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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1777.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Interleukin-6 Stimulates LDL Receptor Gene Expression via Activation of Sterol-Responsive and Sp1 Binding Elements

Hedi Gierens; Markus Nauck; Michael Roth; Renana Schinker; Christine Schürmann; Hubert Scharnagl; Gunther Neuhaus; Heinrich Wieland; Winfried März

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

	Abstract

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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

	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 primates^{3 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.

	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 CG 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.

	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).

View larger version (16K): [in this window] [in a new window]   Figure 1. Binding (A), internalization (B), and degradation (C) of 125I-labeled LDL in HepG2 cells stimulated with IL-6. After preincubation with low-serum medium for 24 hours, cells were stimulated with IL-6 at concentrations from 1.56 to 25 ng/mL for 24 hours. Cells were incubated with 125I-labeled LDL for 1 hour at 4°C (binding) and for 4 hours at 37°C (internalization and degradation). The data shown have not been corrected for nonspecific binding, internalization, and degradation (usually <15%). Each data point represents the mean±SEM from triplicates. Statistically significant differences (P<0.05) vs control are illustrated by the solid symbols. 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).

View larger version (43K): [in this window] [in a new window]   Figure 2. Northern blot analysis of total RNA of HepG2 cells stimulated with IL-6. Cells were preincubated with low-serum medium for 24 hours and incubated with IL-6 (25 ng/mL) for up to 9 hours. Northern blot of total RNA (20 µg per lane) was sequentially hybridized with 32P-labeled probes for human LDL-R and GAPDH. One representative autoradiogram of 3 independent experiments is shown. The average of the levels of LDL-R mRNA normalized to the corresponding GAPDH mRNA of the IL-6–treated cells at zero time point was set as 1. Each data point represents the mean±SEM (3 experiments) of the hybridization signals of LDL-R mRNA signals normalized to GAPDH mRNA. *P<0.05 vs control.

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).

View larger version (24K): [in this window] [in a new window]   Figure 3. LDL-R promoter activity in HepG2 cells stimulated with IL-6. After preincubation with low-serum medium for 24 hours, HepG2 cells were transiently transfected with the LDL-R promoter constructs and pRL-SV40 and stimulated with IL-6 in the presence or absence of LDL (100 µg/mL) or with lovastatin (10-6 mol/L) for 24 hours before lysis and determination of luciferase activities. The level of LDL-R promoter activity, relative to transfection, of the control cells was set as 1. The data are represented as mean±SEM of at least 3 independent experiments, each performed in quadruplicate. *P<0.05 vs control.

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.

View larger version (20K): [in this window] [in a new window]   Figure 4. Promoter activity assay of a construct harboring 2 tandem copies of repeat 3 of LDL-R (pLDLRR33) in HepG2 cells stimulated with IL-6. After preincubation with low-serum medium for 24 hours, HepG2 cells were transiently cotransfected with pLDLRR33 and pRL-SV40 for 4 hours. Cells were subsequently stimulated with IL-6 in the presence or absence of sterols (10 µg/mL cholesterol and 1 µg/mL 25-OH cholesterol) for 24 hours or with lovastatin (10-6 mol/L), and luciferase activities were determined. The promoter activity, relative to transfection, of the control cells was set as 1. Data represent mean±SEM of 4 independent experiments, each performed in quadruplicate. *P<0.05 vs control.

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).

View larger version (64K): [in this window] [in a new window]   Figure 5. Electrophoretic mobility shift analysis with repeat 2 (Rep.2) of the LDL-R promoter and nuclear proteins derived from HepG2 cells stimulated with IL-6. Con indicates control. After preincubation with low-serum medium for 24 hours, HepG2 cells were stimulated with IL-6 (25 ng/mL) for 30, 60, 90, 120, and 180 minutes, followed by extraction of nuclear proteins. To investigate the influence of excessive sterols, cells were incubated with sterols (10 µg/mL cholesterol and 1 µg/mL 25-OH cholesterol) 12 hours before stimulation. 32P-labeled oligonucleotides of Rep.2 of the LDL-R promoter were incubated with nuclear extracts, and free and bound probes were electrophoretically separated in a nondenaturing polyacrylamide gel.

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.

View larger version (80K): [in this window] [in a new window]   Figure 6. Electrophoretic mobility shift analysis with repeat 3 (Rep. 3) of the LDL-R promoter and nuclear proteins derived from HepG2 cells stimulated with IL-6. After preincubation with low-serum medium for 24 hours, HepG2 cells were stimulated with IL-6 (25 ng/mL) for 30, 60, 90, and 120 minutes, followed by extraction of nuclear proteins. 32P-labeled oligonucleotides of repeat 3 of the LDL-R promoter were incubated with nuclear extracts, and free and bound probes were electrophoretically separated in a nondenaturing polyacrylamide gel.

	Discussion

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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.

	Acknowledgments

 
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.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Casey LC, Balk RA, Bone RC. Plasma cytokine and endotoxin levels correlate with survival in patients with the sepsis syndrome. Ann Intern Med. 1993;119:771–778[Abstract/Free Full Text]
2. Miyao Y, Yasue H, Ogawa H, Misumi I, Masuda T, Sakamoto T, Morita E. Elevated plasma interleukin-6 levels in patients with acute myocardial infarction. Am Heart J. 1993;126:1299–1304[Medline] [Order article via Infotrieve]
3. Rosenson RS. Myocardial injury: the acute phase response and lipoprotein metabolism. J Am Coll Cardiol. 1993;22:933–940.[Abstract]
4. Sammalkorpi K, Vlatonen V, Kerttuba Y, Nikkila E, Taskinen M. Changes in serum lipoprotein pattern induced by acute infection. Metabolism. 1988;37:859–865.[Medline] [Order article via Infotrieve]
5. Akgun S, Ertel NH, Mosenthal A, Oser W. Postsurgical reduction of serum lipoproteins: interleukin-6 and the acute-phase response. J Lab Clin Med. 1998;131:103–108.[Medline] [Order article via Infotrieve]
6. Hardardottir I, Grunfeld C, Feingold KR. Effects of endotoxin and cytokines on lipid metabolism. Curr Opin Lipidol. 1994;5:207–215.[Medline] [Order article via Infotrieve]
7. Stoudemire JB, Garnick MB. Effects of recombinant human macrophage colony-stimulating factor on plasma cholesterol levels. Blood. 1991;77:750–755.[Abstract/Free Full Text]
8. Schectman G, Kaul S, Mueller RA, Borden EC, Kissebah AH. The effect of interferon on the metabolism of LDLs. Arterioscler Thromb. 1992;12:1053–1062.[Abstract/Free Full Text]
9. Murthy S, Mathur S, Bishop WP, Field EJ. Inhibition of apolipoprotein B secretion by IL-6 is mediated by EGF or an EGF-like molecule in CaCo-2 cells. J Lipid Res. 1997;38:206–216.[Abstract]
10. Brown MS, Goldstein JL. A receptor-mediated pathway for cholesterol homeostasis. Science. 1986;232:34–47.[Free Full Text]
11. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature. 1990;343:425–430.[Medline] [Order article via Infotrieve]
12. Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell. 1997;89:331–340.[Medline] [Order article via Infotrieve]
13. Sudhof TC, Van der Westhuyzen DR, Goldstein JL, Brown MS, Russell DW. Three direct repeats and a TATA-like sequence are required for regulated expression of the human low density lipoprotein receptor gene. J Biol Chem. 1987;262:10773–10779.[Abstract/Free Full Text]
14. Dawson PA, Hofmann SL, van der Westhuyzen DR, Sudhof TC, Brown MS, Goldstein JL. Sterol-dependent repression of low density lipoprotein receptor promoter mediated by 16-base pair sequence adjacent to binding site for transcription factor Sp1. J Biol Chem. 1988;263:3372–3379.[Abstract/Free Full Text]
15. Yokoyama C, Wang X, Briggs MR, Admon A, Wu J, Hua X, Goldstein JL, Brown MS. SREBP-1, a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene. Cell. 1993;75:187–197.[Medline] [Order article via Infotrieve]
16. Briggs MR, Yokoyama C, Wang X, Brown MS, Goldstein JL. Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter, I: identification of the protein and delineation of its target nucleotide sequence. J Biol Chem. 1993;268:14490–14496.[Abstract/Free Full Text]
17. Wang X, Sato R, Brown MS, Hua X, Goldstein JL. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell. 1994;77:53–62.[Medline] [Order article via Infotrieve]
18. Hua X, Sakai J, Ho YK, Goldstein JL, Brown MS. Hairpin orientation of sterol regulatory element-binding protein-2 in cell membranes as determined by protease protection. J Biol Chem. 1995;270:29422–29427.[Abstract/Free Full Text]
19. Stopeck AT, Nicholson AC, Mancini FP, Hajjar DP. Cytokine regulation of low density lipoprotein receptor gene transcription in HepG2 cells. J Biol Chem. 1993;268:17489–17494.[Abstract/Free Full Text]
20. Liu JW, Streiff R, Zhang YL, Vestal RE, Spence MJ, Briggs MR. Novel mechanism of transcriptional activation of hepatic LDL receptor by oncostatin M. J Lipid Res. 1997;38:2035–2048.[Abstract]
21. Kishimoto T, Akira S, Narazaki M, Taga T. Interleukin-6 family of cytokines and gp130. Blood. 1995;86:1243–1254.[Free Full Text]
22. Heinrich PC, Dufhues G, Flohe S, Horn F, Krause E, Krüttgen A, Legres L, Lenz D, Lütticken C, Schooltink H, Stoyan T, Conradt HS, Rose-John S. Interleukin-6, its hepatic receptor and the acute phase response in liver. In: Sies H, Flohhe L, Zimmer G, eds. Molecular Aspects of Inflammation. Berlin, Germany: Springer; 1991:129–145.
23. Goldstein JL, Brown MS. Binding and degradation of low density lipoproteins by cultured human fibroblasts. J Biol Chem. 1974;249:5153–5162.[Abstract/Free Full Text]
24. Goldstein JL, Basu SK, Brown MS. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol.. 1983;98:241–260.[Medline] [Order article via Infotrieve]
25. März W, Baumstark MW, Scharnagl H, Ruzicka V, Buxbaum S, Herwig J, Pohl T, Russ A, Schaaf L, Berg A, et al. Accumulation of ‘small dense’ low density lipoproteins (LDL) in a homozygous patients with familial defective apolipoprotein B-100 results from heterogenous interaction of LDL subfractions with the LDL receptor. J Clin Invest. 1993;92:2922–2933.
26. Schreiber E, Matthias P, Muller MM, Schaffner W. Rapid detection of octamer binding proteins with ‘mini-extracts,’ prepared from a small number of cells. Nucleic Acids Res. 1989;17:6419.[Free Full Text]
27. Mazzone T, Basheeruddin K, Ping L, Schick C. Relation of growth- and sterol-related regulatory pathways for low density lipoprotein receptor gene expression. J Biol Chem. 1990;265:5145–5149.[Abstract/Free Full Text]
28. Sjottem E, Andersen C, Johansen T. Structural and functional analyses of DNA bending induced by Sp1 family transcription factors. J Mol Biol. 1997;267:490–504.[Medline] [Order article via Infotrieve]
29. Testa M, Yeh M, Lee P, Fanelli R, Loperfido F, Berman JW, LeJemtel TH. Circulating levels of cytokines and their endogenous modulators in patients with mild to severe congestive heart failure due to coronary artery disease or hypertension. J Am Coll Cardiol. 1996;28:964–971.[Abstract]
30. Grunfeld C, Feingold KR. Metabolic disturbances and wasting in the acquired immunodeficiency syndrome. N Engl J Med. 1992;327:329–337.[Medline] [Order article via Infotrieve]
31. Moorby CD, Gherardi E, Dovey L, Godliman C, Bowyer DE. Transforming growth factor-beta 1 and interleukin-1 beta stimulate LDL receptor activity in Hep G2 cells. Atherosclerosis. 1992;97:21–28.[Medline] [Order article via Infotrieve]
32. Graham A, Russell LJ. Stimulation of low-density lipoprotein uptake in HepG2 cells by epidermal growth factor via a tyrosine kinase-dependent, but protein kinase C-independent, mechanism. Biochem J. 1994;298:579–584.
33. Streicher R, Kotzka J, Muller-Wieland D, Siemeister G, Munck M, Avci H, Krone W. SREBP-1 mediates activation of the low density lipoprotein receptor promoter by insulin and insulin-like growth factor-I. J Biol Chem. 1996;271:7128–7133.[Abstract/Free Full Text]
34. Sanchez HB, Yieh L, Osborne TF. Cooperation by sterol regulatory element-binding protein and Sp1 in sterol regulation of low density lipoprotein receptor gene. J Biol Chem. 1995;270:1161–1169.[Abstract/Free Full Text]
35. Li C, Kraemer FB, Ahlborn TE, Liu J. Induction of low density lipoprotein receptor (LDLR) transcription by oncostatin M is mediated by the extracellular signal-regulated kinase signaling pathway and the repeat 3 element of the LDLR promoter. J Biol Chem. 1999;274:6747–6753.[Abstract/Free Full Text]

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