Madin-Darby canine kidney II cells: a pharmacologically validated system for NPC1L1-mediated cholesterol uptake.
Absorption of dietary cholesterol in the proximal region of the intestine is mediated by Niemann-Pick C1-like protein (NPC1L1) and is sensitive to the cholesterol absorption inhibitor ezetimibe (EZE). Although a correlation exists between EZE binding to NPC1L1 in vitro and efficacy in vivo, the precise nature of interaction(s) between NPC1L1, EZE, and cholesterol remain unclear. Here, we analyze the direct relationship between EZE analog binding to NPC1L1 and its influence on cholesterol influx in a novel in vitro system. Using the EZE analog [(3)H]AS, an assay that quantitatively measures the expression of NPC1L1 on the cell surface has been developed. It is noteworthy that whereas two cell lines (CaCo-2 and HepG2) commonly used for studying NPC1L1-dependent processes express almost undetectable levels of NPC1L1 at the cell surface, polarized Madin-Darby canine kidney (MDCKII) cells endogenously express 4 x 10(5) [(3)H]AS sites/cell under basal conditions. Depleting endogenous cholesterol with the HMG CoA reductase inhibitor lovastatin leads to a 2-fold increase in the surface expression of NPC1L1, supporting the contention that MDCKII cells respond to changes in cholesterol homeostasis by up-regulating a pathway for cholesterol influx. However, a significant increase in surface expression levels of NPC1L1 is necessary to characterize a pharmacologically sensitive, EZE-dependent pathway of cholesterol uptake in these cells. Remarkably, the affinity of EZE analogs for binding to NPC1L1 is almost identical to the IC(50) blocking cholesterol flux through NPC1L1 in MDCKII cells. From a mechanistic standpoint, these observations support the contention that EZE analogs and cholesterol share the same/overlapping binding site(s) or are tightly coupled through allosteric interactions.
Weinglass
Adam B
AB
Department of Ion Channels, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, USA. adam_weinglass@merck.com
Köhler
Martin G
MG
Nketiah
Emmanuel O
EO
Liu
Jessica
J
Schmalhofer
William
W
Thomas
Anu
A
Williams
Brande
B
Beers
Lindsey
L
Smith
Lauren
L
Hafey
Mike
M
Bleasby
Kelly
K
Leone
Joseph
J
Tang
Yui Sing
YS
Braun
Matthew
M
Ujjainwalla
Feroze
F
McCann
Margaret E
ME
Kaczorowski
Gregory J
GJ
Garcia
Maria L
ML
D000818:Animals; D001384:Azetidines / Q000737:chemistry / Q000378:metabolism*; D018938:Caco-2 Cells; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D003001:Cloning, Molecular; D004285:Dogs; D000069438:Ezetimibe; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012855:Sitosterols / Q000378:metabolism; D013449:Sulfonamides / Q000737:chemistry; D014162:Transfection; D014316:Tritium; D047090:beta-Lactams / Q000378:metabolism
D001384:Azetidines; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C410800:NPC1L1 protein, human; C482444:NPC1L1 protein, rat; D012855:Sitosterols; D013449:Sulfonamides; D047090:beta-Lactams; D014316:Tritium; C025473:gamma-sitosterol; D002784:Cholesterol; D000069438:Ezetimibe