Bioflavonoid Quercetin Inhibits Interleukin-1-Induced Transcriptional Expression of Monocyte Chemoattractant Protein-1 in Glomerular Cells via Suppression of Nuclear Factor-κB

Mesangial Cells and Isolated Glomeruli

Mesangial cells (SM43) were established from isolated glomeruli of a male Sprague Dawley rat and identified as being of mesangial cell phenotype as described before (19). Cells were maintained in DMEM/Ham's F-12 medium (Life Technologies, Gaithersburg, MD) supplemented with 100 U/ml penicillin G, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, and 10% fetal calf serum (FCS). Media containing 1% FCS were generally used for experiments. Normal glomeruli were isolated on ice from adult male Sprague Dawley rats (250 to 300 g body wt, four rats) by the conventional sieving method (20) and used for experiments.

Stable Transfectants

SM/JUNDN1 cells in which AP-1 is selectively inactivated were established by stable transfection of SM43 mesangial cells with a dominant-negative mutant of c-jun, TAM-67 (21). TAM-67 is a deletion mutant that is lacking amino acids 3 to 122 of c-Jun (22). The protein encoded by this truncated c-jun gene retains the DNA binding and leucine zipper domains but lacks the transactivating domain. Overexpression of TAM-67, therefore, inhibits AP-1-mediated transactivation via blocking formation or binding of functional AP-1 complexes in a dominant-negative manner (22). SM/JUNDN1 cells exhibit depressed activity of AP-1 under both basal and stimulated conditions (21).

SM/IκBαM cells in which NF-κB is selectively inactivated were established as follows. pLIκBαMSN (23), which introduces a superrepressor mutant of IκBα (IκBαM) and a neomycin phosphotransferase gene (neo), was transfected into a helper-free ecotropic packaging line, ΩE (19). Stable transfectants were selected in the presence of neomycin analogue G418 (500 μg/ml). Conditioned media of the transfectants were used as sources of the IκBαM retrovirus. In the presence of 10 μg/ml polybrene, SM43 mesangial cells were exposed to diluted retrovirus, as described before (19). Stable infectants were selected in the presence of G418 (750 μg/ml), and SM/IκBαM cells were established. SM/IκBαM cells exhibit blunted activation of NF-κB when stimulated by proinflammatory cytokines IL-1β and TNF-α (24).

As a control, mock-transfected mesangial cells SM/Neo that express neo alone were created, as described previously (25).

Pharmacologic Manipulations

Confluent mesangial cells cultured in the presence of 1% FCS for 24 to 48 h were treated by recombinant human IL-1β (10 ng/ml; Otsuka Pharmaceutical Co., Tokushima, Japan) for 8 to 24 h. To examine effects of pharmacologic agents, mesangial cells were pretreated with quercetin (1 to 100 μM; Sigma Immunochemicals, St. Louis, MO), c-Jun/AP-1 inhibitor curcumin (10 to 20 μM; Sigma) (25), or NF-κB inhibitors N-acetylcysteine (5 to 10 mM; Sigma) (26) and MG132 (25 to 50 μM; Peptide Institute, Osaka, Japan) (27) for 1.5 h and stimulated by IL-1β for 8 to 24 h.

Isolated glomeruli were suspended in 1% FCS, stimulated by IL-1β (10 ng/ml) for 6 h in the presence or absence of quercetin (50 μM) or MG132 (50 μM), and subjected to Northern blot analysis.

Northern Blot Analysis

Total RNA was extracted by a single-step method (28) and subjected to Northern blot analysis, as described before (14). In brief, RNA samples were electrophoresed on 1.2% agarose gels containing 10% formaldehyde and transferred onto nitrocellulose membranes. As probes, a mouse JE/MCP-1 cDNA (29) and a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (30) were labeled with ³²P-dCTP using the random priming method. The membranes were hybridized with probes at 65°C overnight in a solution containing 4×SSC (600 mM sodium chloride, 60 mM sodium citrate), 5× Denhardt's solution, 10% dextran sulfate, 50 μg/ml herring sperm DNA, and 50 μg/ml poly(A), washed at 50°C, and exposed to Kodak XAR films at -80°C.

Transient Transfection

AP-1 binds to the particular cis element TRE and triggers transcription of target genes. To evaluate the activity of AP-1 in mesangial cells, a transient transfection assay was used (31). In brief, using the calcium phosphate coprecipitation method, mesangial cells cultured in 24-well plates (1.0 to 1.2 × 10⁵/well) were transfected with a reporter plasmid pTRE-LacZ (a gift from Dr. A. Alberts, Imperial Cancer Research Fund, United Kingdom) (32) or a control plasmid pCI-βgal (a gift from Promega, Madison, WI) at 0.3 to 0.5 μg per well. pTRE-LacZ introduces a β-galactosidase gene (lacZ) under the control of TRE. pCI-βgal introduces lacZ under the control of the immediate-early enhancer/promoter of human cytomegalovirus. After incubation for 48 h in 10% FCS with or without quercetin (50 μM), cells were subjected to 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) assay, as described below. To examine the effect of quercetin on the activity of TRE in IL-1-stimulated cells, transfected cells were incubated in the presence of 1% FCS for 48 h, pretreated with or without quercetin for 1.5 h, and stimulated by IL-1β (10 ng/ml) for 24 h. Activity of AP-1 was evaluated by counting X-gal-positive cells in each well (25). That is, the number of X-gal-positive cells transfected with pTRE-LacZ was normalized by the number of positive cells transfected with the control plasmid pCI-βgal. Assays were performed in quadruplicate.

Activity of NF-κB was similarly assessed by the transient transfection assay. As described above, mesangial cells were transfected with pCI-βgal, a κB reporter plasmid pHIVLTRβ-gal, or its control construct pmuHIVLTRβ-gal (33) (gifts from Dr. A. Rattner, The Weizmann Institute of Science, Rehovot, Israel). pHIVLTRβ-gal introduces lacZ under the control of the HIV promoter that contains two κB motifs. The control plasmid pmuHIVLTRβ-gal contains a κB-mutated HIV promoter. NF-κB activity was evaluated by the number of X-gal-positive cells in each group, which was normalized by the number of positive cells transfected with the control plasmid pCI-βgal. Each normalized value of the pHIVLTRβ-gal transfection was then subtracted by the normalized value of the pmuHIVLTRβ-gal transfection, and the resultant value was used as an indicator of NF-κB activity (25). Assays were performed in quadruplicate. The transfection efficiency achieved in these studies was approximately 0.1 to 0.4%.

X-Gal Assay

X-gal assay was performed, as described before (34). In brief, cells were fixed in 0.5% glutaraldehyde, 2 mM MgCl₂, and 1.25 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetra-acetic acid in phosphate-buffered saline at room temperature for 10 min and then incubated at 37°C for 2 to 4 h in a substrate solution containing 1 mg/ml X-gal, 20 mM K₃Fe(CN)₆, 20 mM K₄Fe(CN)₆·3H₂O, 2 mM MgCl₂, 0.01% sodium desoxycholate and 0.02% NP-40 in phosphate-buffered saline.

Electrophoretic Mobility Shift Assay

Confluent mesangial cells cultured in 1% FCS for 24 h were pretreated with or without quercetin for 1.5 h and stimulated by IL-1β for 4 and 24 h. After the treatment, cells were harvested on ice, lysed in lysis buffer (10 mM Hepes, pH 7.9, containing 1.5 mM MgCl₂, 10 mM KCl, 50 μM dithiothreitol, 100 μM phenanthroline, 1 μg/ml pepstatin, 100 μM 1-trans-epoxysuccinyl-leucylamide(4-guanidino)butane, 100 μM 3,4-dichloroisocoumarin, 10 mM NaF, 100 μM sodium orthovanadate, 25 mM β-glycerophosphate, and 0.2% [vol/vol] NP-40) for 10 min on ice, and centrifuged. The pellets were resuspended in extraction buffer (20 mM Hepes, pH 7.9, containing 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM ethylenediaminetetra-acetic acid, 25% [vol/vol] glycerol, and 100 μM dichloroisocoumarin), incubated for 15 min at 4°C, and centrifuged. The supernatants containing nuclear protein were used for electrophoretic mobility shift assay to evaluate NF-κB activity (35).

As probes, double-stranded wild-type NF-κB (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) and mutant NF-κB (5′-AGT TGA GGC GAC TTT CCC AGG C-3′) oligonucleotides were purchased from Promega. These oligonucleotides were radiolabeled with [γ-³²P]ATP by T4 polynucleotide kinase. DNA-binding reactions were performed in binding reaction buffer (10 mM Tris/HCl, pH 7.5, 100 mM NaCl, 1 mM ethylenediaminetetra-acetic acid, 4% [vol/vol] glycerol, 5 mM dithiothreitol, 100 μg/ml nuclease-free bovine serum albumin) containing the nuclear extract (15 μg), ³²P-labeled oligonucleotide probe (0.14 pmol), and poly(dI-dC) (6 μg). After incubation for 15 min at room temperature, samples were electrophoresed on native 5% acrylamide gels. After the electrophoresis, gels were dried and exposed to x-ray film. To confirm the specificity of the reaction, competition assays were performed with 100-fold excess of unlabeled wild-type oligonucleotides that were added to the reaction mixtures 5 min before the addition of the labeled NF-κB probe. A negative control experiment without nuclear extract was also performed in parallel.

Statistical Analyses

Data were expressed as means ± SEM. Statistical analyses were performed using the nonparametric Mann-Whitney U test to compare data in different groups. A P value <0.05 was used to indicate a statistically significant difference.

Inhibition of Cytokine-Triggered MCP-1 Expression by Quercetin

Mesangial cells were pretreated with quercetin (50 μM) for 1.5 h and stimulated by IL-1β (10 ng/ml). Under the basal culture condition (1% FCS), mesangial cells exhibited only slight expression of MCP-1 mRNA (0.8 kb). When stimulated by IL-1β, expression of MCP-1 was markedly induced. Pretreatment with quercetin inhibited both its basal and inducible expression (Figure 1A). The suppressive effect of quercetin was dose-dependent. Modest repression of MCP-1 was observed at concentrations of 5 to 10 μM. Substantial suppression was observed at concentrations higher than 25 μM (Figure 1B).

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Figure 1.

Inhibition of cytokine-triggered expression of monocyte chemoattractant protein-1 (MCP-1) by quercetin in cultured mesangial cells and isolated glomeruli. (A) Rat mesangial cells (SM43) were pretreated with (+) or without (-) quercetin (50 μM) for 1.5 h and stimulated by interleukin-1β (IL-1β; 10 ng/ml) for 8 h. Northern blot analysis was performed on the expression of MCP-1. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a loading control. (B) Mesangial cells were pretreated with serial concentrations of quercetin (1 to 100 μM) and stimulated by IL-1β. Expression of MCP-1 was examined by Northern blot analysis. (C) Isolated normal rat glomeruli were suspended in 1% fetal calf serum (FCS), stimulated by IL-1β (10 ng/ml) for 6 h in the presence (+) or absence (-) of quercetin (50 μM), and subjected to Northern blot analysis.

The effect of quercetin on the expression of MCP-1 was further examined using isolated glomeruli. Isolated glomeruli incubated in 1% FCS exhibited modest MCP-1 expression. Stimulation of glomeruli by IL-1β markedly enhanced the induction of MCP-1 mRNA. Consistent with the result in cultured mesangial cells, quercetin abrogated the IL-1-induced expression of MCP-1 in isolated glomeruli (Figure 1C).

Effect of Quercetin on the Activity of AP-1 and NF-κB

Several signaling molecules including NF-κB and AP-1 have been proposed as transcriptional regulators for MCP-1. To examine the possibility that quercetin inhibits MCP-1 expression via suppression of these transcription factors, reporter assays were performed. Serum-stimulated mesangial cells were transiently transfected with either a κB reporter plasmid or a TRE reporter plasmid, treated with or without quercetin, and activity of NF-κB and AP-1 was evaluated. Under serumstimulated culture conditions, mesangial cells exhibit constitutive activity of NF-κB and AP-1 (21,25). Treatment with quercetin significantly inhibited the activity of both NF-κB (33 ± 6% versus untreated control [100%], mean ± SEM, P < 0.05) and AP-1 (49 ± 4% versus untreated control [100%]) (Figure 2A).

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Figure 2.

Effect of quercetin on the activity of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB). (A) Mesangial cells cultured in 24-well plates were transiently transfected with an NF-κB reporter plasmid pHIVLTRβ-gal or an AP-1 reporter plasmid pTRE-LacZ. After the transfection, cells were incubated in 10% FCS in the presence (+) or absence (-) of quercetin (50 μM) for 48 h and subjected to 5-bromo-4-chloro-3-indolyl β-D-glactopyranoside (X-gal) assay. The activity of AP-1 and NF-κB was evaluated as described in Materials and Methods. Assays were performed in quadruplicate. Data are shown as means ± SEM. ^*P < 0.05. (B) Mesangial cells transfected with reporter plasmids were incubated in the presence of 1% FCS for 48 h, pretreated with or without quercetin for 1.5 h, and stimulated by IL-1β (10 ng/ml) for 24 h. Activity of AP-1 and NF-κB was evaluated by X-gal assay. Assays were performed in quadruplicate. ^*P < 0.05. (C) Mesangial cells were pretreated with (+) or without (-) quercetin for 1.5 h and stimulated by IL-1β for 4 and 24 h. After the treatment, cells were subjected to electrophoretic mobility shift assay. Probes: +, wild-type NF-κB oligonucleotide; M, mutant NF-κB oligonucleotide; +C, wild-type NF-κB oligonucleotide + unlabeled oligonucleotide (100-fold excess). Negative control is a sample without nuclear extract. Nonspecific bindings detected by the mutant probe are indicated on the right by arrows.

We next examined whether IL-1β induces activation of NF-κB and AP-1, and if so, how quercetin modulates the activation of these transcription factors. Mesangial cells transfected with reporter plasmids were cultured in 1% FCS, pretreated with quercetin, and stimulated by IL-1β. X-gal assay showed that IL-1β significantly induced activation of NF-κB (431 ± 41% versus untreated control [100%]) (Figure 2B, left) but did not upregulate the activity of AP-1 (121 ± 11% versus untreated control [100%]) (Figure 2B, right). Treatment with quercetin dramatically diminished the activation of NF-κB in IL-1-stimulated mesangial cells from 431 ± 41% (IL-1β alone) to 85 ± 15% (quercetin + IL-1β), when compared with untreated control (100%) (Figure 2B, left).

The suppressive effect of quercetin on the NF-κB activation was further confirmed by electrophoretic mobility shift assay. Mesangial cells were pretreated with or without quercetin for 1.5 h and stimulated by IL-1β for 4 and 24 h. As shown in Figure 2C, the DNA binding activity of NF-κB induced by IL-1β was significantly inhibited by quercetin. The mutant probe and cold competition demonstrated that the band observed is NF-κB-specific. Four hours after the stimulation, the inhibitory effect of quercetin was partial. However, after 24 h, the DNA binding activity observed in IL-1-stimulated cells was completely abrogated in the cells treated with quercetin.

Roles of NF-κB and AP-1 in the IL-1-Triggered MCP-1 Expression

The roles of NF-κB and AP-1 in the expression of MCP-1 were examined using pharmacologic inhibitors. Mesangial cells were pretreated with an inhibitor of c-Jun/AP-1, curcumin (25), or an inhibitor of NF-κB, MG132 (27), and stimulated by IL-1β. As shown in Figure 3A, induction of MCP-1 by IL-1β was substantially diminished by MG132 (25 to 50 μM). Similar suppression was also observed by another NF-κB inhibitor, N-acetylcysteine (10 mM) (data not shown). In contrast, the induction of MCP-1 was not obviously affected by the treatment with the AP-1 inhibitor curcumin (10 to 20 μM) (Figure 3A), which effectively inhibits expression of the AP-1-dependent genes gelatinase B and stromelysin in mesangial cells (25,36).

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Figure 3.

Effects of pharmacologic inhibition of NF-κB and AP-1 on the cytokine-induced expression of MCP-1. (A) Mesangial cells were pretreated with the c-Jun/AP-1 inhibitor curcumin (10 to 20 μM) or the NF-κB inhibitor MG132 (25 to 50 μM) for 1.5 h and stimulated by IL-1β (10 ng/ml) for 24 h. Expression of MCP-1 was examined by Northern blot analysis. (B) Isolated glomeruli were suspended in 1% FCS, stimulated by IL-1β (10 ng/ml) for 6 h in the presence (+) or absence (-) of MG132 (50 μM), and subjected to Northern blot analysis.

The crucial role of NF-κB in the cytokine induction of MCP-1 was further examined using isolated glomeruli. Normal glomeruli were stimulated by IL-1β for 6 h in the presence or absence of MG132 and subjected to Northern blot analysis. Consistent with the result in mesangial cells, MG132 abolished the induction of MCP-1 in isolated glomeruli (Figure 3B).

The roles of NF-κB and AP-1 were further investigated using mesangial cells that stably express a super-repressor mutant of IκBα and a dominant-interfering from of c-Jun. As reported previously, SM/IκBαM cells exhibit blunted activation of NF-κB when stimulated by IL-1β and TNF-α (24). SM/JUNDN cells show attenuated activity of AP-1 under both basal and stimulated conditions (21). Untransfected SM43 mesangial cells, mock-transfected SM/Neo cells, SM/IκBαM cells, and SM/JUNDN cells were treated with or without IL-1β, and expression of MCP-1 was evaluated by Northern blot analysis. Compared with SM43 and SM/Neo cells, SM/IκBαM cells exhibited blunted expression of MCP-1 in response to IL-1β (Figure 4). In contrast, SM/JUNDN cells showed the same level of MCP-1 mRNA as that observed in control mesangial cells.

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Figure 4.

Effects of genetic inactivation of NF-κB and AP-1 on the cytokine-induced expression of MCP-1. Untransfected SM43 mesangial cells (SM) and transfectants stably expressing a neomycin phosphotransferase gene (neo) alone (SM/Neo), neo and a dominant-negative mutant of c-jun (SM/JUNDN), and neo and a super-repressor mutant of IκBα (SM/IκBαM) were treated with (+) or without (-) IL-1β(10 ng/ml) for 8 h. Northern blot analysis was performed on the expression of MCP-1.