Niemann-Pick C1-Like 1 Overexpression Facilitates Ezetimibe- Sensitive Cholesterol and -Sitosterol Uptake in CaCo-2 Cells

Previous in vivo studies including those with knockout mice suggested that Niemann-Pick C1-like 1 (NPC1L1) plays an essential role in the intestinal absorption of cholesterol. To characterize the mechanism of cholesterol uptake mediated by NPC1L1, an in vitro system reflecting the function of this transporter needs to be established. In the present study, we constructed NPC1L1 overexpressing CaCo-2 cells as an in vitro model and characterized the transport properties of NPC1L1. Immunohistochemical staining revealed that CaCo-2 cells express NPC1L1 on the apical membrane. It was also demonstrated that the uptakes of both cholesterol and -sitosterol are increased by NPC1L1 overexpression. In addition, the uptake of cholesterol was increased in a dose-dependent manner by an increase in the content of taurocholate in micelles, whereas micellar phosphatidylcholine showed a negative correlation with cholesterol uptake. Furthermore, it was confirmed that sterol uptake increased by NPC1L1 overexpression was inhibited by ezetimibe. We could thus establish an in vitro intestinal model to study the mechanism of NPC1L1-dependent sterol uptake and to screen drug candidates whose target is NPC1L1. A high plasma cholesterol level is one of the risk factors for lifestyle-related diseases such as arteriosclerosis and cardiovascular diseases. Cholesterol homeostasis depends on the balance between de novo syntheses and catabolism, excretion into the bile, and intestinal absorption from the diet. Up to now, many drugs including fibrates and statins have been developed and used to reduce plasma cholesterol levels. Among these drugs, ezetimibe is a novel drug that selectively blocks the absorption of cholesterol in the small intestine (van Heek et al., 2001; Patel et al., 2003). The pharmacological target of ezetimibe was demonstrated recently. In 2004, it was reported that Niemann-Pick C1-like 1 (NPC1L1) knockout mice exhibit a reduction in intestinal cholesterol absorption, and the degree of this reduction is almost the same as that observed in wild-type mice treated with ezetimibe (Altmann et al., 2004; Davis et al., 2004). In addition, it was demonstrated that the intestinal absorption of sitosterol was reduced in NPC1L1 knockout mice (Davis et al., 2004). The fact that NPC1L1 is highly expressed in the small intestine, particularly in the jejunum where sterol absorption predominantly takes place, and the fact that this transporter is localized on the apical surface of intestinal cells in rodents and humans (Altmann et al., 2004; Davis et al., 2004; Sane et al., 2006) are consistent with the finding that this transporter is involved in the intestinal absorption of cholesterol. In contrast with these in vivo observations, few in vitro experimental models to examine the intestinal absorption of sterols have been established. In the present study, we constructed CaCo-2 cells stably expressing rat NPC1L1 as an in vitro model and characterized the transport properties of NPC1L1-mediated transport of sterols. In particular, we have focused on whether the uptakes of cholesterol and sitosterol depend on NPC1L1 expression and the concentrationdependent effect of ezetimibe on the NPC1L1-mediated uptake of sterols. In addition, we were interested in the effect of the micellar component (content of bile acids and phospholipids in micelles) on the uptake of cholesterol, because in vivo cholesterol absorption is affected by the micellar component. Materials and Methods Materials. [7(n)-H]Cholesterol (7.0 Ci/mmol) was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). [22,23H] -Sito-sterol (50 Ci/mmol) was purchased from American RadioY.Y. and T.T. contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.114181. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: NPC1L1, Niemann-Pick C1-like 1; PCR, polymerase chain reaction; BSA, bovine serum albumin; TBS-T, Tris-buffered saline containing 0.05% Tween 20; ABCG5, ATP-binding cassette, subfamily G, member 5; ABCG8, ATP-binding cassette, subfamily G, member 8; ANOVA, analysis of variance. 0022-3565/07/3202-559–564$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 2 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 114181/3175621 JPET 320:559–564, 2007 Printed in U.S.A. 559 http://jpet.aspetjournals.org/content/suppl/2006/11/29/jpet.106.114181.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A uust 0, 2017 jpet.asjournals.org D ow nladed from labeled Chemicals (St. Louis, MO). Cholesterol was purchased from Wako Pure Chemicals (Osaka, Japan). Sodium taurocholate, Lphosphatidylcholine, and -sitosterol were purchased from SigmaAldrich (St. Louis, MO). Ezetimibe was purchased from Sequoia Research Products Ltd (Pangbourne, UK). CaCo-2 cells were purchased from Cell Bank, RIKEN BioResource Center (Ibaraki, Japan). pcDNA3.1( ) vector was purchased from Invitrogen (Carlsbad, CA). HindIII and XbaI restriction enzymes were purchased from Takara (Shiga, Japan). Anti-HA tag antibody [HA-probe (Y-11): sc805] was purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). All other chemicals used were commercially available and of reagent grade. Construction of NPC1L1-Overexpressed CaCo-2 Cells. NPC1L1 cDNA was amplified by PCR from total RNA of rat intestine. The complete NPC1L1 cDNA (GenBank accession number AY437867) was amplified with the HindIII site at the 5 -end and with the XbaI site and HA tag (YPYDVPDYA) sequence attached at the 3 -end by PCR and then inserted into the pcDNA3.1( ) vector plasmid. NPC1L1 in pcDNA3.1( ) was transfected into CaCo-2 cells grown on a six-well plate with FuGene 6 (Roche Diagnostics, Indianapolis, IN) according to the user’s manual. Then, CaCo-2 cells were selected by culturing in the presence of 500 g/ml G418 sulfate