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Original Research |
Expression by Preventing NF-
B Activation
Graduate Center for Nutritional Sciences (Y.Z., L.H.C.), Internal Medicine (S.J.-B., S.B.), University of Kentucky, Lexington, KY
Address reprint requests to: Dr. Linda Chen, Graduate Center for Nutritional Sciences, 204 Funkhouser Building, University of Kentucky, Lexington, KY 40506-0054. Phone: (859) 257-3288. E-mail: lchen{at}uky.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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(TNF-) production in mice and human subjects; however, the mechanisms remain unclear. Nuclear factor-
B (NF-B) is a transcription factor that plays an important role in controlling the expression of pro-inflammatory genes including TNF-. Activation of NF-B has been shown to mediate the maximal expression of TNF- in human monocytes. NF-B is kept in an inactive form in the cytoplasm by IB, the inhibitory subunit of NF-B complex. Phosphorylation and subsequent degradation of IB lead to NF-B activation.
Objectives: The effect of eicosapentaenoic acid (EPA), a major n-3 fatty acid in fish oil, on the lipopolysaccharide (LPS)-induced expression of TNF- and activation of NF-B were investigated. The mechanism underlying EPA modulation of NF-B activation was also studied.
Methods: Human monocytic THP-1 cells were pre-incubated with EPA and stimulated with LPS. The levels of secreted TNF- were determined by ELISA. The DNA binding activity of NF-B was analyzed by EMSA. The degradation and phosphorylation of IB- were examined by Western blot analysis.
Results: TNF- production and expression induced by LPS were significantly decreased in cells pre-incubated with EPA. LPS-induced NF-B activation, translocation of p65 subunit to the nucleus, phosphorylation and degradation of IB- were partially prevented by EPA.
Conclusions: The results suggest that suppression of the TNF- expression by EPA is partly attributed to its inhibitory effect on NF-B activation. EPA appears to prevent NF-B activation by preventing the phosphorylation of IB-.
Key words: eicosapentaenoic acid, fish oil, LPS, TNF-, NF-B, IB-
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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Tumor necrosis factor- (TNF-), a cytokine produced primarily in monocytes and macrophages, is one of the principal mediators of inflammation [4]. It has been implicated in the pathobiology of many human diseases including AIDS, rheumatoid arthritis, cancer cachexia, diabetes, inflammatory bowel disease and multiple sclerosis [5–7]. Suppression of elevated TNF- levels has been proposed as one of the strategies to slow down the progression of these diseases [5]. Lipopolysaccharide (LPS), a surface component of gram-negative bacteria released upon host infection, is a major mediator of tissue injury and shock. LPS is one of the strongest stimuli that induce TNF- production, and the adverse effects of LPS are partly mediated by TNF- [8–10].
The promoter region of TNF- gene contains several potential regulatory elements including B. Binding of the transcription factor, nuclear factor-B (NF-B), to B site has been shown to mediate the maximal LPS-induced TNF- expression in human monocytic cells [11–15]. NF-B, a ubiquitous transcription factor, is kept in an inactive form in the cytoplasm by IB, the inhibitory subunit of NF-B complex [16]. IB is phosphorylated by IB kinase (IKK) in response to external stimuli [17,18]. Phosphorylation and subsequent ubiquitination of IB lead to its degradation. The released NF-B moves to the nucleus, binds to target DNA elements and activates the transcription of target genes [19–21]. Because inappropriate activation of NF-B is associated with a wide range of human diseases including septic shock, inflammatory bowel disease, autoimmune arthritis, asthma, AIDS, glomerulonephritis, lung fibrosis, atherosclerosis and cancer [22,23], NF-B has been considered as an important target for therapeutic intervention.
N-3 fatty acid supplementation has been reported to decrease TNF- production in human and mice, yet the mechanisms remain unclear [24–26]. Previous studies in our laboratory have shown that fish oil supplementation inhibited TNF- production as well as NF-B activation induced by virus infection in a mouse model of AIDS [27,28]. Therefore, EPA may decrease TNF- expression by inhibiting the activation of NF-B. The objective of the present study was to investigate the effect of EPA on LPS-induced NF-B activation in order to elucidate the mechanisms underlying the modulation of TNF- expression by EPA. The effects of EPA on LPS-induced NF-B DNA binding activity, B directed gene expression and phosphorylation of IB- were investigated in human monocytic cell line THP-1. These cells display many characteristics similar to human monocytes and have long been used to study the expression of TNF- [11,14,29].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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B-luciferase and pSV-ß-galactosidase were gifts from Dr. Fajun Yang. Antibody for phosphorylated IB- was purchased from New England Biolab (Beverly, MA). Antibody for p65 was purchased from Rockland (Gilbertsville, PA). Other antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were purchased from Sigma unless they are stated otherwise.
Cell Culture
THP-1 cell line was kindly provided by Dr. John S. Thompson, Department of Internal Medicine, University of Kentucky. Cells were maintained in RMPI 1640 medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS), as suggested by ATCC, in a 5% CO2 humid atmosphere at 37°C. EPA sodium salt was dissolved in RPMI 1640 medium with 5% FBS to make a stock solution. Cells with a density of 1 x 106 were incubated with 60 µM of EPA and stimulated with LPS.
Enzyme-Linked Immunosorbent Assay (ELISA)
TNF- was determined by using a commercial ELISA kit (Biosource International, Camarillo, CA) following the procedure provided. The color developed was measured by a microplate reader (Bio-kinetic Reader EL312, Bio-Tek instruments Inc.). The concentrations of TNF- were read from the standard curve.
Quantification of mRNA
Total RNA was isolated by using a commercial kit (Qiagen, Valencia, CA) following the protocol provided. Levels of TNF- mRNA and ß-actin mRNA were determined by using commercial kits (Endogen, Woburn, MA) following the procedures described by Van Arsdell et al. [30]. First, two target-specific oligonucleotide probes were hybridized to a splice junction present in the mRNA of interest. The 3'-end of the invader probe overlapped the region of the signal probe that hybridized to the target mRNA. The overlapping nucleic acid complex was recognized by an enzyme called Cleavase VII that cleaved the signal probe and released its 5' fragment, which was then captured on a streptavidin-coated plate via a biotinylated capture oligo. The complex formed by the capture oligo and signal probe fragment generated a primer-template substrate for DNA polymerase, which extended the signal probe fragment with fluorescein-dUTP. The fluorescein-dUTP was detected by an anti-fluorescein alkaline phosphatase-conjugated antibody and a chemifluorescent substrate. The number of target mRNA transcript in the sample was determined by comparing the fluorescent signal generated by the sample to a standard curve.
Cytosolic and Nuclear Extracts
Cytosolic and nuclear extracts were prepared as described by Finto et al. [31]. Cells were washed in cold phosphate-buffered saline and resuspended in 500 µL of cold buffer (10 mM HEPES, 2 mM MgCl2, 0.1 mM EDTA, 10 mM KCl, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/mL leupepstatin, 1 µg/mL pepstatin and 1 µg/mL leucine thiol, 0.1% IgePal CA 630, pH 7.9) and left on ice for 30 minutes. The samples were then mixed and centrifuged at 5,000 rpm for 30 minutes. The supernatant (cytosolic extract) was kept at -70°C. The pellet was resuspended in 150 µL of cold saline buffer [20 mM HEPES, 50 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl2, 300 mM NaCl, 25% (w/v) glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/mL leupepstatin, 1 µg/mL pepstatin and 1 µg/mL leucine thiol, 0.2% IgePal CA 630, pH 7.9] and left on ice for 1 hour. After centrifuging at 15,000 rpm for 15 minutes at 4°C, the supernatant (nuclear extract) containing the nuclear proteins was collected and stored at -70°C.
Western Blot Analysis
Protein in the sample was first resolved by SDS-polyacrylamide gel electrophoresis, then transferred electrophoretically to nitrocellulose membrane and subsequently incubated with the primary antibody. For detection, the nitrocellulose filter was incubated with a horseradish peroxidase coupled secondary antibody, followed by an enhanced chemiluminescence substrate reaction using ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ). Equal loading and transferring were determined by staining the membranes with 0.1% Ponceau S. solution.
Electrophoretic Mobility Shift Assay (EMSA)
DNA binding activity of NF-B was characterized by EMSA [28]. Nuclear extracts containing 5 to 10 µg of protein were incubated for 30 minutes at room temperature with 0.2 ng of 32P-labeled oligonucleotide probe, 10 mm Tris pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, 1 mM MgCl2, 1 mM EDTA, 4% glycerol (w/v) and 0.08 mg/ml sonicated salmon sperm DNA. The oligonucleotide probes used in the EMSA contained consensus-binding sequence for NF-B, 5'-AGT TGA GGG GAC TTT CCC AGG C-3'. DNA-protein complex with a total volume of 20 µL was resolved on a nondenaturing 6% (v/v) polyacrylamide gel and run for about one hour at 200 V in 0.25 X TBE (2.5 mM Tris, 2.5 mM H3BO3, 2 mM EDTA, pH 8.5). The gel was then dried and autoradiographed using Kodak X-ray film. For competition assays, unlabeled probes were added in excess (50 times) in the binding buffer. The mutant oligonucleotide, 5'-AGT TGA GGC GAC TTT CCC AGG C-3', has a "G"
"C" substitution that does not form a DNA-NF-B protein complex [16]. Probes were labeled with [
32P]dATP. The labeled probes were purified using Nick spin columns from Amersham Pharmacia Biotech (Piscataway, NJ). Antibodies were added in the reaction buffer for the supershift assay.
Transfection
THP-1 cells were transfected with pNF-B-Luc that contains a firefly luciferase gene as the reporter gene. The efficiency of transfection was monitored by co-transfection of cells with a pSV-ß-galactosidase control vector. Transient transfection was performed using DEAE-Dextran. THP-1 cells were incubated in 1 ml of serum-free medium containing 5 µL of DEAE-Dextran reagent and 4 µg of plasmid DNA at 37°C with 5% CO2. RPMI 1640 medium supplemented with 10% FBS was added after 30 minutes. Cells pretreated with or without EPA were stimulated with LPS for 6 hours. The cell extracts were prepared using the reporter lysis buffer (Promega, Madison, WI) and stored at -70°C before analyzing the enzyme activity. The activities of luciferase and ß-galactosidase were determined by using the assay buffers purchased from Promega (Madison, WI) following the protocols provided.
Statistical Analysis
Data were analyzed using one-way or two-way ANOVA by SAS program. When analysis of variance indicated significant differences, the treatment means were compared in pairs using Fisher’s least significant difference procedure [32]. Statistical probability of p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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Production production was detected in either the control or EPA pre-treated cells. Addition of LPS from 0.05 to 1.00 µg/ml to cell culture increased TNF- production in a concentration-dependent manner. Pretreatment with 60 µM EPA significantly decreased LPS-induced TNF- production at each LPS concentration (Fig. 1). TNF- production was decreased to similar extent by EPA pre-treatment when cells were stimulated with LPS for various time periods (6, 12 and 24 hours) (data not shown).
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Expression, the mRNA levels of TNF- were determined with or without pre-incubation with EPA. In a time course experiment, the mRNA levels of TNF- were increased with time and reached the peak at 90 minutes after LPS stimulation (Fig. 2). The mRNA levels of TNF- in cells pretreated with EPA were significantly lower than those of control cells at both 90 and 240 minutes.
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mRNA mRNA, cells pre-incubated with or without EPA were stimulated with LPS and then treated with actinomycin D, which inhibits RNA polymerase II-dependent transcription. The mRNA levels of TNF- in THP-1 cells were determined at different time points after actinomycin D was added. As shown in Fig. 3, TNF- mRNA decayed at the same rate in the control and EPA-treated cells, demonstrating that EPA did not affect the stability of TNF- mRNA.
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B DNA Binding ActivityB activation, THP-1 cells were pre-incubated with or without EPA, and then treated with LPS. DNA binding activity of NF-B was determined in the nuclear extract by EMSA. Without stimulation, no DNA binding activity of NF-B was detected in control cells or cells pre-incubated with EPA (Fig. 4A). The DNA binding activity of NF-B was induced by LPS, and was partially inhibited by pre-incubating cells with EPA prior to LPS stimulation. The specificity of the NF-B binding was confirmed by the competition assay (Fig. 4B). In addition, to confirm that the band in LPS-stimulated cells was indeed NF-B, the nuclear extracts were incubated with the antibodies of p50 and p65 subunit, respectively, and the supershift assay was performed. Fig. 4C verifies that the LPS-activated NF-B complex contained p65 and p50 subunits.
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B to NucleusB from the cytoplasm to the nucleus, cells were pre-incubated with or without EPA, then treated with LPS for various time periods. The amount of NF-B subunit p65, which contains the transcriptional activation domain, was determined in the nuclear extracts by Western blot analysis. Fig. 5 shows that upon LPS stimulation, p65 subunit of NF-B was increased in the nucleus with time, indicating that p65 subunit was translocated from the cytoplasm to the nucleus. Pre-incubating cells with EPA partially decreased the translocation of p65 subunit to the nucleus. Measurements of band densities indicated that the levels of nuclear p65 in EPA-pretreated cells were decreased by 25% and 32% when compared to control cells at 30 minutes and 1 hour, respectively.
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B-Directed Luciferase Expression Induced by LPSB activity, THP-1 cells were transfected with pNF-B-Luc vector in which the enhancer element is located at the upstream of a firefly luciferase gene. Cells were pre-incubated with or without EPA before stimulation with LPS. Luciferase activity was increased upon LPS stimulation as a result of NF-B activation and the subsequent induction of luciferase gene (Fig. 6). Pre-treating cells with EPA inhibited LPS-induced luciferase activity, indicating a lower NF-B activity.
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B- and Degradation of IB-B- and phosphorylated IB- (p-IB-) were determined in the cytosolic extract by Western blot. The levels of p-IB- were increased and the levels of IB- were decreased with time upon LPS stimulation. With EPA pre-treatment, the levels of LPS-induced p-IB- was significantly suppressed (Fig. 7B) by 40% and by 50% at 15 minutes and 30 minutes, respectively. Measurement of band densities indicated that EPA pretreatment inhibited LPS-induced decrease of IB- by 18% at 30 minutes (Fig. 7A). Therefore, EPA might prevent IB- degradation and NF-B translocation by preventing the phosphorylation of IB-.
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DISCUSSION |
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production in THP-1 cells was prevented by pre-treating cells with EPA. The results are consistent with other reports that dietary n-3 fatty acid supplementation decreases the TNF- production in murine marcophages [24,33] and human peripheral-blood mononuclear cells [26,34]. To find out whether the effect of EPA on LPS-induced TNF- production in THP-1 cells is specific for this fatty acid, different types of long chain fatty acids were examined for their capability to modulate LPS-induced TNF- production in THP-1 cells [35]. Our results showed that docosahexanoic acid (22:5, n-3) decreased TNF- production to the same extent as EPA (by 40%). Stearic acid (20:0) had no effect on TNF- production. Arachidonic acid (20:4, n-6), linoleic acid (18:3, n-6) and oleic acid (18:1) decreased TNF- production slightly (by 15–20%), but to a significantly lesser extent when compared to EPA or DHA [35]. Therefore, the significant down-regulation of TNF- production appears to be specific for n-3 fatty acids.
Our results showed that EPA supplementation decreased the levels of LPS-induced TNF- mRNA as well. The AU-rich sequence at 3'-untranslated region of TNF- mRNA has been suggested to play a role in the destabilization of TNF- mRNA [36,37]. It is possible that EPA treatment promotes this destabilization and decreases the steady-state mRNA levels of TNF- by increasing TNF- mRNA degradation. Our result that EPA did not affect the stability of TNF- mRNA indicates that EPA pretreatment most likely decreased mRNA levels of TNF- by inhibiting its formation rather than increasing its degradation. Therefore, EPA may modulate TNF- expression at the transcriptional level.
NF-B is one of the transcription factors that are critical for LPS-induced TNF- expression [11–15]. We previously observed that NF-B DNA binding activity induced by murine leukemia virus infection was suppressed by supplementing fish oil to mice [28]. In this study we observed that LPS-induced DNA binding activity of NF-B was decreased in THP-1 cells pre-incubated with EPA. Similar effects of EPA were reported in mouse marcophage cell line, RAW 264.7 cells [38]. The result in the present study that LPS-induced expression of B-directed luciferase was decreased by pre-incubating cells with EPA further supports that EPA prevents LPS-induced NF-B activation. Recently, suppressed NF-B activation in mice fed a diet enriched with n-3 fatty acids was reported [39]. These results demonstrated that EPA might prevent the expression of TNF- partly by blocking the activation of NF-B.
NF-B is kept in an inactive form by the inhibitory subunit IB in most cell types [16]. Phosphorylation of IB by IKKs leads to its ubiquitination and subsequent degradation, resulting in the translocation and activation of NF-B [17–21]. Our results demonstrated that THP-1 cells pre-incubated with EPA had significantly lower levels of phosphorylated IB- upon LPS stimulation. However, EPA was only able to slightly prevent the decrease of IB levels. First, the amount of IB- is much more abundant compared to the amount of phosphorylated IB-, therefore, it is more difficult to observe the difference providing that EPA is a weak modulator of NF-B activation. Secondly, the IB- levels shown in Fig. 7A represents the total amount of pre-existed and newly synthesized IB-. Since IB- gene is up-regulated by the activation of NF-B (40,41), a decrease in NF-B activation by EPA may result in a slow regeneration of IB- that diminishes the difference of IB- levels between the control cells and EPA-treated cells.
In summary, the results demonstrated that the prevention of LPS-induced NF-B activation by EPA was associated with the prevention of IB- phosphorylation and degradation. Therefore, EPA may suppress NF-B activation through inhibiting the phosphorylation of IB. To clarify the mechanisms underlying EPA modulation of IB- phosphorlation, it will be necessary to investigate the effects of EPA on IKK activity. Since the transcriptional activity of NF-B is also regulated by the phosphorylation of p65 [42,43], it would be interesting to find out whether EPA directly modulates NF-B activity by altering the phosphorylation of the NF-B subunit.
The molecular pathways affected by EPA, which leads to its inhibition on NF-B activation, remain to be understood. EPA may exert its effect on NF-B activation via blocking upstream signals leading to IKK activation. In fact, it has been reported that CD14 expression stimulated by LPS is down-regulated by EPA [44]. Previous findings in our laboratory demonstrated that supplementing fish oil to mice restored the tissue antioxidant defense systems that were suppressed by infection with murine leukemia virus [45]. Numerous studies have shown that NF-B is a redox-sensitive transcription factor [46,47]. A low concentration of hydrogen peroxide can activate NF-B and various antioxidants can prevent NF-B activation [48–50]. LPS stimulation of cells can cause a 6–10 fold increase in the release of reactive oxygen species (ROS) including superoxide and hydrogen peroxide [51,52]. In a CD14-independent pathway, Rac1-dependent ROS formation is shown to mediate LPS-induced IKK activation and induction of NF-B [51]. Pre-treating cells with antioxidants reduces LPS-induced NF-B transcription activity [51,52] and the activities of upstream Rac1 and protein tyrosine kinases [53,54]. Therefore, it is possible that EPA may prevent NF-B activation and TNF- production via reducing ROS production induced by LPS.
This study focused on the effect of EPA on the activation of NF-B, one of the key transcriptional factors that regulate LPS-induced TNF- expression. Since the expression of TNF- involves multiple transcriptional factors, such as activator protein-1 (AP-1) and cyclic AMP responsive element binding protein (CREB) [11], the possibility that EPA may affect the activities of other transcriptional factors cannot be excluded.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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B sites have been found in the promoters and enhancers of numerous genes including cytokines, acute phase proteins and adhesion molecules [19,20]. Inappropriate regulation of NF-B is linked to a wide range of human diseases including septic shock, atherosclerosis, autoimmune arthritis, asthma, glomerulonephritis, and lung fibrosis [22,23]. The results suggest that EPA prevents the LPS-induced TNF- expression by preventing the activation of NF-B, and that EPA prevents NF-B activation through decreasing phosphorylation of IB-. It has been suggested that diets with increased ratio of n-3 to n-6 PUFA may provide a condition favorable to effective drug treatment, since current Western diet with low ratio may be suboptimal for the effective drug therapies that require inhibition of pro-inflammatory cytokine such as TNF- [34]. Such a community-wide strategy may decrease the emergence of inflammatory diseases and thrombic episodes as well as decrease their severity [34]. Thus, EPA or fish oil may have its special value as a food component or supplement for prevention or dietary intervention of many inflammatory diseases.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSCONCLUSIONSACKNOWLEDGMENTSREFERENCES |
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Received December 23, 2002. Accepted August 12, 2003.
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