© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Interleukin-6 Stimulates LDL Receptor Gene Expression via Activation of Sterol-Responsive and Sp1 Binding Elements
From the Department of Clinical Chemistry (H.G., M.N., R.S., C.S., H.S., H.W., W.M.), University Hospital Freiburg, Freiburg, Germany; the Department of Research (M.R.), University Hospital Basel, Basel, Switzerland; and the Faculty of Biology (H.G., G.N.), Institute for Biology II/Cell Biology, Albert Ludwigs University Freiburg, Freiburg, Germany.
Correspondence to Dr Markus Nauck, University Hospital Freiburg, Department of Clinical Chemistry, Hugstetter Strasse 55, 79106 Freiburg, Germany. E-mail msnauck{at}med1.ukl.uni-freiburg.de
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Abstract—Inflammatory or malignant diseases are associated with elevated levels of cytokines and abnormal low density lipoprotein (LDL) cholesterol metabolism. In the acute-phase response to myocardial injury or other trauma or surgery, total and LDL cholesterol levels are markedly decreased. We investigated the effects of the proinflammatory cytokine interleukin (IL)-6 on LDL receptor (LDL-R) function and gene expression in HepG2 cells. IL-6 dose-dependently increased the binding, internalization, and degradation of 125I-LDL. IL-6–stimulated HepG2 cells revealed increased steady-state levels of LDL-R mRNA. In HepG2 cells transiently transfected with reporter gene constructs harboring the sequence of the LDL-R promoter extending from nucleotide -1563 (or from nucleotide -234) through -58 relative to the translation start site, IL-6 dose-dependently increased promoter activity. In the presence of LDL, a similar relative stimulatory effect of IL-6 was observed. Studies using a reporter plasmid with a functionally disrupted sterol-responsive element (SRE)-1 revealed a reduced stimulatory response to IL-6. In gel-shift assays, nuclear extracts of IL-6–treated HepG2 cells showed an induced binding of SRE binding protein (SREBP)-1a and SRE binding protein(SREBP)-2 to the SRE-1 that was independent of the cellular sterol content and an induced binding of Sp1 and Sp3 to repeat 3 of the LDL-R promoter. Our data indicate that IL-6 induces stimulation of the LDL-R gene, resulting in enhanced gene transcription and LDL-R activity. This effect is sterol independent and involves, on the molecular level, activation of nuclear factors binding to SRE-1 and the Sp1 binding site in repeat 2 and repeat 3 of the LDL-R promoter, respectively.
Key Words: transcription factors • cholesterol • lipoproteins • receptors • hypocholesterolemia
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Introduction |
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Elevated levels of interleukin (IL)-6 and other cytokines play a key role in the acute-phase reaction evoked by myocardial infarction, surgery, or infection.1 2 The acute-phase response induced by these disease states produces hypocholesterolemia in humans and nonhuman primates3 4 5 ; this effect on lipoprotein metabolism appears to be mediated by cytokines. Systemic infusion of cytokines lowers serum cholesterol in animals and humans.6 7 Cytokines may affect plasma levels of cholesterol and lipoproteins by modulating the synthesis and secretion of apolipoproteins, lipolytic enzyme activities, or the expression of lipoprotein receptors.8 9
One of the major determinants of plasma cholesterol levels is the activity of the LDL receptor (LDL-R). The LDL-R mediates the cellular uptake and degradation of plasma LDL. It is expressed in almost all organs and cell types. However, more than two thirds of the LDL-Rs in humans are present in the liver.10
Expression of the LDL-R gene is predominantly controlled at the transcriptional level by feedback repression depending on intracellular sterol content.11 12 Transcription of the LDL-R gene is controlled by 3 imperfect direct repeats, designated repeats 1 to 3, within the LDL-R promoter.13 Repeats 1 and 3 represent binding elements for Sp1 or other members of the GC-box transcription factor family and appear to be constitutively positive elements.14 Repeat 2, sterol-responsive element (SRE)-1, is directly responsible for sterol regulation by acting as a conditionally positive element after binding of sterol regulatory element binding proteins (SREBPs).15 16 SREBP-1 and SREBP-2 are 2 members of the family of basic helix-loop-helix leucine zipper (bHLH-ZIP) nuclear proteins, which are highly homologous to each other. Both are synthesized as 125-kDa precursors that are embedded in the membranes of the endoplasmic reticulum and the nuclear envelope. In the absence of sterols, the amino-terminal part of the SREBPs harboring the DNA binding and transcription regulatory domains is proteolytically cleaved from its carboxy-terminal membrane-spanning domain.17 18 The truncated amino-terminal parts of the SREBPs translocate to the nucleus and bind to SRE-1 in target genes, resulting in enhanced gene expression.
A number of proinflammatory cytokines, such as tumor necrosis
factor (TNF)-
, IL-1ß, and oncostatin M (OM), have been shown to
increase LDL-R gene expression. Modulation of LDL-R transcription by
these cytokines appears to occur through different mechanisms.
TNF- and IL-1ß are capable of increasing LDL-R gene transcription
only when cells are deprived of sterol, suggesting that both
cytokines regulate LDL-R gene expression by a sterol-dependent
mechanism.19 In contrast, OM, a cytokine
predominantly produced by activated T cells and
macrophages, stimulates LDL-R gene expression in HepG2 cells
independent of the intracellular cholesterol
level.20
IL-6 is a proinflammatory cytokine released by monocytes, endothelial cells, fibroblasts, and other cells during trauma, injury, and infection. It is a member of a family of cytokines including leukemia-inhibiting factor (LIF), OM, and IL-11. The IL-6 receptor consists of 2 types of subunits, the ligand-binding glycoprotein gp80 and the signal transducer gp130.21 On binding to its receptor, IL-6 induces the synthesis of acute-phase plasma proteins in liver cells.22
In the present study, we examine the action of IL-6 on LDL metabolism and LDL-R gene expression in HepG2 cells. We demonstrate that IL-6 induces LDL-R mRNA and activity in HepG2 cells. Furthermore, our findings indicate that the effect of IL-6 involves the synergistic activation of SRE-1 and Sp1 binding proteins, independent of the cellular sterol content.
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Methods |
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Cell Culture and Reagents
HepG2 cells were grown as a monolayer culture in DMEM supplemented with 10% (vol/vol) FCS (Seromed) and 20 U/mL penicillin/streptomycin (Seromed) in a humidified 5% CO2 atmosphere.
The reporter plasmids pLDLR-CAT1563 and pLDLR-CAT234 were kindly provided by Dr D. Russell, University of Texas, Dallas.
Recombinant human IL-6 was obtained from R&D Systems; antibodies for the supershift experiments SREBP-2(N19), Sp1(PEP2), and Sp3(D-20)-G, from Santa Cruz Biotechnology Inc; anti–SREBP-1(IgG2A4), from Pharmingen; double-stranded poly(dI-dC) · poly(dI-dC) as sodium salt, from Amersham Pharmacia; and human recombinant Sp1 protein, from Promega.
At 80% confluence, cells were transferred into the quiescent state by preincubation for 24 hours in medium supplemented with 0.1% (vol/vol) FCS (low-serum medium). Incubations with IL-6 were performed in low-serum medium.
Binding, Internalization, and Degradation of
125I-Labeled LDL
Human LDL (1.019<density<1.063 kg/L) was isolated by
preparative ultracentrifugation and
iodinated by use of the iodine monochloride
method.23 HepG2 cells were grown in 24-well polystyrene
plates and treated with IL-6 at different concentrations for 24 hours.
Binding, internalization, and degradation of
125I-labeled LDL were measured according to the
procedure described by Goldstein et al24 with slight
modifications.25 Values were corrected for protein
concentrations measured according to the Lowry method with BSA as a
standard (Bio-Rad).
Northern Blot Analysis
HepG2 cells were plated in 25-cm2
polystyrene flasks, and stimuli were added as indicated. Total RNA was
extracted by using the TRIzol reagent (GIBCO-BRL). RNA was
electrophoresed (20 µg per lane) in 0.8% agarose/1.8 mol/L
formaldehyde gels and transferred onto Hybond-N+ nylon membranes
(Amersham Pharmacia) by capillary blotting. The membranes were
hybridized for 1 hour at 60°C with 1 mg salmon sperm DNA (Stratagene)
in Quick-Hyb hybridization buffer (Stratagene) and sequentially probed
with a specific polymerase chain reaction–generated cDNA probe
corresponding to nucleotides 87 to 578 of the human LDL-R
cDNA and with a 0.5-kb cDNA probe for human GAPDH. The probes were
labeled by random priming (Redi prime II, Amersham Pharmacia) with the
use of 30 µCi [-32P]dCTP (Amersham
Pharmacia) and purified by Sephadex G-50 chromatography
(Microspin G-50 columns, Amersham Pharmacia). The membranes were
exposed for autoradiography at -80°C.
Plasmid Construction
The plasmid pLDLR1563 was constructed by subcloning the 1506-bp
fragment extending from nucleotides -1563 through -58
relative to the translation initiation site of the human LDL-R gene
obtained by HindIII digestion of pLDLR-CAT1563 into
HindIII-digested pGL3-Ba basic vector (Promega). The
construct pLDLR234, harboring nucleotides -234 through
-58 relative to the translation start site of the human LDL-R
promoter, was analogously generated from HindIII digestion
of pLDLR-CAT234.13 The plasmid pLDLR234 was also used
as template for generating the repeat-2 mutant pLDLR234/-152G, in
which a C
G mutation at nucleotide -152 in the core of
repeat 2 was introduced via splicing overlap extension, changing the
sequence of repeat 2 from AAAATCACCCCACTGC to
AAAATCACCGCACTG. The reporter gene construct pLDLRR33,
harboring 2 neighboring repeat 3 elements upstream from the native
TATA-like element of the LDL-R promoter, was obtained by polymerase
chain reaction amplification with the plasmid pLDLR234 as template and
the sense primer
5'-CCCAAGCTTAAACTCCTCCCCCTGCAAACTCCTCCCCCTGC- TAG-3',
containing the 2 repeat 3 elements (italic letters) and additional
bases at the 5' end (underlined sequence) to generate a
HindIII restriction site.
Promoter Assay
HepG2 cells were plated in 48-well polystyrene plates. Cells
were transiently transfected with the reporter gene constructs (0.5
µg per well) by lipofection (Tfx50, Promega) for 4 hours. To
normalize for transfection efficacy, a control plasmid harboring the
Renilla luciferase gene driven by the viral SV40 promoter (pRL-SV40,
Promega) was cotransfected. Transfected cells were treated with
low-serum medium containing IL-6 at different concentrations in the
presence or absence of sterols for 24 hours and lysed, and firefly and
Renilla luciferase activities were determined by use of the Dual
Luciferase Assay (Promega) on a luminometer (Lumat LB9501, EG&G
Bertold).
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Analysis
HepG2 cells were seeded in 25-cm2
polystyrene flasks. After stimulation with IL-6 (25 ng/mL), nuclear
extracts were prepared according to the method of Schreiber et
al,26 with slight modifications in buffer compositions
(buffer A contained 10 mmol/L HEPES, 10 mmol/L KCl, 1.5
mmol/L MgCl2, 0.1 mmol/L EDTA, 0.1
mmol/L EGTA, and 1 mmol/L dithiothreitol; buffer C contained
20 mmol/L HEPES, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L
EGTA, and 1.5 mmol/L MgCl2; both were
supplemented with proteinase inhibitors [Complete, Roche
Biochemicals]). To investigate the effect of sterol-suppressive
conditions, cells were preincubated for 12 hours in the presence or
absence of 10 µg/mL cholesterol and 1 µg/mL 25-OH
cholesterol in low-serum medium. Protein concentrations
were typically 3 to 5 µg/µL.
Oligonucleotides, containing repeat 2 or repeat 3 of the human LDL-R promoter and a 5'-TTGG extension (MWG Biotech), were annealed and labeled by random priming as described above. The oligonucleotide sequences were as follows, with the consensus binding sites underlined: repeat 2, 5'-TTGGAAAATCACCC-CACTGCAA-3'; repeat 3, 5'-TTGGCTGCAAACTCCTCCCCCTG-CTAG-3'.
Each binding reaction contained 4 to 8 µg nuclear protein and 0.5 to 1 ng 32P-labeled double-stranded oligonucleotide probe (5x104 to 10x104 cpm) in a final volume of 20 µL. Nuclear proteins were incubated for 30 minutes at room temperature in binding buffer: the buffer for repeat 2 contained 10 mmol/L HEPES, 0.5 mmol/L MgCl2, 80 mmol/L KCl, 1 mmol/L dithiothreitol, 10% glycerol (vol/vol), 0.3 mg/mL BSA, 50 ng/mL poly(dI-dC) · poly(dI-dC),20 and for repeat 3 contained 12.5 mmol/L HEPES, 6 mmol/L MgCl2, 5.5 mmol/L EDTA, 50 mmol/L KCl, 0.5 mmol/L dithiothreitol, 10% glycerol (vol/vol), 0.25 mg/mL BSA, and 50 ng/mL poly(dI-dC) · poly(dI-dC).16 ) The reaction mixtures were electrophoresed at 200 V for 2 hours at 10°C in 4% nondenaturing polyacrylamide gels containing 0.5x TBE (25 mmol/L Tris, 200 mmol/L glycine, and 0.75 mmol/L EDTA, pH 8.0). Gels were then exposed for autoradiography at -80°C. In competition analyses, nuclear extracts were incubated with 40-fold molar excess of unlabeled oligonucleotide probe 15 minutes before the addition of labeled oligonucleotides. For supershift analyses, antibodies were added to the reaction mixture 60 minutes before the addition of labeled oligonucleotides at 4°C.
Statistical Analysis
Experimental data are presented as mean±SEM and were
statistically analyzed by 1-factor ANOVA and least significant
difference a posteriori contrast.
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Results |
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IL-6 increased the binding of 125I-labeled LDL at 4°C to HepG2 cells in a dose-dependent manner (Figure 1A
). Maximum stimulation was
1.4-fold at 25 ng/mL IL-6. The increase in binding was accompanied by
enhanced internalization (Figure 1B) and degradation of
125I-LDL (Figure 1C). At 25 ng/mL IL-6,
maximum stimulation of internalization and degradation was 1.4-fold and
1.3-fold, respectively. For comparison, lovastatin
(10-6 mol/L), a potent
3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase
inhibitor, induced the binding of
125I-labeled LDL 
2-fold in our assay (data not
shown).
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indicates control; •/
, 1.56 ng/mL IL-6;
/
, 6.25 ng/mL IL-6; and 
/
, 25 ng/mL IL-6.
Transcription of the LDL-R gene responds to stimuli initiating cell
cycle traverse, which could be mediated by SRE-1.27
Growing cells create an additional need for cholesterol to
support the synthesis of membranes and express maximum LDL-R
activity,24 so that the IL-6–induced increase in LDL-R
activity could result from a mitogenic effect of IL-6. In
contrast to medium supplemented with 10% FCS, IL-6 did not induce cell
proliferation under conditions used to study LDL-R expression in a
nonradioactive cell proliferation assay (CellTiter96, Promega; see
Figure
I, which can be accessed online at
http://atvb.ahajournals.org).
To assess whether the IL-6–induced increase in LDL-R activity was due
to an increase in the steady-state level of LDL-R mRNA, HepG2 cells
were stimulated with IL-6 (25 ng/mL) for up to 9 hours, and LDL-R mRNA
was measured by Northern blot analysis. IL-6 treatment produced
a rapid increase in LDL-R mRNA in HepG2 cells with a maximum of 5-fold
at 2 hours (Figure 2).
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The determination of the decay of LDL-R mRNA in IL-6–stimulated HepG2
cells by Northern blot analysis with an incubation of HepG2
cells with IL-6 (25 ng/mL) for 2 hours and subsequent incubation with
actinomycin D (5 µg/mL) revealed that blockade of transcription with
actimomycin D reduced the level of LDL-R mRNA over time in cells
incubated in the presence or absence of IL-6. The half-lives of LDL-R
mRNA did not significantly differ in the presence and absence of IL-6
and were 1 hour and 1.5 hours, respectively (Figure II, which can be
accessed online at http://atvb.ahajournals.org). Hence, the induction
of LDL-R mRNA in HepG2 by IL-6 was not due to increased mRNA stability,
and the observed upregulation occurred at the transcriptional
level.
We also examined the role of de novo synthesis of protein in the
IL-6–mediated stimulation of LDL-R mRNA. Consistent with
previous observations,19 treatment of HepG2 cells with
cycloheximide alone increased LDL-R mRNA by 2-fold (Figure III,
which can be accessed online at http://atvb.ahajournals.org).
Cycloheximide pretreatment (5 µg/mL) did not prevent the upregulation
of LDL-R mRNA by IL-6, indicating that the IL-6–dependent induction of
LDL-R gene transcription does not require additional protein
synthesis.
To identify the elements of the LDL-R promoter responsible for the
upregulation of LDL-R mRNA, we generated reporter gene constructs
consisting of parts of the 5'-flanking region of the LDL-R gene and the
firefly luciferase gene. In the construct pLDLR1563, the luciferase
gene was placed under control of a LDL-R promoter fragment extending
from nucleotides -1563 through -58 relative to the start
site of translation. The construct pLDLR234 contained positions -234
through -58 of the LDL-R promoter. This fragment harbors repeats 1
through 3, known to be essential for the regulation of LDL-R gene
transcription.13 The basal activity of the construct
pLDLR234 was significantly higher than the activity of pLDLR1563,
suggesting the presence of a negative regulator between positions -234
and -1563. HepG2 cells were transiently transfected with either of
these constructs and stimulated with increasing concentrations of IL-6
for 24 hours before luciferase activity was determined. IL-6 stimulated
both promoter constructs to a similar extent, reaching a maximum of
2.4-fold at 25 ng/mL IL-6 (Figure 3).
For comparison, incubation of pLDLR1563-transfected HepG2 cells with
lovastatin
(10-6 mol/L) increased
firefly luciferase activity 5-fold. To test whether the stimulatory
effect of IL-6 was suppressed by the addition of sterols, HepG2 cells,
transiently transfected with either reporter gene construct, were
cultured in medium containing 0.1% FCS, supplemented with human LDL
(100 µg/mL). As shown in Figure 3, the addition of LDL
downregulated LDL-R promoter activity to 40% of sterol-depleted
cells, indicating that LDL-R gene expression was subjected to feedback
regulation by sterols in HepG2 cells. IL-6 was still capable of
stimulating the LDL-R promoter activity at a similar magnitude
(2-fold) in cells cultured in the presence of sterols compared with
the appropriate control cells (Figure 3).
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The finding that IL-6 was equally effective in activating pLDLR1563 and
pLDLR234 indicates that the cis-acting elements essential to
mediate the IL-6 signal are located within 177 bp upstream from the
start site of translation. This region contains a TATA-like sequence
and binding sites for the transcription factors SREBP (repeat 2) and
Sp1 (repeats 1 and 3). To separately examine the role of these
regulatory elements in the LDL-R promoter, 2 luciferase reporter
constructs were generated. In the first, pLDLR234/-152G, a point
mutation was introduced in repeat 2 (CG at position -152). This
mutation is known to abolish high-level transcription of the LDL-R in
the absence of sterols, inasmuch as repeat 2, which carries this
mutation, is not able to bind SREBPs.16 The basal
activity of this construct did not significantly differ from the
activity of the wild-type construct pLDLR234. After transient
transfection of the mutant LDL-R construct and stimulation for 24
hours, the stimulatory effect of IL-6 was decreased to 1.5-fold (Figure 3) compared with the constructs harboring the respective
wild-type promoter region (2.4-fold, Figure 3). This indicates
that SRE-1 participates in the IL-6–mediated stimulation but also
suggests that other promoter elements may concomitantly be
involved.
For this reason, we wished to examine the role of the Sp1 binding site
in repeat 3 in the IL-6–induced stimulation of the LDL-R promoter.
Mutations in either repeat 1 or 3, abolishing Sp1 binding, markedly
reduce the transcription of LDL-R in sterol-depleted and in
sterol-suppressed cells, indicating that intact Sp1 binding sites are
required for basal and induced LDL-R promoter
activity.13 14 We produced a reporter gene construct,
pLDLRR33, harboring 2 tandem copies of repeat 3. The basal activity of
this construct was lower than the activities of the constructs
pLDLR1563 and pLDLR234. The activity of this construct was increased to
1.9-fold of control at 25 ng/mL IL-6 (Figure 4). As expected, the activity of pLDLRR33
was not regulated by cholesterol. These data provide
evidence that the effect of IL-6 on the LDL-R promoter is mediated in
part by repeat 3.
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Electrophoretic mobility shift assays were performed to examine whether
IL-6 stimulation leads to an enhanced binding of nuclear proteins to
repeat 2 or 3 in the LDL-R promoter. Incubation of nuclear extracts of
IL-6–treated HepG2 cells with a 32P-labeled
oligonucleotide containing repeat 2 showed that IL-6
increased the formation of a repeat 2/protein complex. By supershift
analysis, these proteins were identified as SREBP-1a and
SREBP-2 bound to repeat 2 (Figure 5, lanes 5 and 6). Complex formation
started within 30 minutes of incubation, was maximal after 60 minutes,
and declined thereafter (Figure 5, lanes 7 through 11). Binding
of nuclear proteins to repeat 2 was also detected when cells were
incubated with an excess of sterols (10 µg/mL chlesterol
and 1 µg/mL 25-OH cholesterol), showing that this
activation of repeat 2 was sterol independent (Figure 5, lanes
12 through 16). Binding was specific, because the formation was
inhibited by competition with 40-fold molar excess of unlabeled repeat
2 oligonucleotide (Figure 5, lane 3).
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Three complexes of nuclear proteins with repeat 3 were detected on IL-6
stimulation. All of them were competed by the addition of 40-fold molar
excess of unlabeled repeat 3 oligonucleotide (Figure 6, lane 3). The slowly migrating complex
comigrated with recombinant human Sp1 bound to repeat 3 (Figure 6, lane 7). Antibody supershift analyses (Figure 6, lanes 4 through 6) identified these proteins as Sp1 (slowly
migrating complex) and Sp3 (fast migrating complex) homodimers and
Sp1/Sp3 heterodimer (intermediate complex). Binding of these 2 members
of the Sp1 family of transcription factors28 to the Sp1
binding sequence in repeat 3 occurred as early as 90 minutes after the
addition of IL-6, declining thereafter (Figure 6, lanes 9
through 13). These data are in line with the promoter assays and show
that IL-6 stimulates the binding of nuclear proteins to the
sterol-responsive element in repeat 2 and to the Sp1 binding site in
repeat 3. Thus, the stimulatory effect of IL-6 on the LDL-R appears to
involve at least 2 members of the Sp1 family of transcription factors
(Sp1 and Sp3) and SREBP-1a and SREBP-2.
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Elevated plasma levels of proinflammatory cytokines, including IL-6, TNF-
, IL-1ß, and interferons, are found in
inflammatory, infectious, and malignant diseases and have been
implicated in the abnormal lipid metabolism in these
disease states.29 30 Our data clearly show that one
mechanism by which IL-6 affects lipid metabolism is the
upregulation of the LDL-R in hepatic cells, which may eventually lead
to a decrease in LDL cholesterol plasma levels. Stimulatory
effects of IL-1ß and TNF- on LDL-R activity in liver cells have
previously been documented.19 31 Our report demonstrates a
similar effect of IL-6, the main mediator of the acute-phase reaction
in the liver. Interestingly, these cytokines appear to regulate
hepatic LDL-R expression via different mechanisms. Whereas TNF- and
IL-1ß differ in that only TNF- requires de novo synthesis of
protein to enhance LDL-R expression, both are not capable of
stimulating receptor expression in sterol-loaded cells.19
In contrast, IL-6 was able to induce LDL-R gene expression to a similar
extent compared with the expression in unstimulated control conditions,
irrespective of the presence of high concentrations of LDL (Figure 3). Thus, IL-6 exerts its effect on the LDL-R gene in a
sterol-independent manner, as has previously been shown for epidermal
growth factor (EGF), OM, and insulin.20 32 33 Using reporter gene constructs, we located the cis-acting elements necessary to mediate the response to IL-6 to a 177-bp fragment of the LDL-R promoter. Although IL-6 was capable of overriding the suppressive effects of LDL on LDL-R promoter activity, we wished to address the possibility that IL-6 regulation is mediated through SRE-1 in repeat 2. We generated a promoter construct, pLDLR234/-152G, in which the binding site for SREBP was completely inactivated.16
As expected, the mutant promoter was not responsive to sterols. The
IL-6–dependent induction of this promoter was reduced by 40% compared
with the wild-type promoter, regardless of whether or not sterols were
present (Figure 3). This suggests that SRE-1 is required for
maximum response to IL-6.
However, the finding that IL-6 was still able to stimulate
pLDLR234/-152G points to the possibility that IL-6 might also act
through Sp1 binding elements in repeats 1 and 3 of the LDL-R promoter.
Dawson et al14 and others20 have shown that a
nonfunctional repeat 3 leads to a loss of basal transcription.
Consequently, we examined the effect of IL-6 on repeat 3 by using a
promoter harboring a tandem copy of repeat 3 and the TATA-like sequence
of the LDL-R promoter (pLDLRR33) as described by others.20
The expression of this construct was inducible with IL-6 (although to a
lesser extent than the wild-type construct) and, as expected, did not
depend on cholesterol levels within the cell (Figure 4). These results indicate that the IL-6–dependent signal is
partly mediated by sterol-independent activation of repeat 3 in the
LDL-R promoter.
It has been shown that the transcription factors SREBP-1 and Sp1
contribute synergistically to the activation of the LDL-R promoter.
Initially, SREBP stimulates the binding of Sp1 to its adjacent
recognition site, and then both proteins activate transcription
more efficiently than either alone.34 Thus, the diminished
promoter activity of our repeat 2 mutant (pLDLR234/-152G) could be due
to the loss of synergistic action of transcription factors binding to
repeats 2 and 3 rather than to a stimulatory effect on repeat 3.
Therefore, we separately investigated the binding of nuclear proteins
to repeats 2 and 3 in gel-shift assays with nuclear extracts of
IL-6–stimulated HepG2 cells. With repeat 2 used as a labeled probe,
IL-6 induced the formation of one DNA/protein complex that reached a
maximum concentration after 60 minutes of incubation (Figure 5).
The increased binding of nuclear protein to repeat 2 was of the same
magnitude even in the presence of sterols. These nuclear proteins were
identified as the members of the SREBP family, SREBP-1a and SREBP-2.
Gel-shift experiments using repeat 3 as a labeled probe revealed the
formation of 3 DNA/protein complexes that were maximally induced 90
minutes after the addition of IL-6 (Figure 6). Through
supershift analyses, these proteins were identified as Sp1 and
the Sp1-related transcription factor Sp3, respectively. Hence,
IL-6–dependent activation of the LDL-R involves the binding of Sp1 and
Sp3 to repeat 3 and, at the same time, stimulates the binding of
SREBP-1a and SREBP-2 to repeat 2. Interestingly, activation of the
SRE-1 in repeat 2, the element predominantly regulated by sterols, by
IL-6 appears to be unaffected by the cellular content of sterols
(Figure 5). This finding is in contrast to the mechanism of
LDL-R gene activation described for OM. Although IL-6 and OM share the
same signal-transducing protein gp130 and both cytokines
stimulate LDL-R in a sterol-independent manner, OM stimulation
exclusively involves repeat 3 as a downstream target of
mitogen-activated protein kinase kinase
(MEK)/extracellular signal–regulated kinase (ERK)
activation.20 35 A further difference between OM and IL-6
is that only IL-6 directly enhances the binding of Sp1 and Sp3 to
repeat 3, whereas OM does not directly upregulate Sp1 binding
activity.20 Thus, our data stand in contrast to the
speculation by Li et al35 suggesting that IL-6 and OM
upregulate LDL-R by a similar mechanism.
How IL-6 enhances the binding of nuclear proteins to repeats 2 and 3 is not clear at present. Because the effect of IL-6 on LDL-R gene expression does not require de novo synthesis of protein, a rather direct action of IL-6 on nuclear factors that bind to specific elements in the promoter is probable. It is conceivable that one or more kinases activated by IL-6 may alter the phosphorylation state of the nuclear proteins binding to the SRE-1 and Sp1 binding sites, thereby modulating their potency to stimulate LDL-R transcription. Alternatively, proteins that are involved in the maturation and degradation of these nuclear factors may be regulated by IL-6–activated kinases.
In summary, the present study has demonstrated that IL-6 activates LDL-R transcription by enhancing the binding of SREBP-1a and SREBP-2 as well as the binding of Sp1 and Sp3 to their cognate DNA sequence in repeat 2 and repeat 3 of the LDL-R promoter. Consequently, the LDL-R activity on the surface of liver cells is enhanced, leading to an increased uptake of LDL from the circulation. These data are consistent with the hypothesis that hypocholesterolemia after myocardial injury, surgery, or infection is partly due to an enhanced catabolism of LDL in the liver.
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Acknowledgments |
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This work has been supported by the "Zentrale Klinische Forschung," University Hospital Freiburg. We thank Sabine von Karger, Ulrike Stein, Sibylle Rall, and Judith Asal for excellent technical assistance.
Received November 27, 1999; accepted April 12, 2000.
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