Mechanism of inhibitory effect of atorvastatin on resistin expression induced by tumor necrosis factor-α in macrophages
© Shyu et al; licensee BioMed Central Ltd. 2009
Received: 21 December 2008
Accepted: 27 May 2009
Published: 27 May 2009
Atorvastatin has been shown to reduce resistin expression in macrophages after pro-inflammatory stimulation. However, the mechanism of reducing resistin expression by atorvastatin is not known. Therefore, we sought to investigate the molecular mechanisms of atorvastatin for reducing resistin expression after proinflammatory cytokine, tumor necrosis factor-α (TNF-α) stimulation in cultured macrophages. Cultured macrophages were obtained from human peripheral blood mononuclear cells. TNF-α stimulation increased resistin protein and mRNA expression and atorvastatin inhibited the induction of resistin by TNF-α. Addition of mevalonate induced resistin protein expression similar to TNF-α stimulation. However, atorvastatin did not have effect on resistin protein expression induced by mevalonate. SP600125 and JNK small interfering RNA (siRNA) completely attenuated the resistin protein expression induced by TNF-α and mevalonate. TNF-α induced phosphorylation of Rac, while atorvastatin and Rac-1 inhibitor inhibited the phosphorylation of Rac induced by TNF-α. The gel shift and promoter activity assay showed that TNF-α increased AP-1-binding activity and resistin promoter activity, while SP600125 and atorvastatin inhibited the AP-1-binding activity and resistin promoter activity induced by TNF-α. Recombinant resistin and TNF-α significantly reduced glucose uptake in cultured macrophages, while atorvastatin reversed the reduced glucose uptake by TNF-α. In conclusion, JNK and Rac pathway mediates the inhibitory effect of atorvastatin on resistin expression induced by TNF-α.
Resistin is an adipocyte-secreted molecule induced during adipocyte differentiation. Recombinant resistin up-regulates cytokines and adhesion molecule expression on human endothelial cells [1, 2], suggesting a potential role in atherosclerosis. Resistin has been shown to have potent proinflammatory properties . Resistin promotes endothelial cell activation and causes endothelial dysfunction of porcine coronary arteries . Recently, resistin was found to have a potential role in atherosclerosis because resistin increases MCP-1 and sVCAM-1 expression in vascular endothelial cells and resistin promotes vascular smooth muscle cell proliferation [5, 6]. More recently, resistin was found to be secreted from macrophages in atheromas and promotes atherosclerosis . Plasma resistin levels are correlated with markers of inflammation and are predictive of coronary atherosclerosis in humans, independent of plasma C – reactive protein. Resistin may represent a novel link between metabolic signals, inflammation, and atherosclerosis .
The 3-hydroxy 3-methyl glutaryl-CoA reductase (HMG-CoA reductase) inhibitors or statins have been proved to reduce inflammation and exert beneficial effects in the prevention of atherosclerosis progression . The pleiotropic effect of statins, independent of their lipid-lowering effects have been described to improve endothelial function, stabilize atheroslerotic plaque, inhibit vascular smooth muscle cell proliferation as well as platelet aggregation, and reduce vascular inflammation . Ichida et al reported that atorvastatin decreases resistin expression in adipocytes and monocytes/macrophages . Atorvastatin decreased resistin mRNA expression in a dose- and time-dependent manner. However, the mechanism of reducing resistin expression by atorvastatin is not known. Therefore, we sought to investigate the molecular mechanisms of atorvastatin for reducing resistin expression after proinflammatory cytokine, TNF-α stimulation in macrophages.
Materials and methods
Atorvastatin, a calcium salt of a pentasubstituted pryole, was supplied by Pfizer. A 10-mmole/l stock solution was made in 100% DMSO. Recombinant TNF-α protein and mevalonate were purchased from Sigma; Polyclonal Rac, and polyclonal phospho-Rac1 (Ser71) antibodies from Cell Signaling; Resistin antibody from R&D Systems; Rac 1 inhibitor, PD 98059, SB 203580, and anisomycin from CALBIOCHEM; Resistin siRNA from Invitrogen.
Human peripheral mononuclear cells (PBMCs) were isolated from heparinized whole blood obtained from normal healthy volunteers by Ficoll-Hypaque gradient centrifugation. The cells were washed three times with sterile PBS and resuspended in RPMI 1640 supplemented with 10% fetal calf serum, 2 mmol/l L-glutamate and 1% penicillin/streptomycin. Monocytes were purified from PBMCs by positive selection using anti-CD14 magnetic beads according to the manufacturer's instructions. The cells were cultured for 4 days in RPMI 1640 supplemented with 10% fetal calf serum, 2 mmol/l L-glutamate and 1% penicillin/streptomycin. For experimental use, purified monocytes/macrophages were changed to serum-free RPMI-1640 supplemented with 2 mmol/l L-glutamate and 1% penicillin/streptomycin for 6 h, then treated with either 1 or 10 μmol/l of atorvastatin for 24 and 48 h.
Western blot analysis
Cells were homogenized in modified RIPA buffer. Equal amounts of protein (15 μg) were loaded into a 12.5% SDS-polyacrylamide minigel, followed by electrophoresis. Protein samples were mixed with sample buffer, boiled for 10 min, separated by SDS-PAGE under denaturing conditions, and electroblotted to nitrocellulose membranes. The blots were incubated overnight in Tris-buffered saline (TBS) containing 5% milk to block nonspecific binding of the antibody. Proteins of interest were revealed with specific antibodies as indicated (1:1000 dilution) for 1 hour at room temperature followed by incubation with a 1:5000 dilution of horseradish peroxidase-conjugated polyclonal anti-rabbit antibody for 1 h at room temperature. Signals were visualized by chemiluminenescent detection. Equal protein loading of the samples was further verified by staining monoclonal antibody GAPDH. All Western blots were quantified using densitometry.
RNA isolation and reverse transcription
Total RNA was isolated from cultured macrophages using the single-step acid guanidinium thiocyanate/phenol/chloroform extraction method. Total RNA (1μg) was incubated with 200U of Moloney-Murine Leukemia Virus reverse transcriptase in a buffer containing a final concentration of 50 mmol/L TrisCl (pH 8.3), 75 mmol/L KCl, 3 mmol/MgCl2, 20 U of RNase inhibitor, 1 μmol/L polydT oligomer, and 0.5 mmol/L of each dNTP in a final volume of 20 μL. The reaction mixture was incubated at 42°C for 1 h and then at 94°C for 5 min to inactivate the enzyme. A total of 80 μL of diethyl pyrocarbonate treated water was added to the reaction mixture before storage at -70°C.
A Lightcycler (Roche Diagnostics, Mannheim, Germany) was used for real-time PCR. cDNA was diluted with nuclease-free water. 2 μL of the solution was used for the Lightcycler SYBR-Green mastermix (Roche Diagnostics): 0.5 μmol/L primer, 5 mmol/L magnesium chloride, and 2 μL Master SYBR-Green in nuclease-free water in a final volume of 20 μL. The primer used for mouse resistin was: forward, 5'-d(GTACCCACGGGATGAAGAACC)-3'; reverse, 5'-d(GCAGACCCACAGGAGCAG)-3'. The primer used for human resistin was: forward, 5'-d(TAAGCAGCATTGGCCTGG)-3'; reverse, 5'-d(CTGTGGCTCGTGGGATGT)-3'. GAPDH: forward, 5'-d(CATCACCATCTTCCAGGAGC)-3'; reverse, 5'-d(GGATGATGTTCTGGGCTGCC)-3'. The initial denaturation phase was 10 min at 95°C followed by an amplification phase as detailed below: denaturation at 95°C for 10 sec; annealing at 55°C for 5 sec; elongation at 72°C for 15 sec and for 30 cycles. Amplification, fluorescence detection, and post-processing calculation were performed using the Lightcycler apparatus. Individual PCR products were analyzed for DNA sequence to confirm the purity of the product.
Electrophoretic mobility shift assay (EMSA)
Nuclear protein concentrations from macrophages were determined by Biorad protein assay. Consensus and control oligonucleotides (Santa Cruz Biotechnology Inc.) were labeled by polynucleotides kinase incorporation of [γ32P]-ATP. Oligonucleotides sequences included the activating protein 1 (AP-1) consensus 5'-CGCTTGATGACTCAGCCGGAA-3'. The AP-1 mutant oligonucleotides sequences were 5'-CGCTTGATGACTTGGCCGGAA-3'. After the oligonucleotide was radiolabeled, the nuclear extracts (4 μg of protein in 2 μl of nuclear extract) were mixed with 20 pmol of the appropriate [γ32P]-ATP-labeled consensus or mutant oligonucleotide in a total volume of 20 μl for 30 min at room temperature. The samples were then resolved on a 4% polyacrylamide gel. Gels were dried and imaged by autoradiography. Controls were performed in each case with mutant oligonucleotides or cold oligonucleotides to compete with labeled sequences.
Promoter activity assay
A-741 to +22 bp rat resistin promoter construct was generated as follows. Rat genomic DNA was amplified with forward primer (ACGCGTCTCAGCGGTAGAGCTCTTG) and reverse primer (AGATCTGGAGAAATGAAAGGTTCTTCATC). The amplified product was digested with MluI and BglII restriction enzymes and ligated into pGL3-basic luciferase plasmid vector (Promega Corp. Madison, Wisconsin, USA) digested with the same enzymes. The resistin promoter contains AP-1 conserved sites (CT) at -51 to -52 bp. For the mutant, the AP-1 binding sites were mutated using the mutagenesis kit (Stratagene, La Jolla, California, USA). Site-specific mutations were confirmed by DNA sequencing. Plasmids were transfected into macrophages using a low pressure-accelerated gene gun (Bioware Technologies, Taipei, Taiwan) essentially following the protocol from the manufacturer. Test plasmid at 2 μg and control plasmid (pGL4-Renilla luciferases) 0.02 μg were cotransfected with gene gun in each well, and then replaced by normal culture medium. Following 4 hours of TNF-α stimulation, cell extracts were prepared using Dual-Luciferase Reporter Assay System (Promega) and measured for dual luciferase activity by luminometer (Turner Designs).
Measurement of resistin concentration
Conditioned media from cultured macrophages under TNF-α stimulation and those from control cells were collected for resistin measurement. The level of resistin was measured by a quantitative sandwich enzyme immunoassay technique (R&D Systems). The lower limit of detection of resistin was 5 pg/mL. Both the intra-observer and inter-observer coefficient of variance were < 10%.
Glucose uptake in macrophages
Macrophages were seeded on ViewPlate for 60 min (Packard Instrument Co., Meriden, Connecticut, USA) at a cell density of 5 × 103 cells/well in serum free medium with transferring 5 μg/mL, insulin 5. μg/mL for overnight. Recombinant TNF-α protein or conditioned medium were added to the medium. Glucose uptake was performed by adding 0.1 mmol/L 2-deoxy-D-glucose and 3,33 nCi/mL 2- [1,2-3H]-deoxy-D-glucose for various periods of time. Cells were washed with PBS twice. Non-specific uptake was performed in the presence of 10 μM cytochalasin B and subtracted from all of the measured value. MicroScint-20 50 μl was added and the plate was read with TopCount (Packard Instrument Co.). The radioactivity was counted and normalized to protein amount measured with a protein assay kit.
The data were expressed as mean+S.E.M. Statistical significance was performed with analysis of variance followed by Dunnett's test for experiments consisting of more than two groups and with Student's t test for stretch at 10% and 20%. A value of P < 0.05 was considered to denote statistical significance.
TNF-α increases resistin expression in human macrophages
Atorvastatin inhibited the effect of TNF-α on resistin expression
Rac pathway mediates the inhibitory effect of atorvastatin on resistin expression induced by TNF-α
TNF-α increases AP-1-binding activity and resistin promoter activity
The EMSA assay showed that TNF-α increased AP-1 DNA-protein binding activity (Fig. 7). An excess of unlabeled AP1 oligonucleotide competed with the probe for binding AP1 protein, whereas an oligonucleotide containing a 2-bp substitution in the AP1 binding site did not compete for binding. Addition of SP600125 and atorvastatin 30 min before TNF-α stimulation abolished the DNA-protein binding activity induced by TNF-α. DNA-binding complexes induced by TNF-α could be supershifted by a monoclonal AP-1 antibody, indicating the presence of this protein in these complexes.
TNF-α stimulates secretion of resistin from macrophages and reduces glucose uptake
As shown in Fig. 8A, TNF-α significantly increased the resistin secretion from cultured macrophages from 4 to 24 h. The mean concentration of resistin rose from 98 ± 13 pg/mL before TNF-α stimulation to 542 ± 64 pg/mL after TNF-α stimulation for 18 h (P < 0.01). Pretreatment with atorvastatin or SP600125 significantly attenuated the secretion of resistin induced by TNF-α
Atherosclerosis has been considered an inflammatory disease. Inflammatory mediators such as TNF-α, interleukin-1 and C-reactiv protein paly an important role in atheogenesis. Resistin could stimulate expression of TNF-α, interleukin-1, 6 and 12 in cultured macrophages [3, 11]. We have previously demonstrated a remarkable induction of resistin protein level even after stimulation with low level of TNF-α in vascular smooth muscle cells . In this study, we further demonstrated that resistin protein and mRNA levels can be induced by TNF-α in cultured human macrophages. Macrophages and vascular smooth muscle cells are important components in the atheroma. These findings indicate that resistin is a promising target for controlling atherosclerotic disease.
Biomarkers that integrate metabolic and inflammatory signals are attractive candidates for defining risk of atherosclerotic cardiovascular disease . Hyperresistinemia impairs glucose tolerance and induces hepatic insulin resistance in rodents , whereas mice deficient in resistin are protected from obesity associated insulin resistance . In this study, we also demonstrated that recombinant resistin protein and TNF-α reduced glucose uptake in human macrophages and atorvastatin reversed the abnormal glucose uptake induced by resistin and TNF-α. Resistin may represent a novel link between metabolic signals, inflammation, and atherosclerosis .
Norata et al. have reported that plasma resistin levels are increased in the presence of metabolic syndrome and are associated with increased cardiovascular risk . Lubos et al. have also reported that resistin levels are elevated in patients with acute coronary syndrome and might play a role as a diagnostic marker . Recently, resistin was found to induce lipolysis and re-esterification of triacylglycerol stores and increase cholesteryl ester deposition in human macrophages . Therefore, resistin contributes macrophage form cell formation. Statins have been shown to reduce lipid lowering effects as well as pleiotropic properties. Although statin cannot alter resistin levels in patients with type 2 diabetic and in healthy men [20–22], statins have been shown to reduce resistin expression in human monocytes and adipocytes [10, 23]. These data implicate that statins may control inflammatory responses by inhibiting resistin expression.
Indeed, our study demonstrated that TNF-α induced resistin protein and mRNA expression in human macrophages and atorvastatin decreased TNF-α-induced resistin expression in a dose-dependent manner. The induction of resistin protein by TNF-α was largely mediated by JNK kinase pathway because the specific and potent inhibitors of an upstream JNK kinase, SP600125, inhibited the induction of resistin protein. Atorvastatin also inhibited the phosphorylation of rac induced by TNF-α. In this study, we demonstrated that TNF-α stimulation of AP-1-DNA binding activity required at least phosphorylation of the JNK since JNK inhibitor abolished the AP-1 binding activity. Atorvastatin also attenuated the AP-1 binding activity induced by TNF-α. The promoter activity of wild resistin promoter after TNF-α was significantly higher than that of AP-1 mutant resistin promoter. This finding indicates that TNF-α regulates resistin in human macrophages at transcriptional level and that AP-1 binding sites in the resistin promoter is essential for the transcriptional regulation. Taken together, our results indicate that TNF-α may increase the AP-1 transcriptional activity in macrophages. Resistin induced by TNF-α was largely though JNK, rac and resistin promoter pathways and atorvastatin could inhibit resistin expression through inhibition of rac phosphorylation, reduced AP-1 binding activity and resistin promoter activity.
In our study, we demonstrated that inhibition of the TNF-α-induced resistin expression by atorvastatin was independent of HMG-CoA reductase. Downregulation of resistin expression induced by CRP by simvastatin was independent of HMG-CoA reductase . Rac pathway mediates the signal trasndcution by isoprenoid intermediates, such as farnesylpyrophosphate and geranylgeranyl-pyrophosphate. In this study, we did not test the effect of isoprenoid intermediate on inhibition of TNF-α-induced resistin expression by atorvastatin. We demonstrated that TNF-α and mevalonate induce resisitn at the similar level. However, atorvastatin only blocks TNF-α but not mevalonate induced resistin. TNF-α but not mevalonate induces rac phosphorylation in cultured macrophages. JNK specific inhibitor S600125 blocked both TNF-α and mevalonate induced resistin expression. This data suggests that mevalonate plays a proinflammatory role and atorvastatin attenuates resistin expression induced by TNF-α is independent of HMG-CoA reductase but through inhibition of Rac and JNK pathway.
In conclusion, our study confirmed previous findings that TNF-α could induce resistin expression in human macrophages and atorvastatin could inhibit the resistin expression induced by TNF-α. The inhibitory effect of atorvastatin on TNF-α-induced resistin expression was mediated by rac and resistin promoter. Our findings provide another evidence of pleiotropic effect of statin. Statin therapy may become another therapeutic strategy for controlling resistin-associated pathologic cardiovascular disease in humans.
activating protein 1
electrophoretic mobility shift assay
- HMG-CoA reductase:
3-hydroxy 3-methyl glutaryl-CoA reductase
peripheral mononuclear cells
tumor necrosis factor-α.
This study was sponsored in part from Pfizer Limited, Taiwan and Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
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