NPC1L1 (Niemann-Pick C1-like 1) mediates sterol-specific unidirectional transport of non-esterified cholesterol in McArdle-RH7777 hepatoma cells

Abstract

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 beta-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 Ca(2+), 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.

Figure 1. Cholesterol depletion enhances NPC1L1-dependent selective FC uptake

EGFP control or L1-EGFP stable cells lines were either kept in Medium A or cholesterol-depleted in Medium B containing 2% MβCD for 1 h. Thereafter, cells were provided with 100 μg/ml of either human HDL or LDL labelled with [¹⁴C]FC or [³H]COE for a period spanning 0–4 h as described in the Materials and methods section. Cell-associated radioactivity is expressed as d.p.m./mg of cellular protein. Data represent means±SEM of one of two independent experiments (n=4). The asterisk (*) represents significant differences between EGFP versus L1-EGFP cells within each MβCD treatment group, P<0.05.

Figure 2. Expression of NPC1L1-EGFP does not alter oleic acid or β-sitosterol uptake

EGFP control or L1-EGFP stable cell lines were either kept in Medium A or cholesterol depleted in Medium B containing 2% MβCD for 1 h. Thereafter, cells were provided with [³H]oleic acid (A), β-[³H]sitosterol alone (B), or β-[³H]sitosterol and [¹⁴C]cholesterol together (C and D) as described in the Materials and methods section, and cell-associated radioactivity was determined after a short time course. (C) Represents dual sterol (β-[³H]sitosterol and [¹⁴C]cholesterol) uptake in cells maintained in Medium A, which exhibit perinuclear/ERC localization of NPC1L1. (D) Represents dual sterol (β-[³H]sitosterol and [¹⁴C]cholesterol) uptake in cells after 1 h treatment with 2% MβCD in Medium B to induce membrane localization of NPC1L1-EGFP. Data are expressed as d.p.m./mg of cellular protein and represent means±S.E.M. for one of two independent experiments (n=4 for A and B; n=3 for C and D). * Represents significant differences between EGFP versus L1-EGFP cells within each MβCD treatment group, P<0.05.

Figure 3. Expression of NPC1L1-EGFP does not alter cellular cholesterol efflux

EGFP control or L1-EGFP stable cells lines were labelled with [¹⁴C]cholesterol for 48 h. Thereafter, cells were either (A) kept in medium A to preserve perinuclear/ERC localization of NPC1L1 or (B) cholesterol-depleted with 2% MβCD in Medium B for 1 h to induce membrane localization of NPC1L1–EGFP. Thereafter, cells were washed and the efflux of cellular [¹⁴C]cholesterol in the absence (no acceptor) or presence of ApoA-I (20 μg/ml), HDL (100 μg/ml), or FBS (5%) cholesterol acceptors was measured after 4 h incubation as described in the Materials and methods section. Results are expressed as percentage efflux and represent means±S.E.M. for one of three independent experiments (n=4). Values not sharing a common superscript differ significantly (P< 0.05).

Figure 4. NPC1L1 does not augment cholesterol loading when it resides intracellularly

EGFP control or L1-EGFP stable cells lines were grown in 150-mm-diameter dishes and maintained in Medium A to preserve perinuclear/ERC localization of NPC1L1. For cholesterol loading (Chol. Load), cells were grown in Medium A supplemented with 10 μg/ml cholesterol/MβCD for 4 h. All cells were then washed twice with PBS, total cellular lipids were extracted, and TC (A), FC (B) and EC (C) mass was measured by using the enzymatic assays described in the Materials and methods section. Results are expressed as μg of lipid/mg of cellular protein and represent means±S.E.M. for one of two independent experiments (n=4). Values not sharing a common superscript differ significantly (P<0.05).

Figure 5. Cholesterol-depletion-driven membrane localization of NPC1L1 facilitates mass cholesterol uptake

EGFP control or L1-EGFP stable cells lines were grown in 150-mm-diameter dishes and maintained in Medium A to preserve perinuclear ERC localization of NPC1L1. A subset of dishes was changed to Medium B containing 2% MβCD for 1 h (1h MβCD) to induce membrane localization of NPC1L1–EGFP. Thereafter, the dishes either received serum-free medium with or without 10 μg/ml cholesterol/MβCD for 2 h (2h Chol. Load) to replete cellular cholesterol. All cells were then washed twice with PBS, total cellular lipids were extracted and total cholesterol (TC) (A), FC (B) and EC (C) masses were measured using enzymatic assays as described in the Materials and methods section. Results are expressed as μg of lipid/mg of cellular protein and represent means±S.E.M. of one of two independent experiments (n=4). Values not sharing a common superscript differ significantly (P<0.05).

Figure 6. Role of cell-signalling mediators and the cytoskeleton in NPC1L1-dependent cholesterol uptake

(A) EGFP control or L1-EGFP stable cells lines were pretreated for 1 h with protein kinase inhibitors: 200 nM calphostin C (Cal. C), 10 μM H-89 (H-89), 100 ng/ml pertussis toxin (PTX) or 50 μM BAPTA-AM. (B) EGFP control or L1-EGFP stable cells lines were pretreated for 1 h with 20 μg/ml nocodazole or 10 μg/ml cytochalasin D. (C) EGFP control or L1-EGFP stable cells lines were pretreated for 1 h with 10 μM monensin or K⁺-depleted as described in the Materials and methods section. Thereafter, cells were either kept in Medium A (−MβCD) or cholesterol depleted by addition of 2% MβCD in Medium B (+MβCD) for 1 h in the presence of indicated inhibitors. Then cells were provided with [¹⁴C]FC in a BSA/ethanol suspension for a period spanning 90 min in the presence of the indicated inhibitors to determine cholesterol uptake. Cell-associated radioactivity is expressed as d.p.m./mg of cellular protein and results are means±S.E.M. for one of two independent experiments (n=2–4). Values not sharing a common superscript differ significantly (P<0.05).