BBAMCB-57220; No. of pages: 9; 4C: 3, 5, 7
Biochimica et Biophysica Acta xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbalip
ABCA1-dependent mobilization of lysosomal cholesterol requires functional
Niemann–Pick C2 but not Niemann–Pick C1 protein☆
Emmanuel Boadu a, Randy C. Nelson b, Gordon A. Francis a,⁎
a
b
Department of Medicine and UBC James Hogg Research Centre, Providence Heart and Lung Institute, University of British Columbia, Vancouver, Canada
Group on the Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Canada
a r t i c l e
i n f o
Article history:
Received 4 October 2011
Received in revised form 28 October 2011
Accepted 28 November 2011
Available online xxxx
Keywords:
ABCA1
Lysosome
Cholesterol
Niemann–Pick disease type C
High density lipoprotein
HDL
a b s t r a c t
Niemann–Pick disease type C (NPC) is caused by mutations leading to loss of function of NPC1 or NPC2 proteins, resulting in accumulation of unesterified cholesterol in late endosomes and lysosomes. We previously
reported that expression of the ATP-binding cassette transporter A1 (ABCA1) is impaired in human NPC1−/−
fibroblasts, resulting in reduced HDL particle formation and providing a mechanism for the reduced plasma
HDL cholesterol seen in the majority of NPC1 patients. We also found that treatment of NPC1 −/− fibroblasts
with an agonist of liver X-receptor corrects ABCA1 expression and HDL formation and reduces lysosomal cholesterol accumulation. We have confirmed that ABCA1 expression is also reduced in NPC2 −/− cells, and found
that α-HDL particle formation is impaired in these cells. To determine whether selective up-regulation of
ABCA1 can correct lysosomal cholesterol accumulation in NPC disease cells and HDL particle formation, we
produced and infected NPC1 −/− and NPC2 −/− fibroblasts with an adenovirus expressing full-length ABCA1
and enhanced green fluorescent protein (AdABCA1-EGFP). ABCA1-EGFP expression in NPC1−/− fibroblasts
resulted in normalization of cholesterol efflux to apolipoprotein A-I (apoA-I) and α-HDL particle formation,
plus a marked reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes. In
contrast, AdABCA1-EGFP treatment of NPC2 −/− fibroblasts to normalize ABCA1 expression had no effect on
cholesterol efflux to apoA-I or accumulation of excess cholesterol in lysosomes, and only partially corrected
α-HDL formation by these cells. These results suggest that correction of ABCA1 expression can bypass the
mutation of NPC1 but not NPC2 to mobilize excess cholesterol from late endosomes and lysosomes in NPC
disease cells. Expression of ABCA1-EGFP in NPC1 −/− cells increased cholesterol available for esterification
and reduced levels of HMG-CoA reductase protein, effects that were abrogated by co-incubation with
apoA-I. A model can be generated in which ABCA1 is able to mobilize cholesterol, to join the intracellular regulatory pool or to be effluxed for HDL particle formation, either directly or indirectly from the lysosomal
membrane, but not from the lysosomal lumen. This article is part of a Special Issue entitled Advances in
High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945–2010).
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
The Niemann–Pick type C1 and C2 proteins (NPC1 and NPC2) function within late endosomes and lysosomes to make cholesterol derived from hydrolysis of lipoprotein cholesteryl esters or from
membrane turnover available to the rest of the cell [1,2]. Previous
and recent work has provided evidence that NPC2 binds cholesterol
[3,4], and delivers it within the lysosomal lumen to NPC1 in the lyososomal membrane for subsequent egress from this compartment [5–7].
Abbreviations: ApoA-I, apolipoprotein A-1; ABCA1, ATP-binding cassette transporter A1; EGFP, enhanced green fluorescent protein; HDL, high density lipoprotein; LDL,
low density lipoprotein; LXR, liver X receptor; NPC, Niemann–Pick type C.
☆ This article is part of a Special Issue entitled Advances in High Density Lipoprotein
Formation and Metabolism: A Tribute to John F. Oram (1945–2010).
⁎ Corresponding author.
E-mail address: [email protected] (G.A. Francis).
Deficiency of either NPC2 or NPC1 therefore results in sequestration of
excess unesterified cholesterol within late endosomes/lysosomes, impaired delivery of cholesterol to the endoplasmic reticulum for esterification or regulation of cholesterol homeostatic genes including
HMG-CoA reductase and the LDL receptor, and impaired generation
of oxysterols [8–10]. Deficiency of activity of either protein leads to a
similar and frequently fatal neurodegenerative and hepatic disorder,
Niemann–Pick disease type C [1].
We previously reported impaired cholesterol-dependent regulation of the key protein regulating new high density lipoprotein
(HDL) formation, the ATP-binding cassette transporter A1 (ABCA1),
in patients with NPC disease [11]. This finding is consistent with impaired oxysterol generation, the key regulator of liver X receptor
(LXR)-dependent activation of ABCA1 expression, in both NPC1 −/−
and NPC2 −/− cells [10], and provides a likely mechanism for the
low plasma HDL-C seen in the majority of NPC disease patients
[11,12]. We subsequently found that treatment of NPC1 −/− cells
1388-1981/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbalip.2011.11.013
Please cite this article as: E. Boadu, et al., ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann–Pick C2 but
not Niemann–Pick C1 protein, Biochim. Biophys. Acta (2012), doi:10.1016/j.bbalip.2011.11.013
2
E. Boadu et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx
with a nonoxysterol agonist of LXR corrected ABCA1 and ABCG1 expression, lipid efflux to apoA-I, and HDL formation, and could bypass
the NPC1 mutation to reduce accumulation of late endosome/
lysosome cholesterol [13]. In the present studies we aimed to determine whether an increase in ABCA1 expression alone has a similar effect in NPC1 −/− cells, as well as the effect in NPC2 −/− cells. We
confirmed that ABCA1 expression is also impaired in NPC2 −/− cells.
Overexpression of ABCA1 using adenoviral gene delivery was able
to correct cholesterol efflux to apoAI and HDL particle formation,
and to markedly reduce lysosomal cholesterol accumulation in
NPC1- but not NPC2-deficient cells. In addition, ABCA1 expression in
the absence of apoA-I increased the intracellular cholesterol pool regulating cholesteryl ester formation and reduced HMG-CoA reductase
protein in NPC1 −/− cells. We conclude that ABCA1 can bypass mutations in NPC1 to mobilize cholesterol either directly or indirectly from
the lysosomal membrane, but cannot overcome mutations in NPC2 to
mobilize cholesterol from the lysosomal lumen.
2. Materials and methods
2.1. Materials
Fatty acid-free bovine serum albumin (hereafter just referred to as
albumin) and filipin were purchased from Sigma-Aldrich (St. Louis,
MO). Bovine serum albumin, Dulbecco's modified Eagle's medium
(DMEM) and lipoprotein-deficient serum (LPDS) were from Hyclone
(South Logan, UT). [cholesteryl-1,2,6,7- 3H]cholesteryl linoleate
(84 Ci/mmol) was from PerkinElmer Life Sciences (Boston, MA).
2.2. Cloning of ABCA1
Expression pcDNA3.1 plasmid encoding full-length murine ABCA1, a
kind gift from Dr. Nan Wang, Columbia University [14], was amplified
by PCR with the primers (5′-AGA GAG CTC GAG TCT AGA CCA CCA
TGG CTT GTT GGC CTC AGT TA-3′ containing XhoI and XbaI sites and
5′-GAG AGT CCG CGG TAC ATA ACT TTC TTT CAC TTT CTC AT-3′
containing Sac II site) and pfu polymerase (Invitrogen, Burlington,
Canada). The reaction mixture was prepared in duplicate and
contained 10 ng of cDNA and 12.5 pmol of each primer. The reaction
mixture was preheated at 85 °C for 5 min prior to the addition of
pfu polymerase, followed by initial denaturation at 95 °C for 2 min,
25 times cycling of cDNAs at 95 °C for 30 s, 58 °C for 30 s and 72 °C
for 8 min, and final extension at 72 °C for 10 min. The products
were pooled together, purified by QIAquick PCR Purification Kit
(Invitrogen), cloned into pEGFP-N1 vector (Clontech, Palo Alto, CA) in
frame with EGFP gene using XhoI and Sac II restriction endonucleases,
and the construct confirmed by sequencing.
2.3. Recombinant adenovirus preparation
We used E1/E3 deleted replication defective human adenovirus
serotype 5 Adeno-x Expression System 1 (Clontech) to generate recombinant ABCA1-EGFP-expressing adenovirus. Generation of adenovirus was performed according to the protocol described by the
manufacturer with some modifications. ABCA1-EGFP cDNA construct
cloned into pShuttle2 vector yields an expression cassette of 8.3 kb,
which exceeds the manufacturer's suggested insertion capacity of
Adeno-x viral DNA (i.e., 8.0 kb). Because of this, we modified the
transfer region of pShuttle2 vector to a smaller region that is 320
bases less than the original pShuttle2 vector by PCR with primers
(5′-ACA CAC AGA TCT TTA CAT AAC TTA CGG TAA ATG G-3′
containing Bgl II site and 5′-ACA CAC AGA TCT GCA GAT GAG TTT
GGA-3′ containing Bgl II site) and pfu polymerase (Fig. 1). The PCR
reaction mixture contained 10 ng of pShuttle2 and 12.5 pmol of
each primer; the reaction conditions with 30 cycles were identical
to those described above for cloning of ABCA1. The PCR product was
purified by QIAquick PCR Purification Kit, and cloned into Bgl II
restriction endonuclease digested pShuttle2 vector that had its Bgl II
restriction endonuclease digestion sites dephosphorylated with
Shrimp alkaline phosphatase (Fermentas, Burlington, Canada).
ABCA1-EGFP construct was subcloned from pEGFP-N1 plasmid
into the Not I and Xba I sites of modified pShuttle2 to construct the
transfer plasmid pShuttle2-ABCA1-EGFP. EGFP was subcloned into
Nhe I and Not I sites of modified pShuttle2 to make pShuttle2-EGFP
(control). The expression cassette from pShuttle2-ABCA1-EGFP or
pShuttle2-EGFP was ligated with I-Ceu and PI-Sce I digested Adenox viral DNA and subsequently transformed into Max Efficiency
DH5α competent cells (Invitrogen). Recombinants were selected by
ampicillin resistance and confirmed by I-Ceu and PI-Sce I restriction
endonuclease analysis, and named Ad-ABCA1-EGFP and Ad-EGFP, respectively (Fig. 2A). The recombinant plasmid was linearized with Pac
I restriction endonuclease, and 10 μg of the plasmid transfected into
subconfluent HEK293 cells in 60 mm dish using calcium phosphate
(Clontech) according to the manufacturer's instructions. Recombinant adenoviruses obtained after 18 days post-transfection were amplified in HEK293 cells and purified by centrifugation on cesium
chloride gradient.
2.4. Preparation of lipoproteins and ApoA-I
HDL (d = 1.07–1.21 g/ml) and LDL (d = 1.019–1063 g/ml) were
isolated from pooled plasma of healthy volunteers by ultracentrifugation [15]. ApoA-I was purified from delipidated HDL on Q-Sepharose
Fast Flow column using the method of Yokoyama et al. [16]. Radiolabeling of LDL with [1,2,6,7- 3H]cholesteryl linoleate to a specific activity of 16–44 cpm/ng of LDL protein was performed as described by
Sattler and Stocker [17].
2.5. Cell culture
Normal human skin fibroblasts (wild type, CRL-2076) were purchased from the American Type Culture Collection. Human NPC1
null mutant fibroblasts line 93.41 (NPC1 −/−, hereafter referred to as
NPC1 cells) were provided by Dr. W. Garver (Dept. of Pediatrics, The
University of Arizona, Arizona, USA). NPC2 −/− mutant human skin fibroblasts (hereafter referred to as NPC2 cells, NIH 99.04) were
provided by Dr. D. Ory (Center for Cardiovascular Research, Washington University School of Medicine, St. Louis, USA). Wild type and
NPC2 fibroblasts were plated at 30,000 cells/16-mm well or 80,000
cells/35-mm well, and NPC1 fibroblasts were plated at 40,000 cells/
16-mm well or 100,000 cells/35-mm well. The cells were grown to
90% confluence in DMEM containing 10% fetal bovine serum (FBS)
and 1% penicillin–streptomycin. The medium was then replaced
with DMEM containing 10% LPDS for 24 h followed by adenoviral infection. The wild type and NPC1 fibroblasts were infected with recombinant adenovirus at multiplicity of infection (MOI) of 50 and
NPC2 fibroblasts were infected at MOI of 100 in Opti-mem media
without serum for 4 h; these MOIs were found in control experiments
to provide equivalent expression of ABCA1. After the initial infection
period, an equal volume of DMEM/20% LPDS plus antibiotics was
added to the inoculum and the cells cultured for an additional 44 h
prior to cholesterol loading.
2.6. Cholesterol efflux
Following infection, fibroblasts in 16-mm wells were labeled
with LDL-derived cholesterol by incubation with DMEM containing
1 mg/ml albumin (DMEM/albumin) plus 50 μg/ml [1,2,6,7- 3H]cholesteryl linoleate-labeled LDL protein for 24 h. Cells were washed twice
with DMEM and equilibrated for 24 h in DMEM/albumin. After equilibration, the cells were rinsed twice with DMEM and were incubated
with either DMEM/albumin alone or DMEM/albumin plus 10 μg/ml
Please cite this article as: E. Boadu, et al., ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann–Pick C2 but
not Niemann–Pick C1 protein, Biochim. Biophys. Acta (2012), doi:10.1016/j.bbalip.2011.11.013
E. Boadu et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx
Bgl II (35)
Bgl II (1121)
SV40
poly A
Bgl II (330)
Original pShuttle2
pUC ori
Pcmv MCS
(336)
I-Ceu I
3
Kanr
PI-Sce I
Amplification
SV40
poly A
Bgl II (330)
Pcmv MCS
Bgl II (1121)
Insertion
Bgl II (35
Modified pShuttle2
I-Ceu I
SV40
poly A
330)
Bgl II (1121)
pUC ori
Pcmv MCS
Kanr
PI-Sce I
Fig. 1. Modification of pShuttle2 vector. Reduction of the transfer region of pShuttle2 by 320 bases was performed by introducing a new Bgl II site at position 330 by PCR. A forward
primer (5′-ACA CAC AGA TCT TTA CAT AAC TTA CGG TAA ATG G-3′) containing Bgl II site designed to match the initial sequence of Pcmv promoter and a reverse primer (5′-ACA CAC
AGA TCT GCA GAT GAG TTT GGA-3′) matching Bgl II site at position 1121 and pfu polymerase were used by PCR to generate a smaller fragment (Bgl II 330–Bgl II 1121) of the transfer
region of pShuttle2. PCR conditions are described in the Materials and methods. The truncated fragment was cloned into Shrimp alkaline phosphatase dephosphorylated Bgl II sites
of pShuttle2.
human apolipoprotein A-I (apoA-I) for 24 h. The media were collected and the cells washed three times with ice-cold PBS, and solubilized
in 0.1 N NaOH. Efflux media were centrifuged at 2000 × g for 5 min at
4 °C to remove cell debris. Radioactivity in media was determined by
liquid scintillation counting. Protein concentration of cell lysates was
determined using bovine serum albumin as standard [18].
2.7. Cholesterol esterification by ACAT
To assess the effect of ABCA1 expression on the pool of intracellular cholesterol regulating cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT), wild type and NPC1 fibroblasts were
grown in 16-mm wells and the NPC1 cells infected with Ad-ABCA1-
A
C
a
ITR
Ad-EGFP
PCMV IE
EGFP
PCMV IE
ABCA1-EGFP
b
SV40 pA
ITR
Ad-ABCA1-EGFP
B
1
2
3
SV40 pA
D
Anti-ABCA1
250 kD
250 kD
Anti-GFP
25 kD
Anti-GFP
12 kb
3 kb
2 kb
β-actin
Ad-EGFP
Ad-ABCA1-EGFP
+
-
-
+
Fig. 2. Generation of recombinant adenoviral vectors expressing ABCA1-EGFP fusion protein and EGFP. Adenoviral vectors expressing ABCA1-EGFP fusion protein and EGFP (control) were prepared as described in the Materials and methods. (A) The expression of ABCA1-EGFP or EGFP gene was under the upstream control of the human cytomegalovirus
immediate-early gene promoter (PCMV IE) and the downstream control of the SV40 polyadenylation signal. (B) The adenoviral vectors were digested with Pac I to expose the
inverted terminal repeats (ITRs) located at both ends of the adenoviral genome. Lane 1, 1 Kb plus DNA size marker; lane 2, Ad-EGFP; lane 3, Ad-ABCA1-EGFP. (C) Microscopic appearance of EGFP expression in HEK293 cells infected at a multiplicity of infection (MOI) of 2 with either Ad-EGFP (a) or Ad-ABCA1-EGFP (b) for 1 h and cultured for an additional
20 h. Bar = 20 μm. (D) Immunoblot analysis of the infected HEK293 cells confirmed EGFP-tagged ABCA1 protein expression. Nitrocellulose membrane was probed with ABCA1 antibody and then reprobed with GFP antibody.
Please cite this article as: E. Boadu, et al., ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann–Pick C2 but
not Niemann–Pick C1 protein, Biochim. Biophys. Acta (2012), doi:10.1016/j.bbalip.2011.11.013
4
E. Boadu et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx
EGFP as described above. The cells were then incubated in DMEM/
albumin containing 50 μg/ml LDL for 24 h. Cells were rinsed twice
with DMEM and then equilibrated for 24 h in DMEM/albumin.
Following equilibration, the cells were incubated with DMEM/
albumin ± 10 μg/ml apoA-I for 24 h, rinsed with DMEM, and incubated for 1 h at 37 °C with DMEM containing 9 μM [ 14C]oleate
bound to 3 μM BSA [19]. Cells were then placed on ice, washed
twice with cold PBS-BSA and twice with cold PBS, and total lipids
extracted using hexane/2-propanol (3:2 v/v) as described [20].
Lipids were separated by thin-layer chromatography on PE SIL G
plastic-backed plates (Whatman) developed in hexane/diethyl
ether/acetic acid (130:40:1.5 v/v). Lipid spots were detected by
staining in I2 vapor and comigration with standards. After allowing
I2 stain to evaporate, cholesteryl ester spots were taken for determination of radioactivity. The cells were solubilized with 0.1 N
NaOH and used for determination of protein. ACAT activity was determined by normalizing the amount of cholesteryl ester formed to
the corresponding cellular protein.
anti-GFP (which also recognizes EGFP; 1:2000 dilution, Molecular
Probes, Eugene, OR), rabbit anti-HMG-CoA reductase (1:500 dilution,
Upstate Cell Signaling Solutions, Lake Placid, NY) and rabbit anti-βactin (Abcam, Cambridge, MA) as primary antibodies, with goat antirabbit IgG horse-radish peroxidase-conjugated secondary antibody
(1:5000 dilution, Sigma). Proteins were visualized by using enhanced
chemiluminescence reagents from Pierce (Rockford, IL).
2.10. Two-dimensional gel electrophoresis of HDL particles
Media with apoA-I-containing particles generated by cells in 35mm wells were analyzed by non-denaturing 2-dimensional gel electrophoresis as previously described [13], substituting goat antirabbit IgG horse-radish peroxidase-conjugated secondary antibody
(1:5000 dilution, Sigma) and enhanced chemiluminescence reagents
(Pierce) for 125I-labeled donkey anti-rabbit antibody (Amersham Biosciences) and autoradiography.
2.11. Statistical analysis
2.8. Filipin staining
Fibroblasts were grown on coverslips in 35-mm wells and infected
with recombinant adenovirus. The cells were loaded with nonlabeled LDL and incubated with apoA-I as described above. Cells
were then fixed and stained with filipin as described [13].
Results for Figs. 3B, 5 and 8 were analyzed using GraphPad Prism
version 5.0 and are presented as the mean ± S.D. Significant differences between experimental groups were determined using the Student's t test.
3. Results
2.9. Immunoblot analysis
Cells in 35-mm wells were lysed with 100 μl of radioimmunoprecipitation assay (RIPA) buffer [PBS, 0.1% sodium dodecyl
sulfate (SDS), 0.5% sodium deoxycholate, 1% nonidet p40 (NP-40),
pH 7.4] supplemented with Complete Protease Inhibitor Cocktail
tablet (Roche Diagnostics, Indianapolis, IN) for 1 h at 4 °C . Sixty micrograms of total lysate proteins were separated by SDS-PAGE on 5–
15% (w/v) gradient gel, and transferred onto nitrocellulose membrane. Immunoblotting was performed using rabbit anti-human
ABCA1 (1:1000 dilution, Novus Biologicals, Littleton, CO), rabbit
A
3.1. Generation of adenoviral vector expressing ABCA1-EGFP fusion
protein
To determine whether our previous results showing reduced lysosomal cholesterol accumulation and enhanced HDL formation by
NPC1-deficient fibroblasts treated with an LXR agonist [13] could
be reproduced by upregulating ABCA1 alone, we required an efficient method of overexpressing ABCA1 in these cells. Adenoviral
vectors have been vehicles of choice for gene expression in skin
fibroblasts, since nonviral vector-mediated gene transfer using
WT
1
NPC1
1.4
0.7
0.7
NPC2
0.6
0.7
ABCA1
β-actin
LDL
-
+
-
+
-
+
ABCA1 protein (Densitometry units)
B
1.6
1.4
1.2
1
* **
0.8
* **
0.6
0.4
0.2
0
LDL
NPC genotype
WT
+
-
+
NPC1
-
+
NPC2
Fig. 3. Diminished ABCA1 expression in NPC1 and NPC2 fibroblasts. (A) Wild type (WT), NPC1 and NPC2 fibroblasts were grown to 60% confluence in DMEM/10% FBS and then
cultured in DMEM/10% LPDS to confluence. The cells were incubated in DMEM/albumin in the absence or presence of LDL (50 μg/ml) for 24 h, and then equilibrated in DMEM/
albumin for 24 h prior to determination of ABCA1 by immunoblotting. ABCA1 was detected using 60 μg of total cell protein. Numerical values represent the ratio of ABCA1 to βactin intensity, normalized to the ratio in non-LDL loaded wild type fibroblasts set as one. Results are representative of three experiments with similar results. (B) Average
ABCA1 protein levels as determined by immunoblotting in cells incubated in the absence (−) or presence (+) of LDL, relative to non-LDL-loaded wild type cells. Results are averages ± S.D. for 3 experiments. *, significantly lower than non-LDL-loaded wild type fibroblasts, p b 0.05. **, significantly lower than LDL-loaded wild type fibroblasts, p b 0.0001.
Please cite this article as: E. Boadu, et al., ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann–Pick C2 but
not Niemann–Pick C1 protein, Biochim. Biophys. Acta (2012), doi:10.1016/j.bbalip.2011.11.013
E. Boadu et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx
various transfection methods is very inefficient in these cells. We
therefore generated adenovirus expressing ABCA1-EGFP fusion protein or control EGFP alone using the Clontech Adeno-x expression
system 1. Based on the manufacturer's advice that this system has
an insertion capacity of 8.0 kb (http://www.clontech.com/xxclt_
ibcGetAttachment.jsp?cItemId=17524&minisite=10020), we modified the transfer region of pShuttle2 (Fig. 1) and used that to generate adenoviral vectors Ad-ABCA1-EGFP and Ad-EGFP (Fig. 2A).
Digestion of the adenoviral vectors with Pac I restriction endonuclease resulted in the release of a larger fragment and a smaller fragment of 3.0 kb (Fig. 2B), consistent with a previous report [21].
Initial evaluation of the newly generated Ad-ABCA1-EGFP- and AdEGFP-infected HEK293 cells by fluorescence microscopy showed
bright green fluorescence in HEK293 cells (Fig. 2C), indicating the infectivity of the viruses and GFP expression in the cells. Immunoblot
analysis confirmed the expression of ABCA1-EGFP fusion protein by
Ad-ABCA1-EGFP but not Ad-EGFP (Fig. 2D). To the best of our knowledge, there is no prior demonstration of a single adenovirus expressing ABCA1-EGFP fusion protein.
3.2. ABCA1 expression in NPC1 and NPC2 human fibroblasts and increase
with ABCA1-EGFP fusion protein expression
We previously demonstrated low basal and cholesterol-stimulated
ABCA1 protein levels in human NPC1 fibroblasts [11], which were corrected by treatment of the cells with an exogenous LXR ligand TO-
A
901317 [13]. The inability of NPC2-deficient fibroblasts to correctly
deliver cholesterol to the lysosomal membrane for egress and stimulation of oxysterol generation suggests NPC2-deficient cells should also
exhibit impaired ABCA1 expression. We confirmed our previous finding of impaired basal and LDL-stimulated expression of ABCA1 in
NPC1-deficient fibroblasts, and also demonstrate this defect in NPC2deficient cells (Figs. 3 and 4B). Infection of wild type, NPC1 and
NPC2 fibroblasts with Ad-EGFP resulted in a diffuse pattern of EGFP
fluorescence, and a punctuate pattern of fluorescence upon infection
with Ad-ABCA1-EGFP (Fig. 4A), as previously reported in fibroblasts
and other cell types using transfection or adenoviral infection [22–
25]. Expression of ABCA1-EGFP fusion protein was seen in all cell
types following infection with Ad-ABCA1-EGFP (Fig. 4B).
3.3. Enhanced cholesterol efflux in ABCA1-EGFP-expressing NPC1 but not
NPC2 fibroblasts
NPC1 and NPC2 cells were infected with Ad-ABCA1-EGFP and then
incubated with LDL labeled with [ 3H]cholesteryl linoleate prior to incubation with apoA-I to assess cholesterol efflux. Consistent with impaired ABCA1 expression, NPC1 and NPC2 fibroblasts both showed
approximately 50% less efflux of LDL-derived cholesterol to apoA-I
when compared to wild type fibroblasts (Fig. 5). Overexpression of
ABCA1 in NPC1 cells resulted in correction of apoA-I-mediated efflux
of LDL-derived cholesterol to a level similar to wild type fibroblasts
infected with AdABCA1-EGFP. Infection of NPC2 cells with this vector,
NPC2
NPC1
Ad-ABCA1EGFP
Ad-EGFP
WT
5
B
WT
NPC1
NPC2
anti-ABCA1
250 kD
anti-GFP
250 kD
anti-GFP
25 kD
β-actin
LDL Ad-EGFP Ad-ABCA1 -EGFP
+ + +
- + - - +
-
+ + +
- + - - +
-
+ + +
- + - - +
Fig. 4. ABCA1-EGFP expression in wild type, NPC1 and NPC2 fibroblasts. (A) Microscopic appearance of EGFP expression in wild type (WT), NPC1 and NPC2 fibroblasts infected with
Ad-EGFP (control) or Ad-ABCA1-EGFP as described in the Materials and methods. Bar = 10 μm. (B) Immunoblot analysis confirmed that ABCA1-EGFP fusion protein was expressed
in wild type, NPC1 and NPC2 fibroblasts infected with Ad-ABCA1-EGFP, relative to controls expressing EGFP alone. Nitrocellulose membrane was probed with ABCA1 antibody and
then reprobed with antibody against GFP. Data are representative of three experiments with similar results.
Please cite this article as: E. Boadu, et al., ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann–Pick C2 but
not Niemann–Pick C1 protein, Biochim. Biophys. Acta (2012), doi:10.1016/j.bbalip.2011.11.013
6
E. Boadu et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx
[ 3 H]Cholesterol efflux
(cpm x 10 3 /mg cell protein)
18
*
16
14
*
12
10
8
+
6
+
+
4
2
0
WT
WT
+ ABCA1
NPC1
NPC1
+ ABCA1
NPC2
NPC2
+ ABCA1
Fig. 5. ABCA1-EGFP expression increases LDL-derived cholesterol efflux to apoA-I in
NPC1 but not NPC2 fibroblasts. Wild type (WT), NPC1 and NPC2 fibroblasts were
infected with Ad-ABCA1-EGFP (indicated in figure as + ABCA1, open bars) as described
in the Materials and methods. The cells were labeled with [3H]cholesteryl linoleatelabeled LDL, followed by equilibration and incubation with DMEM/albumin containing
10 μg/ml apoA-I for 24 h. Results are expressed as counts/min of [3H]cholesterol per mg
of cell protein in the medium after subtraction of efflux to medium containing albumin
alone. Values are the mean ± S.D. of triplicate determinations and are representative of
two experiments with similar results. +, significantly lower than non-infected wild
type fibroblasts, p b 0.01. *, significantly higher than non-infected wild type fibroblasts,
p b 0.05.
however, resulted in no increase in apoA-I-dependent efflux of cholesterol from LDL. These results suggest correction of ABCA1 expression is able to overcome the defect in lysosomal cholesterol
mobilization in NPC1- but not NPC2-deficient cells.
3.4. Normalization of HDL particle formation by NPC1 but not NPC2 cells
expressing ABCA1-EGFP
We previously reported impaired α-HDL particle formation by NPC1
fibroblasts incubated with apoA-I, consistent with a role of ABCA1 in
generating α-HDL, and correction of formation of α-HDL particles by
NPC1 cells following incubation with exogenous LXR agonist [13]. To
determine the role of correcting ABCA1 expression alone on HDL particle formation by NPC1 and NPC2 cells, we assessed the HDL particles
A
WT
NPC1
NPC2
formed in apoA-I-containing medium by cells before and after infection
with Ad-ABCA1-EGFP using two-dimensional gel electrophoresis. The
medium of NPC1 cells showed near absence α-HDL particles, consistent
with our previous report [13], as did NPC2 cell medium (Fig. 6A). Overexpression of ABCA1 by the adenoviral vector increased α-HDL particle
formation by NPC1 fibroblasts to a similar extent as in wild type cells
(Fig. 6B). α-HDL particle formation by NPC2 cells was increased only
modestly by this treatment, likely because of increased efflux of nonlysosomal rather than lysosomal cholesterol by the increased ABCA1
protein. These results indicate further the importance of lysosomallyderived cholesterol in the generation of HDL particles by ABCA1.
3.5. Mobilization of late endosome/lysosome cholesterol by ABCA1-EGFP
in NPC1 but not NPC2 fibroblasts
To more directly assess the effect of overexpression of ABCA1 on
mobilization of cholesterol from late endosomes and lysosomes,
NPC1 and NPC2 cells infected with control Ad-EGFP or Ad-ABCA1EGFP were then loaded with LDL, incubated for 24 h in the absence
or presence of 10 μg/ml apoA-I, and unesterified cholesterol remaining in the cells was detected using filipin. Incubation of noninfected or Ad-EGFP-infected NPC1 and NPC2 fibroblasts with
albumin alone or albumin plus apoA-I resulted in no change in the intensity of filipin staining (Fig. 7). Expression of ABCA1-EGFP in NPC1
fibroblasts resulted in a marked decrease in filipin staining whether
or not the cells were also incubated with apoA-I. Overexpression of
ABCA1 in NPC2 cells, conversely, had no effect on accumulation of
cholesterol in late endosomes/lysosomes in the absence or presence
of apoA-I. These results further suggest that ABCA1 expression can
overcome the defect in cholesterol mobilization from late endosomes/lysosomes and make this cholesterol available for HDL formation in NPC1 but not NPC2 fibroblasts. They also suggest ABCA1 can
mobilize cholesterol from late endosomes/lysosomes in NPC1 cells
even in the absence of apoA-I as a cholesterol (and phospholipid)
acceptor.
3.6. Effect of ABCA1-EGFP expression on the regulatory pool of cholesterol
in NPC1 fibroblasts
The impaired egress of cholesterol out of late endosomes and lysosomes in Niemann–Pick disease type C1 and C2 cells results in reduced
esterification of cholesterol by acyl-coA:cholesterol acyltransferase
B
NPC1
WT
NPC2
17.0
12.2
10.5
7.1
Pre-β α
Pre-β α
ApoA-I
Pre-β
α
Pre-β
β
α
Pre-β
α Pre-β
α
ApoA-I + Ad-ABCA1-EGFP
Fig. 6. Increased HDL particle formation in NPC1 fibroblasts by ABCA1-EGFP fusion protein. Media from non-infected (A) and Ad-ABCA1-EGFP-infected (B) wild type (WT), NPC1
and NPC2 fibroblasts that had been LDL-loaded, equilibrated and incubated with apoA-I as described in the Materials and methods were analyzed for apoA-I-containing HDL particles by two-dimensional gel electrophoresis. Results are representative of two experiments with similar results.
Please cite this article as: E. Boadu, et al., ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann–Pick C2 but
not Niemann–Pick C1 protein, Biochim. Biophys. Acta (2012), doi:10.1016/j.bbalip.2011.11.013
E. Boadu et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx
Ad-EGFP
Ad-ABCA1-EGFP
NPC2
NPC1
WT
No infection
7
ApoA-I
-
+
-
+
-
+
Fig. 7. ABCA1-EGFP expression decreases late endosome/lysosome cholesterol in NPC1 but not NPC2 fibroblasts. Wild type (WT), NPC1 and NPC2 fibroblasts were infected with
either Ad-EGFP or Ad-ABCA1-EGFP. The cells were then loaded with LDL for 24 h and incubated in DMEM/albumin alone or with 10 μg/ml apoA-I for a further 24 h. Cells were
washed with PBS, fixed, and stained with filipin to measure intralysosomal unesterified cholesterol. Results are representative of three experiments with similar results.
Bar = 20 μm.
14
C-labelled CE (pmol/mg cell protein)
(ACAT), increased expression of HMG-CoA reductase and de novo
cholesterol synthesis, and impaired downregulation of LDL receptor
expression in response to LDL loading when compared to normal
cells [8–10,26]. To determine potential effects of increased mobilization of late endosome/lysosome cholesterol by overexpressing
ABCA1 on the regulatory pool of cholesterol in NPC1 cells, we measured the degree of new cholesteryl ester formation and expression
of HMG-CoA reductase in the absence or presence of apoA-I. NPC1
cells showed reduced cholesterol esterification in the absence of
apoA-I, and no reduction in ACAT-accessible cholesterol following
incubation with apoA-I when compared to normal cells (Fig. 8).
Overexpression of ABCA1 with Ad-ABCAI-EGFP in the absence of
apoA-I resulted in increased cholesteryl ester formation, suggesting
cholesterol mobilized from late endosomes/lysosomes in response to
increased ABCA1 becomes available for esterification in the endoplasmic reticulum. This effect, however, was prevented by the addition of
apoA-I. Consistent with these findings, Ad-ABCA1-EGFP infection of
NPC1 cells reduced HMG-CoA reductase protein levels in the absence
of apoA-I (Fig. 9). Addition of apoA-I removed this suppression of
HMG-CoA reductase, again suggesting the increase in the regulatory
pool of cholesterol induced by increasing ABCA1 was prevented by removal of this cholesterol by apoA-I.
1600
1400
1200
#
1000
800
*
600
*
*
*
400
200
0
WT
WT + apoA-I
NPC1
NPC1+ apoA-I
NPC1 +
ABCA1
NPC1 +
ABCA1 + apoA-I
Fig. 8. ABCA1-EGFP expression in NPC1 cells increases cholesterol available for esterification in the absence of apoA-I. Wild type (WT) fibroblasts or NPC1 fibroblasts with (+
ABCA1, open bars) or without Ad-ABCA1-EGFP infection were loaded with LDL and incubated in the presence of 1 mg/ml albumin alone or plus 10 μg/ml apoA-I (+ apoA-I) for
24 h. The cells were then washed and incubated with [14C]oleate for 1 h, and the cellular cholesteryl [14C]oleate formed as a measure of cholesterol available for esterification
was determined as described in the Materials and methods. Values are the mean ± S.D. of triplicate determinations and are representative of two experiments with similar results.
*, significantly lower than wild type fibroblasts incubated with albumin alone, p b 0.01. #, significantly higher than NPC1fibroblasts uninfected with AdABCA1-EGFP and incubated
with albumin alone, p b 0.01.
Please cite this article as: E. Boadu, et al., ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann–Pick C2 but
not Niemann–Pick C1 protein, Biochim. Biophys. Acta (2012), doi:10.1016/j.bbalip.2011.11.013
8
E. Boadu et al. / Biochimica et Biophysica Acta xxx (2012) xxx–xxx
ABCA1
HMG-CoAR
β-actin
LDL
-
+
+
+
+
ApoA-I
-
-
-
-
+
Ad-EGFP
-
-
+
-
-
Ad-ABCA1
-EGFP
-
-
-
+
+
Fig. 9. ABCA1-EGFP expression in NPC1 cells suppresses HMG-CoA reductase expression in the absence of apoA-I. NPC1 fibroblasts were infected with Ad-EGFP or AdABCA1-EGFP and loaded with LDL for 24 h. The cells were equilibrated in DMEM/
albumin followed by incubation with DMEM/albumin alone or plus 10 μg/ml apoA-I
for 24 h prior to determination of ABCA1 and HMG-CoA reductase by immunoblotting.
Proteins were detected using 60 μg of total cell protein. Results are representative of
three experiments with similar results.
4. Discussion
The results presented here provide several lines of evidence that
overexpression of ABCA1 alone is able to correct the mobilization of
cholesterol from late endosomes/lysosomes and the formation of HDL
particles in NPC1- but not NPC2-deficient human fibroblasts. Cholesterol available for efflux to apoA-I following the loading of cells with LDL
was increased to the same level as seen in normal cells by increasing expression of ABCA1 in NPC1 but not NPC2 cells. This effect was confirmed
by the correction of α-HDL particle formation by NPC1 cells but not
NPC2 cells expressing ABCA1. Late endosome/lysosome cholesterol
was markedly reduced in NPC1 but not NPC2 cells overexpressing
ABCA1. Based on these results, a model can be proposed in which
cholesterol delivered to late endosomal/lysosomal membranes by functional NPC2 can be mobilized either directly or indirectly from this compartment when ABCA1 expression is corrected, even in the absence of
functional NPC1. In the absence of functional NPC2, however, cholesterol is not delivered to the lysosomal membrane and overexpressing
ABCA1 is not able to induce cholesterol egress out of the late endosome/lysosome compartment.
These results are consistent with the demonstration that NPC2
binds cholesterol [3,4,6], and can deliver cholesterol to NPC1 [7] or
to liposomal membranes [3,4,7]. Even in the absence of functional
NPC1, if NPC2 is able to deliver cholesterol to the lysosomal membrane either via mutant NPC1 or the membrane itself, our model suggests this cholesterol then becomes available for removal following
upregulation of ABCA1. Whether or not ABCA1 is removing this
cholesterol from late endosomes/lysosomes directly through retroendocytosis, as some evidence suggests [13,25,27,28], or whether it
indirectly causes a shift in lysosomal cholesterol to the plasma membrane following depletion of plasma membrane cholesterol by ABCA1
[29], or both, is not yet known. Previous studies have shown that expression of ABCA1-GFP by transfection or adenoviral gene delivery results in ABCA1 localization in the plasma membrane as well as
intracellular compartments including late endosomes and lysosomes
and the Golgi compartment [22–25]. Further experiments are required to determine whether this effect of ABCA1 in NPC1 cells is mediated at least in part by a direct interaction with ABCA1 and the
lysosomal membrane to mobilize cholesterol. In the experiments presented here, apoA-I was not required to see depletion of late endosome/lysosome cholesterol in NPC1 cells following overexpression
of ABCA1 (Fig. 7), suggesting the mobilization of cholesterol by
ABCA1 at late endosomes/lysosomes and/or the plasma membrane
does not require the presence or co-internalization of apoA-I.
These results are in interesting contrast to findings recently
reported showing the mobilization of lysosomal cholesterol from
both NPC1- and NPC2-deficient cells using cyclodextrins. In vivo studies in NPC1- [30] [31] and NPC2-deficient mice [31] found treatment
with 2-hydroxypropyl-β-cyclodextrin (HPCD) prolonged lifespan,
corrected cholesterol regulation [30] and reduced intraneuronal
cholesterol content [31] in these animals. Cell culture studies also
showed reduced lysosomal cholesterol in both NPC1- and NPC2deficient fibroblasts treated with either HPCD [32,33] or methyl-βcyclodextrin [33], as well as correction of cholesterol esterification
by ACAT in both cell types. Cyclodextrins are thought to be acting
by either direct mobilization of cholesterol within endocytic organelles [33], or by inducing calcium-dependent exocytosis from lysosomes [34], bypassing both NPC2 and NPC1 defects. In contrast, our
findings suggest overexpressed ABCA1 can bypass a defect of NPC1
to mobilize lysosomal membrane cholesterol, but does not mobilize
cholesterol directly from the lysosomal compartment in the absence
of NPC2 function.
Our results showing impaired ABCA1 expression in NPC2 cells, as
also recently reported [35], underscore further the importance of
lysosomally-derived cholesterol in the regulation of ABCA1. In addition to our original observation of this in NPC disease [11], we have
found a similar defect in the lysosomal acid lipase deficiency cholesteryl ester storage disease [36], where the slowed hydrolysis of cholesteryl esters in lysosomes reduces the rate of flux of cholesterol
from this compartment. In addition to regulating ABCA1 expression,
the lack of major increase in HDL formation despite overexpressing
ABCA1 in NPC2 cells (Fig. 6) suggests lysosomally-derived cholesterol
forms a very large fraction of the cholesterol substrate used by ABCA1
for HDL formation. As seen for the cylcodextrins, and in the absence of
apoA-I, cholesterol mobilized by ABCA1 from the late endosome/lysosome compartment is not only destined for cholesterol efflux but can
also join the regulatory pool of cholesterol regulating cholesterol synthesis and esterification (Figs. 8 and 9). It is interesting to note than in
2003 Vaughan and Oram reported a similar increase of cholesterol esterification in baby hamster kidney cells expressing ABCA1, in the absence of any lysosomal cholesterol trafficking defect [37].
In summary, the current findings indicate that correction of the
ABCA1 expression defect in NPC disease cells can overcome mutations in NPC1 but not NPC2 to mobilize lysosomal cholesterol, by a
mechanism apparently quite different from that mediated by cyclodextrins. Treatments such as LXR agonists that correct the defect in
ABCA1 expression in NPC disease cells would therefore be expected
to have potentially similar effects as cyclodextrins in the majority of
NPC disease patients.
Acknowledgements
The authors thank Leanne Bilawchuk and Teddy Chan for excellent
technical assistance. This work was funded by CIHR operating grant
MOP-79532 to GAF.
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