Received for publication November 27, 2007.
Revised January 9, 2008.
Accepted for publication January 9, 2008.

MDCKII cells: a pharmacologically validated system for NPC1L1-mediated cholesterol uptake

Abstract

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). While a correlation exists between EZE binding to NPC1L1 in vitro and in vivo efficacy, 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, [³H]AS, an assay that quantitatively measures the expression of NPC1L1 on the cell surface has been developed. Interestingly, while 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 10⁵ [³H]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 IC₅₀ blocking cholesterol flux through NPC1L1 in MDCKII cells. Mechanistically, these observations support the contention that EZE-analogs and cholesterol share the same/overlapping binding site(s) or are tightly coupled through allosteric interactions.

Key words: Func. analysis receptor/ion channel mutants, Receptor binding studies, Cholesterol metabolism/lipoproteins

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