toxin-sensitive G-protein-coupled receptors. Collectively, these findings support the notion that NPC1L1 can selectively recognize non-esterified cholesterol and promote its unidirectional transport into hepatoma cells.
Key words: cholesterol efflux, ezetimibe, Niemann–Pick C1-like 1 (NPC1L1), sitosterol, sterol uptake.
Abbreviations used: ABC, ATP-binding cassette; Apo, apolipoprotein; BAPTA-AM, 1,2-bis-(2-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid tetrakis(acetoxymethyl ester); BCA, bicinchoninic acid; CE, cholesteryl ester; CETP, cholesterol ester transfer protein; COE, cholesterol oleoyl ether; DMEM, Dulbecco's modified Eagle's medium; EC, esterified cholesterol; EGFP, enhanced green fluorescent protein; ERC, endocytic recycling compartment; FC, free (non-esterified) cholesterol; GPCR, G-protein-coupled receptor; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LDLR, low-density-ipoprotein receptor; NPC1, Niemann–Pick C1; NPC1L1, Niemann–Pick C1-like 1; MbCD, methyl-b-cyclodextrin; PKA, protein kinase A; PKC, protein kinase C; SR-BI, scavenger receptor class B type I; SSD, sterol-sensing domain; TC, total cholesterol.
1To whom correspondence should be addressed (email lyu@wfubmc.edu).
Received 31 January 2007/1 May 2007; accepted 24 May 2007
Published as BJ Immediate Publication 24 May 2007, doi:10.1042/BJ20070168
© The Authors Journal compilation © 2007 Biochemical Society
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