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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1901-1909.)
© 1997 American Heart Association, Inc.

Articles

Dysregulation of Monocytic Nuclear Factor-B by Oxidized Low-Density Lipoprotein

Korbinian Brand; Tamara Eisele; Ursula Kreusel; Michael Page; Sharon Page; Monika Haas; Astrid Gerling; Christian Kaltschmidt; Franz-Josef Neumann; Nigel Mackman; Patrick A. Baeuerle; Autar K. Walli; ; Dieter Neumeier

From the Institute of Clinical Chemistry and Pathobiochemistry (K.B., T.E., U.K., M.P., S.P., M.H., D.N.) and the Department of Internal Medicine I (F.J.N.), Klinikum rechts der Isar, Technical University Munich (Germany); the Institute of Clinical Chemistry (A.G., A.K.W.), Klinikum Grosshadern, Ludwig-Maximilians University Munich (Germany); the Institute of Biochemistry (C.K.), Albert-Ludwigs University, Freiburg, Germany; the Departments of Immunology and Vascular Biology (N.M.), the Scripps Research Institute, La Jolla, Calif; and Tularik Inc (P.A.B.), South San Francisco, Calif.

Correspondence to Dr Korbinian Brand, Institute of Clinical Chemistry and Pathobiochemistry, Klinikum rechts der Isar, Technical University Munich, Ismaninger Str 22, D-81675 München, Germany.

	Abstract

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Abstract Nuclear factor-B (NF-B)/Rel transcription factors may be involved in atherosclerosis, as is suggested by the presence of activated NF-B in human atherosclerotic lesions. The aim of the present study was to investigate the effects of oxidized LDL (oxLDL) on the NF-B system in human THP-1 monocytic cells as well as adherent monocytes. Our results demonstrate that short-term incubation of these cells with oxLDL activated p50/p65 containing NF-B dimers and induced the expression of the target gene IL-8. This activation of NF-B was inhibited by the antioxidant and H₂O₂ scavenger pyrrolidine dithiocarbamate and the proteasome inhibitor PSI. The oxLDL-induced NF-B activation was accompanied by an initial depletion of IB- followed by a slight transient increase in the level of this inhibitor protein. In contrast, long-term treatment with oxLDL prevented the lipopolysaccharide-induced depletion of IB-, accompanied by an inhibition of both NF-B activation and the expression of tumor necrosis factor- and interleukin-1ß genes. These observations provide additional evidence that oxLDL is a potent modulator of gene expression and suggest that (dys)regulation of NF-B/Rel is likely to play an important role in atherogenesis.

Key Words: nuclear factor-B • oxidized LDL • reactive oxygen intermediates • IB- • monocytes • macrophages

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The pleiotropic transcription factor nuclear factor-B (NF-B)/Rel is present in the cytosol in an inactive state bound to inhibitory proteins collectively termed IB.^{1 2} The prototypic NF-B dimer consists of the subunits p50 and p65, although other subunits such as c-Rel have been described.^{3 4} Several IB proteins have been identified, including IB- and IB-ß.^{5 6} Activation of NF-B is induced by a variety of agents including inflammatory or lymphoproliferative cytokines, reactive oxygen intermediates, microbial pathogens, and viruses.^{5 7} This activation of NF-B involves phosphorylation of IB and proteolysis of this inhibitor by a large protease complex, the proteasome, followed by nuclear translocation of the dimer.^{4 5} In the nucleus, activated NF-B interacts with regulatory B DNA elements in promoters and enhancers, thereby controlling the inducible gene transcription of a plethora of genes involved in inflammation and proliferation.^{5 7}

It has been suggested that NF-B may play an important role in atherosclerosis by modulating gene expression in cells participating in lesion formation.^{8 9 10} We have recently demonstrated the presence of activated NF-B in monocyte/macrophages, smooth muscle cells, and endothelial cells of human atherosclerotic lesions in vivo.¹¹ A variety of genes are induced in the atherosclerotic lesion and/or proposed to be involved in atherogenesis¹² that are known to be regulated by NF-B in cultured cells, including the genes encoding TNF-^{5 13} ; IL-1,¹⁴ IL-6,⁵ and IL-8^{5 15} ; the granulocyte/macrophage-colony stimulating factors (G-CSF, M-CSF, and GM-CSF)^{5 16 17} ; monocyte chemotactic protein-1 (MCP-1)^{5 18 19} ; tissue factor^{20 21 22 23} ; several adhesion molecules^{5 24 25 26 27} ; and c-myc.²⁸

oxLDL has been identified as a major component of the early as well as advanced atherosclerotic lesion, and numerous studies have demonstrated a variety of effects of this lipoprotein on different cell functions^{9 29 30} including gene expression in several cell types involved in atherogenesis.^{31 32 33 34 35 36 37} Recent studies indicate that NF-B is involved in mediating the effects of oxLDL on gene expression: However, cultured human endothelial cells are the only cell type in which an oxLDL-induced activation of NF-B has been so far described.^{38 39} In contrast, recent reports have found an inhibition of LPS-induced NF-B activation by long-term treatment with oxLDL in macrophages and smooth muscle cells.^{40 41 42}

The aims of the present study were, therefore, to test whether oxLDL can activate NF-B in cells of the monocytic lineage under certain conditions and to investigate the effects of oxLDL on the NF-B system more thoroughly in order to understand the underlying mechanisms that produce the apparently conflicting results in the literature described above. Using human THP-1 monocytic cells and ex vivo isolated adherent monocytes, we demonstrated that short term exposure to oxLDL activated NF-B and associated target gene expression, whereas after long-term incubation with these lipoproteins an inhibition of the NF-B system was observed. Both opposing effects were further characterized using specific antibodies and inhibitors and by investigation of the fate of the NF-B inhibitor protein IB-. A potential role of aberrant NF-B/Rel regulation by oxLDL in the pathology of atherosclerosis is discussed.

	Methods

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Cell Culture and Reagents
THP-1 monocytic cells (DSM) were maintained in suspension at a density of 2x10⁵ to 1x10⁶/mL in RPMI-1640 (Glutamax-1, low endotoxin; GibcoBRL) containing 7% fetal calf serum (Myoclone super plus, low endotoxin; GibcoBRL) and 100 U/mL penicillin/100 µg/mL streptomycin (GibcoBRL). For some experiments, THP-1 cells were differentiated into macrophage-like cells using phorbol myristate acetate (Sigma) at a concentration of 10^-7 mol/L.⁴³ For the experiments, the cells were incubated in 25 cm² tissue culture flasks (3x10⁶ cells/flask) or plated at a density of 5x10⁶ cells per well in six-well culture dishes as described.⁴³ Peripheral blood mononuclear cells were isolated from blood samples of normal donors by the Ficoll-Hypaque method as described.³³ Monocytes were isolated from mononuclear cells by adherence (3-hour adherence, nonadherent cells were subsequently removed by washing 3 times) to achieve a purity of approximately 90% as determined by flow cytometry. The adherent monocytes were cultured for 20 hours in the same medium as THP-1 cells but with 5% fetal calf serum before the experiment was started. LPS (Escherichia coli 0111:B4) was purchased from Sigma. Viability of cells was monitored by trypan blue dye exclusion. PDTC was obtained from Sigma, PSI from Peptide Institute Inc, and tricyclodecan-9-yl xanthate (D609) from Biomol. A monoclonal mouse anti-human monocyte CD14 antibody was obtained from Dako.

Lipoprotein Isolation, Modification, and Characterization
LDL (d=1.019 to 1.063 g/mL) was isolated from human plasma of normolipidemic healthy volunteers by sequential ultracentrifugation as described³³ and stored in PBS containing 2 mmol/L EDTA. Immediately before oxidation, the EDTA was removed from LDL by passing the lipoprotein through a PD 10 column (Pharmacia). LDL was oxidized in Ham's F-10 medium by exposure to 5 µmol/L CuSO₄ at 37°C.^{44 33} Slightly modified LDL was also obtained by long-term storage at 4°C³⁴ or exposure of the LDL to monocytic cells.⁴⁴ Equivalent effects were found with dialyzed and nondialyzed preparations (data not shown). For the presented experiments, the preparations were dialyzed before they were added to the cultured cells. Acetylated LDL was prepared by treatment of LDL with acetic anhydride as described earlier.⁴⁵ The protein concentration was determined by the Lowry method⁴⁵ and the cholesterol content by the cholesterol-assay from Boehringer Mannheim. Thiobarbituric acid reactive substances (TBARS) were measured as described^{33 46} and the initial TBARS value of native LDL was <0.1 nmol malondialdehyde equivalents/mg of protein (7 preparations). Unless otherwise stated, mildly to moderately oxidized forms of oxLDL were used for the experiments which were modified by short time exposure to CuSO₄ (TBARS values ranging from 1.94 to 5.37 nmol/mg). Precautions taken to prevent endotoxin contamination during lipoprotein isolation and oxidation and experimental procedures included the use of pyrogen-free sterile water, reagents, and culture dishware. Endotoxin contamination was screened by the limulus amoebocyte lysate assay (Labortechnik Peter Schultz) and the Kinetic-QCL-test (BioWhittaker) and only lipoprotein preparations with an endotoxin content of <10 pg/mL were used for the experiments. As an additional control, some preparations were passed through a column containing the Affinity Pak Detoxi-Gel, which removes endotoxin.

Exclusion of Toxic Effects of Lipoproteins
A potential toxic effect of LDL and oxLDL on the cells was investigated by trypan blue exclusion, which demonstrated that >98% of the cells were trypan blue negative in the presence of the lipoproteins. In addition, toxicity of the reagents on the cells was monitored by the WST-1 test (Boehringer Mannheim), which confirmed that the lipoproteins were not toxic under the conditions used in our experiments (Table). Furthermore, the adherence of monocytes in the presence of LDL or oxLDL was not affected up to 48 hours as determined by counting the cells that remained attached (data not shown).

View this table: [in this window] [in a new window]   Table 1. Metabolic Activity of Cells Exposed to Lipoproteins

Electrophoretic Mobility Shift Assay
Nuclear extracts from 5x10⁶ cells were prepared and analyzed as described previously.²² Protein concentrations were determined by the Bradford method (BioRad). The prototypic immunoglobulin -chain oligonucleotide was used as a probe (5'-CAGAGGGACTTTCCGAGA-3')⁵ and labeled by annealing of complementary primers followed by primer extension with the Klenow fragment of DNA polymerase I (Boehringer Mannheim) in the presence of [-³²P]dCTP (>3000 Ci/mmol; DuPont) and deoxynucleoside triphosphates (Boehringer Mannheim). Nuclear extracts (5 µg protein) were incubated with radiolabeled DNA probes (10 ng; 10⁵ cpm) for 30 minutes at room temperature in 20 µL of binding buffer (20 mmol/L Tris-HCl, pH 7.9; 50 mmol/L KCl; 1 mmol/L dithiothreitol; 0.5 mmol/L EDTA; 5% glycerol; 1 mg/mL BSA; 0.2% NP-40; 50 ng of poly(dI-dC)/µL). Samples were run in 0.25x TBE buffer (10x 890 mmol/L Tris; 890 mmol/L boric acid; 20 mmol/L EDTA, pH 8.0) on nondenaturing 4% or 6% polyacrylamide gels at 125 V. Nuclear extracts from LPS-stimulated THP-1 cells and Hela cells were used as positive controls. As an additional control, samples were incubated with an excess (10x, 100x) of nonlabeled B oligonucleotide, which completely abolished binding of the radiolabeled oligonucleotide to the nuclear proteins. To control the nuclear protein content, the nuclear extracts were incubated with a blunt end double-stranded Sp-1 oligonucleotide that was labeled with [-³²P]ATP (>5000 Ci/mmol, DuPont) and T4 polynucleotide kinase (Boehringer Mannheim). Gels were dried and analyzed by autoradiography.

Supershift Analysis
The nuclear extracts were incubated with 2 µL of appropriate TransCruz gel supershift antibodies (Santa Cruz Biotechnology) per 20 µL of reaction volume at 4°C or room temperature for 2 hours. The following rabbit polyclonal supershift antibodies were used: anti-p50 (recognizing part of the basic nuclear location sequence and the N-terminal adjacent 11 amino acids of the p105 precursor), anti-p65 (raised against a peptide corresponding to amino acids 3-19 within the N-terminal region) and anti–c-Rel (recognizing amino acids 152-176 within the N-terminal domain). This incubation step was followed by EMSA as described above.

Immunocytochemistry
Immunocytochemistry was performed to examine the labeling for activated NF-B within cells. The antibody used was -p65MAb (¹¹ Boehringer-Mannheim), which recognizes an epitope on the p65 subunit of NF-B that is masked by the bound inhibitor IB. Therefore, this antibody reacts only with the activated IB released form of NF-B. After incubation with lipoproteins, the cells were harvested by centrifugation, washed three times with PBS, and bound to adhesion slides (BioRad). After fixation with reagent A of the Fix and Perm kit (An der Grub) at a dilution of 1:2 in PBS, nonspecific antibody binding was blocked with 0.2% bovine serum albumin (BSA) and the cells incubated for 2 hours at 4°C with the -p65MAb antibody at a dilution of 1:500 in PBS with 0.25% reagent B (Fix and Perm kit) to aid permeabilization. The cells were then incubated with a biotinylated goat anti-mouse secondary antibody (Dianova) followed by ABC (avidin biotin horseradish peroxidase complex) (Zymed) both for 30 minutes at room temperature, before visualization with benzidine dihydrochloride (BDHC) (0.01% wt/vol). The slides were dehydrated and mounted in DePex (Fluka) before photography.

Transfection of THP-1 Cells
A luciferase reporter plasmid (pGL2 Basic, Promega) containing 420 bp of the 5'-upstream region of the IL-8 gene (pGL2 IL-8) was transiently cotransfected with a Renilla luciferase control plasmid (pRLtK, Promega) into THP-1 cells using a DEAE-dextran–based protocol.²² Cells were plated out after transfection to a density of 2x10⁶/3 ml in a 6-well plate and kept at 37°C, with 5% CO₂. After 2 days the cells were incubated for 5 hours with LDL, oxLDL, or without lipoproteins. A plasmid lacking the 5'-upstream region of the IL-8 gene was used as a specificity control (pGL2 Basic, Promega). Subsequent to stimulation the cells were harvested by centrifugation and washed once with PBS. Lysis was performed in 1x Passive Lysis Buffer (Promega) for 15 minutes at room temperature. The luciferase activity in the resulting protein lysates was measured using the Dual Luciferase Reporter Assay system (Promega), recording the firefly luciferase activity produced by the experimental plasmids and the Renilla luciferase activity produced by the constitutively active cotransfected control plasmid. The results are expressed as normalized (against the value for lysis buffer alone) firefly luciferase RLU divided by the normalized RLU values obtained for the Renilla luciferase, giving comparative data that account for any differences in transfection efficiency.

Polyacrylamide Gel Electrophoresis and Western Blot Analysis
Cytosolic extracts were isolated as described earlier.⁴⁷ Electrophoresis was performed with 9% polyacrylamide gels (0.1% SDS) and carried out at 140 mA for 1.5 hours (separating gel) in a cooled system as previously described.⁴³ The proteins were transferred to a nitrocellulose membrane using the wet blotting technique. After transfer, the nitrocellulose membranes were incubated with a polyclonal antibody raised against the carboxy terminal domain of the inhibitor IB- (Santa Cruz Biotechnology), followed by a peroxidase-conjugated polyclonal goat anti-rabbit IgG antibody (Dianova). Antibody binding to IB- was visualized on x-ray film using the enhanced chemiluminescence technique (Amersham). The protein size was confirmed by molecular weight standards.

Northern Blot Analysis
Total RNA was extracted from 5x10⁶ cells by means of the micro RNA isolation kit (Stratagene). Northern blotting was performed essentially as described.²¹ Five micrograms total RNA was electrophoresed through a denaturing 1.2% formaldehyde gel and capillary blotted overnight onto nylon membranes (Boehringer Mannheim). Hybridization was carried out overnight at 42°C with probes that were labeled using the Multiprime DNA labeling system (Amersham). The blot was washed with increasingly stringent concentrations of SSC at 52°C and exposed to autoradiography film (Du Pont). To account for variability in sample loading, the blot was rehybridized with a GAPDH cDNA probe.

Determination of IL-8
Concentrations of IL-8 in cell supernatants were measured by sandwich-type immunoassay (Quantikine, R&D systems).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Exposure to oxLDL Induces Activation and Nuclear Appearance of NF-B
THP-1 monocytic cells were incubated in the presence of 80 µg protein/mL of either LDL or oxLDL and activation of NF-B was measured by EMSA. In our previous dose-response experiments, it had been shown that this concentration of lipoprotein was effective for the modulation of NF-B target gene expression in THP-1 cells and adherent monocytes.^{15 33} Exposure of cells to copper-modified oxLDL for 1 hour increased NF-B activation in THP-1 monocytic cells (Fig 1A) that remained elevated for up to 12 hours (data not shown, see below). In the same extracts, we also examined the binding of nuclear proteins to an oligonucleotide comprising the Sp-1 consensus sequence, which was not affected by the oxLDL treatment. LDL modified by long-term storage at 4°C or cell-induced oxidation also activated NF-B (data not shown). Little or no NF-B activation was observed in cells incubated with LDL (Fig 1A) or acetylated LDL (data not shown). Similar results were obtained with phorbol ester-treated THP-1 macrophage-like cells in which a 3.0±0.9-fold (n=3) induction of NF-B activity by oxLDL was measured.

View larger version (51K): [in this window] [in a new window]   Figure 1. Activation of NF-B by oxLDL in monocytic cells. A, THP-1 cells were incubated for 1 hour in the absence of lipoproteins or with 80 µg/mL of oxLDL or LDL, respectively. Activation of NF-B was determined by EMSA; bracket indicates the position of NF-B bands. Binding of nuclear proteins to an oligonucleotide containing the Sp-1 consensus sequence was also examined in the same nuclear extracts (double arrow). B, Nuclear appearance of activated NF-B in the presence of oxLDL. Activated NF-B was detected by immunocytochemistry using the monoclonal antibody -p65MAb (dilution 1:500) and BDHC staining, giving a blue-black color in the original micrograph and a granular staining pattern. The activated NF-B appears to be present mostly in the nucleus of oxLDL-treated cells. Magnification x240. Co indicates control.

The nuclear appearance of oxLDL-activated NF-B was also examined by immunocytochemistry. For this purpose, we used -p65MAb, a monoclonal antibody that selectively detects activated NF-B.¹¹ In unstimulated cells, little staining was detected, indicating the presence of only low amounts of active p65-containing NF-B dimers (Fig 1B). In the presence of oxLDL (12-hour incubation), we observed an increased nuclear staining of cells, suggesting increased activation and nuclear translocation of NF-B, which corroborates our results from EMSA analysis. A staining pattern similar to that of the control was observed in the presence of LDL (data not shown). No staining was detected in the absence of the primary antibody or when an isotype control was used (data not shown).

NF-B Activation by oxLDL and Associated Target Gene Expression in Adherent Monocytes
Next, we examined whether oxLDL activates NF-B in ex vivo isolated human adherent monocytes. Human peripheral blood monocytes were isolated, adhered overnight, and subsequently incubated with the lipoproteins for 4 hours. As can be seen in Fig 2A, incubation with 80 µg/mL of oxLDL but not LDL activated NF-B in these cells. We have recently shown that oxLDL induces the expression of IL-8 in adherent monocytes.¹⁵ Therefore, in the same set of experiments, the production of the NF-B target gene IL-8⁵ was monitored. In the presence of oxLDL, a significant increase in the synthesis of IL-8 compared with the control or the LDL-treated sample was observed under the same conditions in which NF-B activation was detected (Fig 2, A and B). A similar induction of IL-8 production by oxLDL was observed in THP-1 cells (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 2. oxLDL induces NF-B activation and associated target gene expression in adherent monocytes. Human adherent monocytes were incubated for 4 hours in the absence of lipoproteins or with 80 µg/mL of LDL or oxLDL, respectively. Activated NF-B was measured by EMSA and is indicated by the bracket (A). In the same set of experiments the production of IL-8 in the supernatant was detected by immunoassay (B). Co indicates control.

IL-8 Promoter–Dependent Transcription Is Induced by oxLDL
To investigate whether oxLDL specifically induced IL-8 promoter-dependent transcription in our system, THP-1 cells were transiently transfected with a luciferase reporter construct containing 420 bp of the 5'-upstream region of the IL-8 gene. Incubation with oxLDL but not LDL significantly induced the transcription of this construct (Fig 3). A control plasmid lacking the IL-8 5'-region was not induced by oxLDL.

View larger version (21K): [in this window] [in a new window]   Figure 3. IL-8 promoter-dependent transcription is induced by oxLDL. THP-1 cells were transiently cotransfected with a luciferase reporter gene construct containing 420 bp of the 5'-region of the IL-8 gene (pGL2 IL-8) or lacking this region (pGL2 Basic) and pRLtk Renilla luciferase control plasmid. Transfected cells were incubated with LDL or oxLDL (80 µg/mL) for 5 hours. Data are expressed as firefly luciferase RLU divided by Renilla-RLU.

OxLDL Induces p50/p65 Containing NF-B Dimers
To identify the NF-B subunits in the protein/DNA complexes induced by oxLDL, polyclonal antibodies against the NF-B subunits p50, p65, and c-Rel were used for supershift analysis of nuclear extracts from THP-1 cells (Fig 4A) and adherent monocytes (Fig 4B). Incubation with anti-p50 almost completely abrogated or supershifted the protein/DNA complexes in EMSA induced by oxLDL, indicating that the majority of the dimers contained a p50 subunit. Likewise, a similar effect was observed with anti-p65, whereas no inhibition was observed with anti–c-Rel. In conclusion, oxLDL appears to induce predominantly p50/p65 containing NF-B dimers in human monocytic cells.

View larger version (33K): [in this window] [in a new window]   Figure 4. Composition of oxLDL-induced complexes. Nuclear extracts harvested from (A) oxLDL-treated THP-1 cells (1-hour incubation) and (B) adherent monocytes (4-hour incubation) were analyzed by supershift analysis. For this purpose, nuclear extracts were preincubated with polyclonal antibodies directed against p50, p65, and c-Rel or with p65 and c-Rel together. Brackets indicate position of the NF-B bands. Arrows mark the positions of the bands shifted by preincubation with anti-p65 or anti-p50. No Ab indicates that no antibody was added for preincubation.

Effects of the Antioxidant PDTC and the Proteasome Inhibitor PSI
Various compounds were tested for their ability to modulate oxLDL-induced activation of the NF-B system. To demonstrate an involvement of ROI in oxLDL-induced signaling and NF-B activation, the cells were incubated with the antioxidant and H₂O₂ scavenger PDTC.^{48 49} As seen in Fig 5A, PDTC blocked the activation of NF-B by oxLDL in a dose-dependent manner. Next, we tested the proteasome inhibitor PSI⁴⁷ to investigate whether IB- proteolysis was involved in oxLDL-induced NF-B activation. In cells pretreated with PSI for 1 hour, the oxLDL-induced NF-B activation was strongly inhibited (Fig 5B). In the same experiments, the level of IB- was examined in cytosolic extracts by Western blot analysis. The data show that the oxLDL-induced activation of NF-B at 1 hour was accompanied by a depletion of IB-, which was prevented by PSI in a similar dose range to that in which NF-B activation was inhibited (Fig 5B). Under the experimental conditions used, the toxicity of PDTC and PSI was excluded by the WST-1 assay (data not shown).

View larger version (25K): [in this window] [in a new window]   Figure 5. Effects of the antioxidant PDTC and the proteasome inhibitor PSI. THP-1 cells were incubated in plain medium or with 80 µg/mL of oxLDL in the absence or presence of different concentrations of PDTC for 6 hours (A) or pretreated with increasing doses of PSI for 1 hour and then stimulated with lipoproteins for 1 hour (B). NF-B was examined by EMSA; brackets indicate activated NF-B. Cytosolic IB- was detected by Western blot analysis and is marked by arrows. Note that in most of the experiments the slower-migrating phosphorylated form of IB- could be detected as marked by the upper arrow of the doublet.

In contrast, neither the xanthate D609, an inhibitor of phosphatidylcholine-specific phospholipase C, which blocks TNF-induced NF-B activation,⁵⁰ nor antibodies against the LPS receptor CD 14 had an effect on oxLDL-induced NF-B activation (data not shown).

Time Course of oxLDL-Induced NF-B Activation and IB- Depletion
In several studies, cells were incubated with oxLDL for a longer period of time before gene expression was examined.^{36 37} Therefore, time course experiments were performed in which the cells were cultivated for up to 24 hours with oxLDL, and the activation of NF-B and the level of IB- was monitored over time. Incubation with oxLDL induced the activation of NF-B, which reached a maximal level at 1 hour, remained elevated at 6 hours, and returned to baseline by 24 hours (Fig 6). Under the same conditions, oxLDL caused an initial depletion of IB- by 1 hour followed by a transient slight increase in the level of this inhibitor by 6 hours. In the presence of LDL or in untreated cells, the IB- levels remained constant during the incubation intervals (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 6. Time course of oxLDL-induced NF-B activation and IB- depletion. THP-1 cells were incubated over 24 hours with 80 µg/mL of oxLDL. NF-B activation was determined by EMSA and cytosolic IB- was detected by Western blot analysis. Data were analyzed by densitometry and are combined on a histogram. NF-B activation is depicted as arbitrary units normalized against Sp-1 binding examined in the same extracts. For the IB- data, the level of this inhibitor in the untreated control (0 hours) was defined as the 100% value.

Loss of Inducible NF-B Activation and Target Gene Expression After Long-term Exposure to oxLDL
It has been reported that a longer preincubation with oxLDL inhibits the inducible expression of the NF-B target genes TNF- and IL-1ß.^{36 37} To test whether long-term exposure to lipoproteins can alter the induced NF-B activation in our system, the cells were cultured in the absence or presence of LDL or oxLDL for 24 hours followed by a 1-hour stimulation with 1 µg/mL LPS, a very potent stimulus for NF-B activation in cells of the monocytic lineage. In cells preincubated with LDL, the LPS-induced NF-B activation was not affected (Fig 7A). Preincubation with oxLDL, however, significantly reduced the activation of NF-B by LPS. Little or no NF-B activity was observed in cells exposed to LDL or oxLDL for 24 hours (data not shown). In the same extracts, we also investigated the binding of nuclear proteins to an Sp-1 oligonucleotide, which was not affected by long-term treatment with oxLDL (Fig 7A).

View larger version (42K): [in this window] [in a new window]   Figure 7. Long-term exposure to oxLDL inhibits LPS-induced NF-B activation and target gene expression. A, Inhibition of LPS-induced NF-B activation by oxLDL. THP-1 cells were preincubated in medium alone, 80 µg/mL LDL, or oxLDL for 24 hours followed by a 1-hour stimulation with 1 µg/mL LPS. Activated NF-B, examined by EMSA, is indicated by a bracket. Binding of nuclear proteins to an oligonucleotide containing the Sp-1 consensus sequence was also examined in the same nuclear extracts (double arrow). B, Inhibition of LPS-induced TNF- and IL-1ß mRNA expression by oxLDL. The cells were incubated as mentioned in A, with the only exception that total RNA was harvested for Northern blot analysis after a 3-hour stimulation with LPS. To account for variability in sample loading, the blot was rehybridized with a GAPDH cDNA probe.

Next, we examined whether long-term treatment with oxLDL affects the expression of the NF-B target genes TNF- and IL-1ß. The cells were preincubated with either LDL or oxLDL for 24 hour followed by stimulation with LPS for 3 hours, and total RNA was harvested for Northern blot analysis. As expected, stimulation with LPS induced mRNA expression for the two NF-B target genes TNF- and IL-1ß (Fig 7B). Exposure of cells to oxLDL but not LDL significantly inhibited the LPS-induced expression of both genes. These data demonstrate an inhibition of NF-B by long-term exposure to oxLDL associated with a negative regulatory effect on NF-B target gene expression in monocytic cells.

Long-term Exposure to oxLDL Prevents the Efficient Activation-Induced Depletion of IB-
The LPS-induced NF-B activation and the expression of TNF- and IL-1ß was inhibited in cells exposed to oxLDL for 24 hours (see Fig 7B). Therefore, the fate of the inhibitor IB- was examined in cells preincubated with oxLDL for 24 hours and then stimulated with LPS for 1 hour. As expected, incubation with LPS leads to a rapid proteolytic removal of IB- within 1 hour in the absence of lipoproteins (Fig 8). This effect, however, was not evident in cells pretreated with oxLDL. In contrast, the LPS-induced removal of IB- was only slightly affected by the presence of LDL (Fig 8B). Therefore, preincubation with oxLDL appears to prevent an efficient depletion of IB- after stimulation of cells.

View larger version (18K): [in this window] [in a new window]   Figure 8. Long-term exposure to oxLDL prevents the efficient activation-induced depletion of IB-. A, THP-1 cells were preincubated in medium alone or 80 µg/mL oxLDL for 24 hours followed by a 1-hour stimulation with 1 µg/mL LPS. Cytosolic IB- was determined by Western blot analysis and is indicated by the arrows. The slower migrating phosphorylated form of IB- that could be detected in most of the experiments is marked by the upper arrow of the doublet. The positions of the molecular weight (MW) marker proteins are indicated. B, Cells were preincubated in the absence of lipoproteins or with 80 µg/mL LDL or oxLDL for 24 hours and then stimulated with 1 µg/mL LPS for 1 hour. Cytosolic IB- was measured as described in A.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
An increasing body of data indicates that lipoproteins influence a variety of cellular functions, not just lipid metabolism.^{9 12 29} Many of the lipoprotein-induced effects on signal transduction and transcriptional regulation have not yet been characterized. Several studies suggest that oxLDL may play an important role in gene regulation by modulating the NF-B system.

In our study, a short time exposure to mildly oxLDL activated transcription factor NF-B in human THP-1 cells and adherent monocytes, whereas a longer preincubation (24 hours) with these lipoproteins inhibited the LPS-induced NF-B activation. In endothelial cells it has been shown that short-term exposure to minimally or mildly oxLDL activates NF-B.^{38 39} In human and mouse macrophages, a long-term incubation (22 to 24 hours) with mildly and highly oxLDL inhibits this transcription factor.^{40 42} In one of these studies, the appearance of an oxLDL-induced NF-B band was observed that was not characterized further.⁴⁰ In human smooth muscle cells, mildly and highly oxLDL had no effect on the DNA-binding activity of NF-B.⁴¹ However, a 2.5-hour preincubation with extensively oxLDL significantly inhibited the LPS-induced activation of this transcription factor, whereas mildly oxLDL showed only a weak effect.⁴¹ All these data, taken together with our results, indicate that various cell types in culture apparently react differently to oxLDL and/or that the biological effects of mildly oxLDL and highly oxLDL are different. For instance, the longer preincubation period required to inhibit the LPS-induced NF-B activation in our system compared with the relatively short exposure time found to be effective in smooth muscle cells⁴¹ could be due to differences between monocytic and smooth muscle cells but also a result of the fact that we used mildly oxLDL.

The oxLDL-induced activation of NF-B in our study was accompanied by a dramatic increase in the production of the NF-B target gene, IL-8.⁵ Using reporter constructs, we demonstrated that oxLDL significantly induced IL-8 promoter-dependent transcription. These data suggest that the NF-B activated by oxLDL induces transcription of the IL-8 gene, leading to the upregulation of IL-8 expression in monocytic cells. In addition, it has been demonstrated that oxLDL stimulates or enhances the expression of the NF-B–regulated genes IL-1ß³¹ and tissue factor³³ in cells of monocytic origin.

ROI such as H₂O₂ have recently been implicated as one of the intracellular second-messenger molecules that induce NF-B activation.^{5 7 48 51} Cell treatment with H₂O₂ has been demonstrated to activate NF-B and induce B-dependent gene expression.⁴⁹ Cell lines stably overexpressing the H₂O₂ degrading enzyme catalase are deficient in activation of NF-B in response to TNF and okadaic acid.⁵² In contrast, stable overexpression of cytoplasmic superoxide dismutase, which enhances the production of H₂O₂ from superoxide, potentiated NF-B activation.⁵² In our study, the activation of NF-B by oxLDL could be inhibited by the antioxidant and H₂O₂ scavenger PDTC in a dose-dependent manner. Therefore, it is interesting to hypothesize that oxLDL-induced signaling leads to the production of ROI followed by the activation of NF-B.

The oxLDL-induced activation of NF-B was accompanied by a depletion of IB-, both of which were inhibited by the proteasome inhibitor PSI. This suggests that exposure to oxLDL induces IB- proteolysis leading to NF-B activation. The initial depletion of IB- was followed by a slight and transient increase in the level of this inhibitor in our experiments. It should be mentioned that a number of functional NF-B regulatory sequences have been found in the IB- promoter and that the gene encoding this inhibitor is itself regulated by NF-B.^{5 6} Therefore, the increase in the level of IB- in cells exposed to oxLDL is likely to be the result of NF-B–mediated expression of the IB- gene. Long-term exposure to oxLDL for 24 hours inhibited the LPS-induced NF-B activation and target gene expression in our study. Under these conditions, our data demonstrated that the LPS-induced depletion of IB- was not evident in cells preexposed to oxLDL for 24 hours, whereas in the absence of modified lipoproteins, the expected proteolytic degradation of IB- after activation was observed.^{5 7} This suggests that long-term incubation with oxLDL inhibits pathways for NF-B activation at the level of IB- degradation (eg, inhibition of the proteasome) and/or mechanisms located upstream of this protease step.⁵³

Recently, we have shown the presence of activated NF-B in some but not all macrophages present in the atherosclerotic lesion.¹¹ How is this possible in view of the presence of oxLDL in the lesion, since we have shown in the present study that the long-term exposure of macrophages to oxLDL actually inhibits the NF-B system? Several potential explanations arise: Despite a significant inhibition of LPS-induced NF-B activity by long-term exposure to oxLDL in our study, a modest activation of this transcription factor above the base line level remained under these conditions (ie, in the presence of oxLDL plus LPS). The inhibition of NF-B activation by long-term incubation with oxLDL may also represent an autoregulatory mechanism⁵³ and allow restimulation after a certain time span. Furthermore, it is not known whether oxLDL is able to inhibit the activation of NF-B by all of the numerous known stimuli for this transcription factor in the atherosclerotic lesion including TNF-, IL-1ß, growth factors, thrombin, and fibronectin,^{8 9 10} which may induce different signaling pathways. Furthermore, macrophages should also be able to phagocytose the oxLDL^{12 29} and therefore remove these lipoproteins from their close environment, thereby preventing a long exposure to oxLDL that presumably would be highly toxic. There are also lipid-poor atherosclerotic regions¹² in which the macrophages would be less exposed to oxLDL. Finally, the various subpopulations of macrophages that exist in sites of chronic inflammatory processes such as atherosclerosis may respond differently to oxLDL.^{9 12 29} In conclusion, as in any environment of chronic inflammation, there may be both activating and inhibiting forces in the atherosclerotic lesion which positively as well as negatively (dys)regulate the NF-B-system. Future experiments should address the question to what extent both activation and inhibition of NF-B affect the process of atherogenesis.

	Selected Abbreviations and Acronyms

 

EMSA = electrophoretic mobility shift assay IL = interleukin LPS = lipopolysaccharide NF-B = nuclear factor-B oxLDL = oxidized low-density lipoprotein PDTC = pyrrolidine dithiocarbamate PSI = proteasome inhibitor N-benzyloxycarbonyl-Ile-Glu-(O-t-Bu)-Ala-Leucinal RLU = relative light units ROI = reactive oxygen intermediates TNF = tumor necrosis factor

	Acknowledgments

 
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Br 1026/3-1, SFB 469, Dr K. Brand) and by NIH grant HL-48872 (Dr N. Mackman). We are very grateful to Dr C.A. Banka for helpful discussion, Dr Bruno Bonetti for providing cDNA probes, and Dr N. Marx and C. Rokitta for valuable contributions.

Received September 30, 1996; accepted May 2, 1997.

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