From the Department of Pediatrics, Lipid Research Atherosclerosis
Unit, The Johns Hopkins University School of Medicine, Baltimore,
Maryland 21287 and University of Toronto, Toronto,
Ontario M5G 1X8, Canada
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ABSTRACT |
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The mechanism by which genes involved in
cholesterol biosynthesis and import are preferentially up-regulated in
response to sterol depletion was elucidated with the cloning of sterol
regulatory element binding protein-1 (SREBP-1). SREBP-1 is a
transcription factor whose entry into the nucleus is gated by
sterol-regulated proteolysis. We have investigated the role of tumor
necrosis factor- (TNF-
) as a mediator of SREBP-1 maturation in
human hepatocytes. TNF-
is capable of inducing SREBP-1 maturation in
a time- and dose-dependent manner that is consistent with
the kinetics of TNF-
-mediated activation of neutral sphingomyelinase
(N-SMase). Antibodies to N-SMase inhibit TNF-
-induced SREBP-1
maturation suggesting that N-SMase is a necessary component of this
signal transduction pathway. Ceramide, a product of sphingomyelin
hydrolysis, is also capable of inducing SREBP-1 maturation. The mature
form of SREBP-1 generated by TNF-
, sphingomyelinase or ceramide
treatment translocates to the nucleus and binds the sterol regulatory
element. This promotes transcription of the gene upstream of the sterol regulatory element.
A unique finding of our studies is that ceramide stimulated SREBP-1 maturation even in the presence of cholesterol and 25-hydroxycholesterol both of which are known suppressors of SREBP-1 maturation. Our findings indicate that ceramide-mediated maturation of SREBP-1 maturation is a novel sterol-independent mechanism by which cholesterol homeostasis may be regulated.
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INTRODUCTION |
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The cytokine tumor necrosis factor
(TNF1-) elicits a wide
range of biological effects including inflammatory, cytotoxic,
antiviral, and proliferative processes (1). Despite significant
progress in our understanding of the signal transducing mechanisms
employed by TNF-
(2), they remain incompletely characterized.
Elucidation of these pathways is complicated by the existence of at
least two TNF receptors. These receptors share some common downstream effectors but also signal via receptor specific pathways.
One of the earliest events in TNF signaling is the activation of
neutral sphingomyelinase (N-SMase). Neutral sphingomyelinase is a
membrane bound enzyme that catalyzes the hydrolysis of sphingomyelin to
ceramide and phosphocholine at a pH optima of 7.4 (3). The role of
neutral sphingomyelinase in signal transduction has primarily been
ascribed to its ability to generate the lipid second messenger ceramide. In addition to TNF-, Fas receptor ligand (4, 5), vitamin
D3 (6), interleukin-1
(7), nerve growth factor (8),
anti-CD28 antibodies (9), and
-interferon (10) have all been shown
to increase ceramide levels.
Sphingolipids, including ceramide, are increasingly appreciated as regulators of cell growth and differentiation (8, 11). Other lipids, such as cholesterol, have long been appreciated for their roles in cell physiology and pathophysiology. Cholesterol homeostasis in particular is a tightly regulated process. Dysregulation of cholesterol metabolism can lead to a variety of pathophysiological states including heart disease and stroke (12).
The focus of regulation for cholesterol homeostasis is the low density lipoprotein (LDL) receptor (12). The LDL receptor binds to cholesterol rich particles in the plasma and delivers them to cells. Transcription of the LDL receptor gene is suppressed when sterols accumulate and induced when sterols are depleted. Sterol sensitivity is conferred by a 10-base pair sequence upstream of the LDL receptor gene known as the sterol regulatory element (SRE) (13). The mature form of the sterol regulatory element binding protein-1 (SREBP-1) binds to the SRE and promotes transcription (14).
The activity of SREBP-1 is controlled by an extranuclear sequestration
mechanism. Like nuclear factor-kB, SREBP-1 is synthesized as an
inactive precursor that is proteolytically processed into a mature
transcriptionally active form that translocates to the nucleus. Unlike
nuclear factor-kB, however, SREBP-1 proteolysis is induced by sterol
depletion and suppressed by sterol accumulation. Both molecules are
proteolyzed in response to treatment with TNF-.
Here we report that TNF- is capable of inducing SREBP-1 proteolysis
independent of the presence of sterols. Exogenously supplied sphingomyelinase and ceramide are also capable of inducing SREBP-1 proteolysis in a time- and dose-dependent manner. The
kinetics of SREBP-1 maturation is consistent with those of neutral
sphingomyelinase activation by TNF-
. Further, SREBP-1 maturation can
be blocked with anti-N-SMase antibodies suggesting that neutral
sphingomyelinase is necessary for TNF-
-induced sterol-independent
SREBP-1 cleavage. The product of sterol-independent SREBP-1 proteolysis
is capable of nuclear translocation and binds to the sterol regulatory
element.
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EXPERIMENTAL PROCEDURES |
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Materials--
A continuous line of human hepatocytes designated
HH-25 was prepared from normal human tissue (18). Alpha-modified
minimal essential medium was purchased from Mediatech (Herndon, VA).
Fetal bovine serum was purchased from Hyclone (Salt Lake City, UT). F10
medium and the insulin-transferrin-selenium supplement were purchased
from Life Technologies, Inc. Human recombinant epidermal growth factor,
platelet-derived growth factor, and TNF- were from Upstate
Biotechnology (Lake Placid, NY). C2-ceramide
(N-acetylsphingosine) was obtained from Matreya (Pleasant
Gap, PA). [14C]Sphingomyelin (specific activity 50 mCi/mmol) was from American Radiolabeled Chemicals (St. Louis, MO).
Anti-SREBP-1 antibody was purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Sphingomyelinase from Streptomyces species
and all other reagents were obtained from Sigma.
Cell Culture--
HH-25 cells were grown in alpha-minimal
essential medium supplemented with 100 units/ml penicillin, 100 µg/ml
streptomycin, 10 µg/ml insulin, 0.1 µM selenium, 5.5 µg/ml transferrin, 0.5 µg/ml linoleic acid, and 10% fetal bovine
serum (medium A). The cells were incubated in serum free F10 media for
30 to 60 min prior to initiating treatment with TNF-,
C2-ceramide, or sphingomyelinase.
Cell Fractionation--
Following treatment, the cells were
washed with 5 ml of PBS and pelleted at 1500 × g for
10 min at 4 °C. The pellet was stored at 70 °C and lysed in 0.5 ml buffer A (10 mM HEPES, pH 7.4, 5 mM EDTA,
0.25 mM EGTA, 50 mM NaF, 7 mM
-mercaptoethanol, 0.35 M sucrose, 0.1% Nonidet P-40,
and protease inhibitors, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 10 µg/ml leupeptin, and 5 µg/ml
pepstatin). The lysate was centrifuged at 12,000 × g
for 15 min at 4 °C to prepare a nuclear fraction.
Neutral Sphingomyelinase Assay--
After stimulation with
TNF- for the indicated time intervals, the cells were washed once
with 5 ml of PBS and harvested. The pellet was stored frozen at
70 °C and resuspended in 0.5 ml of buffer B (100 mM
Tris-HCl, pH 7.4, 0.1% Triton X-100, 1 mM EDTA, and
protease inhibitors). The cell suspension was sonicated 3 times (3-s
bursts) using a probe sonicator and centrifuged at 500 × g at 4 °C for 5 min. The supernatant was used as the
enzyme source.
Immunoblotting-- 50 µg of nuclear protein was separated by gel electrophoresis on a 7.5% polyacrylamide gel. Gels were calibrated by high range molecular weight markers (Bio-Rad) which were transferred to a polyvinylidene difluoride membrane and visualized with Coomassie staining. Rabbit polyclonal antibodies against SREBP-1 were used at 0.5 µg/ml according to the instructions of the manufacturer. The antibody was visualized with horseradish peroxidase-conjugated anti-rabbit IgG made in donkey (Amersham) using the enhanced chemiluminescence (ECL) Western blotting detection system kit (Amersham). Polyvinylidene difluoride membranes were exposed to hyperfilm ECL (Amersham) for the indicated time. Immunoblots were quantified via densitometry performed on a PDI densitometer scanner (model 20J7) coupled to a SPARC IRC workstation.
Indirect Immunofluorescence-- Cultured HH-25 cells were grown on coverslips and treated as described. After treatment, the cells were washed 3 times with PBS containing 1 mM MgCl2 and 0.1 mM CaCl2 (solution A). The cells were fixed with 3% paraformaldehyde in solution A for 10 min and permeabilized with 0.5% Triton X-100 in solution A for 6 min at room temperature. The coverslips were then washed 3 times for 5 min with solution A.
Primary antibody (anti-SREBP1) was used at a dilution of 0.5 µg/ml in PBS and applied for 1 h with gentle shaking. The cells were washed as above and a fluorescein isothiocyanate-conjugated anti-rabbit IgG secondary antibody was applied for 30 min according to the instructions of the manufacturer. The coverslips were washed, mounted on microscope slides, viewed, and photographed at the indicated magnification on a Zeiss Axiovert 25 fluorescence microscope.DNA Laddering Assay--
Cells were treated with either TNF-,
sphingomyelinase, or C2-ceramide for 1 h at
concentrations identical to those used in the SREBP-1 maturation
studies. The cells were then washed twice with minimal essential medium
and refed with medium A for 6 h. The cells were harvested and
genomic DNA was prepared as described (22). Genomic DNA was
electrophoresed and stained with ethidium bromide.
Electrophoretic Mobility Shift Assays--
Gel mobility shift
assays were performed as follows. Each 20-µl reaction mixture
contained 8-10 µg of nuclear protein plus a
-32P-labeled 25-base pair oligonucleotide probe
containing the SREBP-binding site (14) in binding buffer (10 mM HEPES, pH 7.5, 0.5 mM spermidine, 0.15 mM EDTA, 10 mM dithiothreitol, 0.35 mM sucrose). The reaction mixture was incubated at room
temperature for 15 min and loaded directly onto a 6.5% polyacrylamide
(49:0.6 acrylamide/bisacrylamide) gel in a buffer of 25 mM
Tris borate (pH 8.0), 0.25 mM EDTA. In some experiments,
antisera specific for SREBP or preimmune sera were added to reaction
mixtures to determine the composition of protein-probe complexes. For
these "supershift" assays, extracts were incubated with 1 µl of
preimmune sera or an equal volume of anti-SREBP antisera at 4 °C for
30 min prior to addition of
-32P-labeled probe. In all
experiments, proteins were separated by electrophoresis at 200 V for
2 h at room temperature. Gels were dried and exposed to Kodak XAR
film with intensifying screens. Assays were repeated with nuclear
extracts obtained from three unique experiments and evaluated by
phosphoimage analysis to ensure reproducibility of results.
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RESULTS |
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The Effect of TNF- on Neutral Sphingomyelinase
Activity--
Neutral sphingomyelinase activity increased rapidly with
the addition of TNF-
(Fig. 1). A
maximal 2.5-fold increase in activity was observed 15 min after TNF-
was added to the cells. The gradual return of N-SMase activity to
control levels within 1 h contrasted the rapid onset of activation
and is reflected in the asymmetric kinetic profile observed.
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Kinetics of SREBP-1
Proteolysis--
Sterol-independent SREBP-1 maturation in
response to TNF- closely paralleled the kinetics of TNF-
induced
N-SMase activation. The mass of the mature form of SREBP-1 was found to
increase 2-fold after 5 min and 3-fold after 15 min of incubation with
TNF-
(Fig. 2). The amount of mature
SREBP-1 returned to control levels within 1 h. This effect could
not be recapitulated with epidermal growth factor or platelet-derived
growth factor treatment (data not shown). The increase in mature
SREBP-1 levels was accompanied by a concomitant decrease in the
intensity of the band corresponding to the precursor form of SREBP-1
see Fig. 2. After 60 min of treatment, significantly less precursor
SREBP-1 is visible.
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Effects of TNF-, Sphingomyelinase, and C2-ceramide
on Apoptosis in Hepatocytes--
To demonstrate that the observed
maturation of SREBP-1 was not an artifact of the more general
phenomenon of apoptosis-induced proteolysis, we performed DNA laddering
assays. The 160-base pair DNA ladder characteristic of cells undergoing
apoptosis was not observed in any of the samples (data not shown).
Effects of TNF-, Sphingomyelinase, and C2-ceramide
Concentration on SREBP-1 Maturation--
The extent of TNF-
induced
SREBP-1 maturation did not vary appreciably with concentration. A
maximal effect was observed with 10 ng/ml of TNF-
(Fig.
3). 250 milliunits of sphingomyelinase activity induced an 80% decrease in the precursor to mature ratio (Fig. 3). As little as 1 µM of C2-ceramide
was effective in producing an 81% maximal effect. The maximal effect,
however, was obtained with a C2-ceramide concentration of
50 µM (Fig. 3).
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The Effect of Anti-N-SMase Antibodies on TNF--mediated SREBP-1
Maturation--
The availability of anti-N-SMase antibodies allowed us
to examine the effects of TNF-
on this pathway independent of
N-SMase activation (10). Polyclonal anti-N-SMase antibodies at a
dilution of 1:200 completely block TNF-
-induced SREBP-1 maturation
(Fig. 4). The suppression of
TNF-
-mediated SREBP-1 maturation was relieved with increasing
antibody dilution. Preincubation with preimmune serum at the same
dilution had no appreciable effect.
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Effects of TNF-, C2-ceramide, and Sphingomyelinase
on the Subcellular Localization of SREBP-1--
To determine if the
SREBP-1 fragment generated by TNF-
, C2-ceramide, or
sphingomyelinase treatment was capable of nuclear translocation, we
pursued immunofluorescence studies. Previous immunofluorescence studies
have relied on the overexpression of precursor and mature forms of
SREBP-1 (14). We were able to visualize endogenous SREBP-1 in treated
and untreated cells with polyclonal antibodies directed against the DNA
binding domain of SREBP-1. Since the DNA binding domain is common to
both the precursor and mature forms, we were able to examine the total distribution of endogenous SREBP-1.
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Electrophoretic Mobility Shift Assays--
Electrophoretic
mobility shift assays were pursued to demonstrate that the mature
SREBP-1 fragment is additionally capable of binding to its consensus
sequence. The amount of electrophoretically retarded probe increases
with time following TNF- treatment (Fig. 6A). The kinetics of this
process is consistent with the activation of N-SMase. The amount of
probe bound increases with sphingomyelinase and ceramide treatment. As
expected, C2-ceramide induces a more rapid accumulation of
active nuclear SREBP-1 than either TNF-
or sphingomyelinase (Fig. 6,
A-C). Antibodies directed toward the DNA binding domain of
SREBP successfully compete with the oligonucleotide probe for binding
(Fig. 6D). Binding of the probe is not titrated by an
unrelated oligonucleotide but is decreased with the addition of a
nonradioactive competing probe (data not shown).
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DISCUSSION |
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The importance of cholesterol homeostasis is underscored by the significant amount of redundancy that has been incorporated into its regulation. Cholesterol homeostasis is regulated at the transcriptional, translational, and enzymatic levels. The observation that feedback inhibition plays an important role in cholesterol homeostasis has stimulated much work in elucidating the exact role of sterols in this process (12). The current data suggest a novel pathway by which SREBP-1 maturation could be effected in a sterol-independent manner.
TNF- is capable of inducing SREBP-1 maturation in a
sterol-independent manner in human hepatocytes. These findings are not a general response to growth factors, as they could not be
recapitulated with epidermal growth factor or platelet-derived growth
factor. The maturation, nuclear translocation, and SRE binding activity of SREBP-1 in response to TNF-
closely paralleled the kinetics of
N-SMase activation. The effect of TNF-
on SREBP-1 maturation could
be reconstituted with exogenously supplied bacterial or human
sphingomyelinase (data not shown) or C2-ceramide but could not be recapitulated with dihydroceramide, phospholipase
A2, or phospholipase D.
Preincubation with anti-N-SMase antibody effectively blocked
TNF--induced SREBP-1 maturation. Inhibition was not observed with
preimmune serum treatment and was relieved with increasing antibody
dilution. Such findings are also confirmed by the ability of the
antibody to inhibit TNF-
-induced increases in cholesterol ester
synthesis and N-SMase-induced increases in 125I-LDL
binding, internalization, and degradation in human fibroblasts (15,
16).
The addition of C2-ceramide, a water soluble ceramide
analog, or bacterial sphingomyelinase mimicked the effect of TNF- on SREBP-1 maturation. In fact, C2-ceramide and
sphingomyelinase induced more extensive SREBP-1 maturation than
TNF-
. This may reflect the presence of a regulatory event upstream
of ceramide generation that is effectively bypassed with exogenous
ceramide or sphingomyelinase. The lack of apparent dose dependence
observed with TNF-
treatment might be attributable to saturable
binding of the TNF-
receptors or an internal regulatory event that
reduces the signaling capacity of the TNF-
receptors.
Previous studies have shown that an increase in SREBP-1 levels
increases LDL receptor levels and sterol biogenesis (13). Our gel
mobility shift experiments (Fig. 6) clearly indicate that TNF-,
N-SMase and C2-ceramide all induce SREBP-1 levels in
hepatocytes. In addition, we have previously shown that TNF-
induces
sterol metabolism in cultured human fibroblasts (15) and LDL receptors (16, 17). Finally, our preliminary data indicate that indeed TNF-
induces LDL receptor mRNA levels in human hepatocytes.
Collectively, these observations suggest that TNF-
-induced increase
in mature SREBP-1 level is accompanied by increased LDL receptors and
sterol metabolism.
TNF-, C2-ceramide, and sphingomyelinase did not induce
apoptosis in our studies demonstrating that in hepatocytes, SREBP-1 maturation is not part of the more general phenomenon of apoptotic protein hydrolysis.
It has been speculated that cells undergoing apoptosis require
cholesterol to maintain the integrity of their plasma membranes (18).
The 160-base pair DNA ladder characteristic of cells undergoing apoptosis generally appears 4-8 h after an apoptosis-initiating stimuli is introduced. The kinetics of SREBP-1 maturation presented in
this study would suggest that SREBP-1 proteolysis is a sufficiently early event to be involved in providing cholesterol to apoptotic cells.
However, there was no evidence of apoptosis in the HH-25 human
hepatocyte cell line used in this study. It is possible that the
sterol-independent induction of SREBP-1 maturation in hepatocytes
is a physiologic process that does not require that apoptosis be
induced. Alternatively, the two pathways may diverge before the cell
has been committed to apoptosis suggesting a manner in which
sterol-independent SREBP-1 proteolysis could be employed independent of
the induction of apoptosis. TNF- is known to activate the apoptotic
protease CPP32, and it has been shown that SREBP-1 is a physiological
substrate (18). In a recent study, a cell-permeable ceramide was shown
to induce the proteolytic cleavage of CPP32, a ced-3 interleukin-1
converting enzyme-like protease but not interleukin-1
converting
enzyme (19). Thus, sterol-independent cleavage of SREBP-1 observed in
our studies with human hepatocytes could also occur by ceramide
generated by the TNF-
-induced N-SMase activation. This phenomenon
may be reconstituted by the exogenous addition of N-SMase and/or
C2-ceramide to the hepatocytes. It is tempting to speculate
that this pathway may represent a role for the apoptotic protease CPP32
in normal cellular homeostasis.
Previous studies have shown that cholesterol efflux from the membrane into the cytosol occurs with increased neutral sphingomyelinase activity (16). This effect is attributed to the depletion of plasma membrane sphingomyelin, which has been shown to exert a stabilizing effect on plasma membrane cholesterol stores through favorable thermodynamic interactions (20-22).
We propose a model (Fig. 7) that
incorporates these data and suggests a mechanism by which TNF- could
initiate SREBP-1 proteolysis. According to this hypothesis, TNF-
binds to one or more of its cell surface receptors and in so doing
promotes the activation of N-SMase. Recently, a candidate protein that
mediates this activation has been cloned (23). N-SMase hydrolyzes
membrane sphingomyelin into ceramide and phosphocholine. Ceramide, in
turn, activates a protease perhaps CPP32 that mediates SREBP-1
maturation. The mature SREBP-1 then migrates into the nucleus as shown
and drives the transcription of genes with an upstream sterol
regulatory element.
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This model may be invoked to explain how sterol homeostasis can occur in the presence of increased cytosolic sterols, which would be predicted to suppress SREBP-1 maturation. The advantage conferred by the participation of neutral sphingomyelinase in cholesterol homeostasis is that it is capable of providing a short term solution to cholesterol starvation through mobilization of plasma membrane cholesterol and can facilitate long term compensatory mechanisms by promoting the maturation of SREBP-1.
Here, we report that TNF- is capable of inducing SREBP-1 proteolysis
independent of the presence of sterols. Exogenously supplied
sphingomyelinase and ceramide are also capable of inducing SREBP-1
proteolysis in a time- and dose-dependent manner. The kinetics of SREBP-1 maturation is consistent with the activation of
neutral sphingomyelinase by TNF-
. Furthermore, our preliminary data
indicates that recombinant human N-SMase can also exert a time- and
concentration-dependent induction of SREBP-1 maturation. In
addition, anti-N-SMase antibodies block SREBP-1 maturation. Taken
together, these findings suggest that neutral sphingomyelinase is
necessary for TNF-
-induced sterol-independent SREBP-1 cleavage.
Our data implicate N-SMase in the TNF- initiated signal transduction
pathway leading to SREBP-1 maturation and provide evidence that
ceramide is the second messenger employed. Further, they suggest a role
for TNF-
in the regulation of cholesterol homeostasis. Additional
studies are required to establish the role of cholesterol in apoptotic
cells and the role of the different tumor necrosis factor receptors in
SREBP-1 maturation. Better understanding of this pathway may yield
novel targets for the pharmacological manipulation of serum cholesterol
levels.
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ACKNOWLEDGEMENTS |
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We thank Hui Han for valuable suggestions, Demmy Adesina for excellent technical assistance, and Irina Dobromilskaya for cell culture assistance. We also thank David Sabatini for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants RO-1 DK-31722 and P50-HL47212 (to S. C.) and a Medical Scientist Training Grant GM-07309 (to J. F. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: CMSC-604, The Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21287-3645.
1 The abbreviations used are: TNF, tumor necrosis factor; N-SMase, neutral sphingomyelinase; LDL, low density lipoprotein; SRE, sterol regulatory element; SREBP-1, SRE binding protein-1; PBS, phosphate-buffered saline; C2-ceramide, N-acetylsphingosine.
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REFERENCES |
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