SB203580

Quercetin disrupts tyrosine-phosphorylated phosphatidylinositol 3-kinase and myeloid differentiation factor-88 association, and inhibits MAPK/AP-1 and IKK/NF-nB-induced inflammatory mediators production in RAW 264.7 cells

Abstract

Quercetin is a major bioflavonoid widely present in fruits and vegetables. It exhibits anti-inflammatory, anti-tumor, antioxidant properties and reduces cardiovascular disease risks. However, the molecular mechanism of action against inflammation in RAW 264.7 cells is only partially explored. Quercetin effect on LPS-induced gene and protein expressions of inflammatory mediators and cytokines were determined. Moreover, involvement of heme-oxygenase-1, protein kinases, adaptor proteins and transcription factors in molecular mechanism of quercetin action against inflammation were examined. Quercetin inhibited LPS-induced NO, PGE2, iNOS, COX-2, TNF-α, IL-1β, IL-6 and GM-CSF mRNA and protein expressions while it promoted HO-1 induction in a dose- and time-dependent manner. It also suppressed I-nB-phosphorylation, NF-nB translocation, AP-1 and NF-nB-DNA-binding and reporter gene transcription. Quercetin attenuated p38MAPK and JNK1/2 but not ERK1/2 activations and this effect was further confirmed by SB203580 and SP600125-mediated suppressions of HO-1, iNOS, and COX-2 protein expressions. Moreover, quercetin arrested Src, PI3K, PDK1 and Akt activation in a time- and dose-dependent manner, which was comparable to PP2 and LY294002 inhibition of Src, PI3K/Akt and iNOS expressions. Quercetin further arrested Src and Syk tyrosine phosphorylations and their kinase activities followed by inhibition of PI3K tyrosine phosphorylation. Moreover, quercetin disrupted LPS-induced p85 association to TLR4/MyD88 complex and it then limited activation of IRAK1, TRAF6 and TAK1 with a subsequent reduction in p38 and JNK activations, and suppression in IKKα/β-mediated I-nB phosphorylation. Quercetin limits LPS-induced inflammation via inhibition of Src- and Syk-mediated PI3K- (p85) tyrosine phosphorylation and subsequent TLR4/MyD88/PI3K complex formation that limits activation of downstream signaling pathways.

Introduction

Activation of innate immune cells (macrophages, neutrophils and dendritic cells) by pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) is detected via pattern recognition receptors including Toll-like receptor (TLR)-4, stimula- tion of which affect every step of the development of inflammation (Kawai and Akira 2010). This process triggers a series of signaling molecules, kinases, transcription factors and inflammatory gene expressions (Mogensen 2009). TLR4 activation by LPS triggers the association of myeloid differentiation primary-response protein 88 (MyD88), which recruits IL-1 receptor-associated kinase 4 (IRAK4), thereby allowing the association and phosphorylation of IRAK-1 (Akira and Takeda 2004). IRAK1 and TNF receptor-associated factor (TRAF)-6 then form a complex with transforming-growth-factor- β-activated kinase-1 (TAK1), TAK1-binding protein-1 (TAB1), and TAB2 which induces the phosphorylation of TAB2 and TAK1 (Akira and Takeda 2004). The complex then associates with the ubiquitin-conjugating enzyme 13 (UBC13) and ubiquitin- conjugating enzyme E2 variant 1 (UEV1A) ubiquitin ligases (Mann 2011) for the ubiquitylation of TRAF6 and activation of TAK1. TAK1 then activates the inhibitory NF-nB kinase (IKK) complex consisting of IKK-α, IKK-β and IKK-γ (Takeda and Akira 2004). The IKK complex phosphorylates InB, leading to its ubiquitylation and subsequent degradation, which allows nuclear factor kappa-B (NF-nB) translocation and its target genes expression (inflamma- tory mediators and cytokines) (Tak and Firestein 2001). TAK1 also phosphorylates mitogen-activated protein (MAP) kinases with sub- sequent AP-1 activation and gene transcription (Gay and Gangloff 2007).

TLR4-mediated signaling also leads to rapid activation of phosphatidylinositol 3-kinase (PI3K) that functions either as a pos- itive or negative regulator of TLR signaling. PI3K has been shown to positively regulate cytokine expression through the formation of a complex between the p85 regulatory subunit, TLR4 and MyD88 (Ojaniemi et al. 2003), and contribute to TLR4-mediated NF-nB acti- vation and cytokine release (Li et al. 2003). Though quercetin is a known inhibitor of PI3K, the link between the anti-inflammatory effect of quercetin and LPS-induced TLR4 activation with subse- quent PI3K stimulation is not well characterized.

The expressions of inflammatory mediators and cytokines are regulated mainly at the transcriptional level, and the major trans- criptional regulators of these genes are NF-nB and activator protein (AP)-1 (Calandra and Roger 2003). Macrophages including RAW 264.7 cells release high amount of inflammatory mediators such as [inducible nitric oxide synthase (iNOS), COX-2], and proin- flammatory cytokines [including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and granulocyte macrophage colony stim- ulating factor (GM-CSF)] in response to LPS stimulation (Middleton et al. 2000), all of which are important markers of inflamma- tion in searching and developing new potential anti-inflammatory drugs. Therefore, modulation of such signaling pathways involving TLR4 and its downstream adapter molecules, kinases, transcription factors and expression of inflammatory mediators using anti- inflammatory agents of dietary origin such as quercetin may interfere the activation state of the TLR4 mediated signaling and limit the progress and development of chronic inflammation.

Since inflammation is linked to a wide range of progressive diseases, including cancer, neurological disease, metabolic disorder and cardiovascular disease (Libby 2007), studies suggest elimi- nation of inflammation by dietary flavonoids such as quercetin, is a major way to prevent various chronic diseases (Pan et al. 2009). Quercetin is a promising anti-inflammatory agent that warr- ants a comprehensive evaluation of its potential as a putative new class of anti-inflammatory drug. However, its mechanism of action and signaling pathways involved against LPS-induced inflammation is only partially determined in RAW 264.7 cells and further exploration is required. Previous reports indicated that quercetin possesses a potent antioxidant, immunomodulatory, anti-inflammatory and antiatherosclerotic properties (Comalada et al. 2006; Russo et al. 2012). It inhibits IKKα and IKKβ (Peet and Li 1999), suppresses NF-nB, STAT1, iNOS, NO and TNFα activation and stimulates HO-1 induction (Chen et al. 2005) and IL-10 secre- tion (Comalada et al. 2006). It has also been reported to inhibit MAPKs, Akt, Src, JAK-1, Tyk2, STAT1 activation (Kao et al. 2010), down-regulated iNOS and COX-2 expression, and decreased IL-1β, IL-6, and TNF-α expression through the suppression of NF-nB p65 nuclear translocation.

However, the modulatory effects of quercetin to LPS-induced TLR4-activation, adapter protein recruitment and the association of upstream kinases are only partially explored. In this study, therefore, we determined quercetin inhibition on LPS-induced Src and Syk mediated PI3K tyrosine phosphorylations and Akt acti- vation. Interestingly, quercetin disrupted p85 subunit association with the adapter protein MyD88 and TLR4 complex with subse- quent attenuation of downstream signaling pathways. Such effects include quercetin mediated HO-1-induction, JNK and p38 inhibi- tion, NF-nB and AP1 attenuation and inflammatory mediators and pro-inflammatory cytokines suppression.

Materials and methods

Reagents

Quercetin (Fig. 1) with the purity of ≥98%, solid (HPLC, Q4951) was procured from (Sigma–Aldrich, USA). Primary antibodies for iNOS, COX-2, IL-1β, β-actin, PARP, HO-1, phospho- PI3K/p85, PI3K, phospho-Src, non-phospho-Src, Syk, phospho- PDK1, PDK1, phospho-Akt, Akt, NF-nB/p65, phospho-NF-nB/p65, InB-α, phospho-InB-α, phospho-p38, p38MAPK, phospho-JNK, JNK, phospho-p44/p42, p44/42, IRAK1, TRAF6 (D21G3), phoshpo- TAK1, TAK1, phospho-IKKα/β, phospho-c-jun, phospho-ATF2 and horseradish peroxidase-conjugated secondary antibody were from Cell Signaling technology (Danvers, USA). TLR4 (M-16), MyD88 (HFL-296) and TRAF6 (H-274) from (Santa Cruz, USA), phospho- rtyrosine from (Millipore, USA), and HO-1 (H4535) and IRAK1 (SAB4501559), SB203580 (S8307), SP600125 (S5567) and PD98059 (P215), LY-294002 hydrochloride (L9908), PP2 (P0042), dimethyl- sulfoxide (DMSO), lipopolysaccharide (LPS) and Griess’s reagent were from (Sigma–Aldrich). Prostaglandin E2 EIA Kit was obtained from (Enzo Life Sciences, USA), and TNF-α, IL-1β, IL-6 and GM CSF MILLIPLEXTM ELISA kit was from (Millipore Corp., USA). All other reagents were of the first grade.

Cell culture

RAW 264.7 murine macrophages from the American (ATCC TIB71) were grown in Dulbecco’s modified Eagle’s medium (Invi- trogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml strepto- mycin under endotoxin-free conditions at 37 ◦C in a 5% CO2 humidified air.

Cell viability assay

The effects of quercetin on cell viability were evaluated using the MTT assay. In brief, RAW 264.7 cells were seeded at a den- sity of 1 × 104 cells/well in a 96 well-plate, and incubated at 37 ◦C for 24 h, and treated with various concentration of quercetin alone or with LPS (100 ng/ml). After 24 h incubation at 37 ◦C, 50 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 0.5 mg/mL) was added to each well and incubated for 4 h. The absorbance of the resulting color, formazan, at 450 nm was assayed using a microplate reader.

Nitric oxide assay

Nitrite, a stable oxidation product of nitric oxide, was assayed as an indicator of NO production. Briefly, RAW 264.7 cells (1 × 106 cells/ml) were preincubated for 24 h, treated with quercetin 30 minutes before LPS (100 ng/ml) stimulation for another 24 h. Then, 100 µl of culture supernatant was mixed with an equal volume of Griess reagent (50 µl 1% sulfanilamide in 5% phosphoric acid and 50 µl 0.1% N-1-napthyl-ethylenediamine dihydrochloride in distilled water). After incubation at room tem- perature for 10 min, absorbance was measured at 540 nm on a microplate reader. Nitrite concentration in the supernatants was calculated against a standard curve prepared with sodium nitrite.

Enzyme-linked immunosorbent assay (ELISA)

RAW 264.7 cells were preincubated with 5–20 µM quercetin for 30 min and then further incubated with LPS (100 ng/ml) for 24 h. The level of TNF-α, IL-1β, IL-6 and GM CSF in the cul- ture medium was measured by MILLIPLEXTM mouse cytokine ELISA kit using antibodies against the indicated proinflammatory cytokines and biotinylated secondary antibodies according to the manufacturer’s instruction (Millipore Corp., St. Charles, MO). In addition, PGE2 contents in the culture medium were measured using prostaglandin E2 Kit following the manufacturer’s instruction (Enzo Life Sciences, Ann Arbor, MI, USA).

Extraction of total RNA and semiquantitative RT-PCR amplification

Total RNA was prepared using easy BLUETM kit (iNtRON Biotech- nology Co., Korea), according to the manufacturer’s protocol and
immediately stored at —70 ◦C until use. Briefly, total RNAs (2 µg) were incubated with oligo-dT18 at 70 ◦C for 5 min, cooled on ice
and then incubated with RT premix (Bioneer Co., Korea) for 90 min at 42.5 ◦C. The reactions were terminated at 95 ◦C for 5 min for the inactivation of reverse transcriptase. The PCR reaction was further conducted using PCR premix (Bioneer Co., Korea) with the appro- priate sense/antisense primer sequences of genes indicated in the following table.

Immunoprecipitation and in vitro Src and Syk kinase activity assays

Cell lysates containing equal amounts of protein (500 µg) from RAW264.7 (107 cells/ml) treated with quercetin (20 µm) 30 min before LPS-stimulation for 15–30 min were precleared with 10 µl of protein A-coupled Sepharose magnetic beads (10%, v/v; ELPIS Biotech, Korea) for 1 h at 4 ◦C. Precleared samples were incubated with 3 µl of anti-Src, anti-Syk, anti-PI3K, anti-MyD88 antibodies overnight at 4 ◦C. Immune complexes were mixed with 10 µl of protein A-coupled Sepharose magnetic beads (10%, v/v) and incu- bated for 4 h at 4 ◦C. The immunoprecipitates were then washed 5 times with immunoprecipitation (IP) buffer (20 mM Tris–HCl, pH 7.4; 2 mM EDTA, 2 mM EGTA, 50 mM β-glycero-phosphate, 5 mM NaF, 1 mM sodium orthovanadate, 1% Triton X-100, 10% glycerol, 10 µg/ml aprotinin, 10 µg/ml pepstatin, 1 mM PMSF, 1 mM ben- zimide and 2 mM hydrogen peroxide). After washing, the beads were boiled in Laemmli sample buffer (2% sodium dodecyl sulfate [SDS], 1% β-mercaptoethanol, 0.008% bromophenol blue, 80 mM Tris (pH 6.8), 1 mM EDTA) and the proteins were resolved by SDS- PAGE and transferred to PVDF membranes, blocked and probed with anti-phosphotyrosine, anti-p85, anti-Src, anti-Syk, anti-TLR4, anti-MyD88 antibodies. Then, immunoblots were visualized as indicated above.

For evaluating Src and Syk kinase activities following immuno- preciptation, the beads were washed five times in IP buffer and once in kinase reaction buffer (100 mM Tris–HCl, pH 7.2; 125 mM Mg (C2H3O2)2; 25 mM MnCl2; 2 mM EGTA; 0.25 mM sodium ortho- vanadate and 2 mM dithiothreitol), and then resuspended in kinase buffer containing 1 µm ATP and 12.5 µCi of γ-32ATP. After 5 min of incubation at 30 ◦C, the reaction was terminated by lysis buffer.

Preparation of nuclear extracts

Nuclear extracts were prepared after cells were scraped from dishes, transferred to microtubes and washed with ice cold PBS. Cells were allowed to swell by adding lysis buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet-P40, 1 mM dithiothreitol and 0.5 mM phenylmethyl- sulfonylfluoride]. The samples were incubated for 10 min on ice and centrifuged for 5 min at 4 ◦C. Pellets containing crude nuclei were resuspended in 50 ml of the extraction buffer con- taining 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol and 1 mM phenylmethyl-sulfonylfluoride, and then incubated for 30 min on ice. Lysates were centrifuged at 15,800 × g for 10 min to obtain the supernatant containing nuclear extracts.

Electrophoretic mobility shift assay

RAW 264.7 macrophages (3 × 106) were treated with quercetin or vehicle and stimulated with LPS for 45 min, washed once with PBS, scraped into cold PBS, and pelleted by centrifugation. Cytosolic and nuclear protein fractions were extracted using Active Motif Nuclear Extraction Kit (Carlsbad, USA). Binding reactions were per- formed at 37 ◦C for 15 min in 20 µl of reaction buffer containing 10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 10% glycerol, 1 µg poly (dI–dC), 1 mM dithiothreitol, and 30,000 cpm 32P-labeled oligonucleotide probes for NF-nB and AP-1. DNA–protein com- plexes were separated from unbound DNA probe on native 6% polyacrylamide gels at 75 V in 0.5× TBE buffer and then transferred to a nylon membrane.

Transient transfection and luciferase assay

Transfection of RAW 264.7 cells with NF-nB and AP-1 pro- moter firefly luciferase construct was performed in triplicate in 24-well plates using lipofectamineTM 2000 (invitrogen, CA, USA). The reporter activity was measured using a luciferase assay sys- tem according to the manufacturer’s instructions. Briefly, 6 h after treatment, cells were washed twice with ice-cold PBS (pH 7.4) and lysed by adding 200 µl of 1× reporter lysis buffer (Promega). After centrifugation at 12,000 g for 10 min at 4 ◦C, the supernatant was analyzed for luciferase activity and normalized to β-galactosidase activity.

Statistical analysis

One-way ANOVA with a post hoc Dunnett multiple comparison Student’s t test was used to determine the statistical significance of differences between values for the experimental and control groups. P values of 0.05 or less were considered as statistically significant. Data represent the means ± SEM of three experiments conducted in triplicates.

Results

The inhibitory effect of quercetin on NO production and iNOS expression Quercetin (Fig. 1), significantly inhibited LPS-evoked NO pro- duction in a concentration-dependent manner and it completely abolished at maximal dose of 20 µM (Fig. 2B), while it did not have cytotoxic effect over the concentration range used in this study (Fig. 2A). In addition, quercetin exhibited a dose-dependent inhibitory effect on LPS-induced iNOS mRNA and protein expres- sions in RAW 246.7 cells (Fig. 2D and E), suggesting that the inhibitory effect of quercetin on LPS-induced NO production is under the control of an inducible enzyme iNOS gene expression.

Quercetin suppresses PGE2 generation, COX-2 mRNA and protein expressions

Since PGE2 is the critical mediator of inflammation and its production depends on the activity of COX-2, we determined the effect of quercetin treatment on PGE2 synthesis and COX-2 activity. As shown in Fig. 2C, quercetin treatment exhibited a remarkable inhibition in PGE2 biosynthesis as compared to the vehicle con- trol and complete blockage was observed in cells treated with 20 µM. Similarly, LPS-induced COX-2 mRNA and protein expres- sions were dose-dependently arrested in response to quercetin treatment (Fig. 2F and G).

Effect of quercetin on pro-inflammatory cytokine gene and protein expressions

Since cytokine activations are critical markers of macrophage response to LPS-stimulation, we further examined whether quercetin affects LPS-induced pro-inflammatory cytokines in RAW 264.7 cells. As shown in Fig. 3, quercetin did inhibit the secre- tion of LPS-activated TNF-α, IL-1β, IL-6 and GM-CSF protein in the culture media. While the concentration-dependent attenuation of TNF-α secretion was relatively weaker, quercetin effects in IL-1β and IL-6 were strong at higher doses of treatments and it blocked GM-CSF secretion across the given dose ranges (Fig. 3A–D), respec- tively. Results in Fig. 4A, B and C, D show that quercetin markedly suppressed the indicated cytokines gene expressions at the mRNA levels in a similar fashion to the effects observed at secreted protein levels, suggesting that quercetin-mediated inhibition of the tested cytokines may be at the transcriptional level.

Effect of quercetin on the LPS-induced activation of NF-нB and AP-1

To determine whether quercetin affects NF-nB activation, LPS- activated IkB-α phosphorylation in RAW 264.7 cells was examined. LPS increased IkB-α phosphorylations (Fig. 5A, upper panel), lead- ing to a reduction in total-IkBα levels (middle panel). However, quercetin time- and dose-dependently inhibited I-nB phosphoryla- tions (Fig. 5A and B). It also inhibited LPS-induced NF-nB activation in a concentration dependent manner (Fig. 5B). Pretreatment of the cells with quercetin reversed LPS effects on total-IkB-α depletion and NF-nB activation (Fig. 5A and B).

To examine whether quercetin attenuates LPS-induced NF-nB and/or AP-1-mediated DNA binding, we conducted EMSA analy- ses. As shown in Fig. 6A and B, quercetin at the dose of 10 and 20 µM demonstrated a selective reduction of NF-nB and AP-1 DNA binding in nuclear proteins from LPS-stimulated RAW 264.7 cells. To further investigate whether quercetin attenuates NF-nB and/or AP-1-mediated promoter activities, we tested luciferase reporter gene transcription. Incubation of transfected RAW 264.7 cells with LPS for 6 h increased luciferase activity. However, the increased NF-nB and AP-1 reporter gene activities were suppressed in a quercetin-sensitive manner (Fig. 6C and D), indicating that inhi- bition of inflammatory mediators and pro-inflammatory cytokines expressions correlate with decreased NF-nB- and AP-1-stimulated DNA binding and promoter activity. Since, the effect of quercetin on AP1 DNA binding is low despite pronounced effect on reporter gene expression, we examined its effect on c-Jun or activating tran- scription factor 2 (ATF2) phosphorylations. Quercetin significantly, inhibited c-Jun and ATF2 phosphorylation (data not shown), which regulates transactivation, but with a limited effect on DNA binding.

Quercetin pretreatment upregulates HO-1 gene and protein expressions

Since studies have shown that flavonoid induced HO-1 expression plays a critical role in mediating antioxidant and anti- inflammatory effects (Yao et al. 2007), we determined whether anti-inflammatory activity of quercetin could be linked to HO-1 induction. As shown in Fig. 7A–D, quercetin effect against inflam- mation was accompanied by HO-1 induction. Western blot analysis revealed that HO-1 protein expression levels were remarkably elevated in a time- and dose-dependent manner in quercetin treated-macrophages (Fig. 7C and D). In the kinetics of HO-1 pro- tein expression, the peak induction was shown between 6 and 12 h (Fig. 7C). Similarly, in the concentration-dependent effects, maximum HO-1 induction was observed at 40 µM of the dose range treated for 12 h (Fig. 7D). In addition, results in Fig. 7A and B further show that quercetin induced HO-1 gene expres- sion in a time- and dose-dependent manner, suggesting that quercetin mediated HO-1 induction may involve the transcriptional regulation.

Quercetin treatment attenuates LPS-stimulated p38 and JNK1/2 MAPK activation

The activation of MAPK is a crucial trigger for HO-1 induction. To characterize signal transduction mechanism(s) through MAPK pathway, we investigated the effect of three well-studied MAPK pathways, i.e. ERK1/2, JNK and p38MAPK involvements in quercetin treated cells. Quercetin markedly suppressed LPS-stimulated JNK and p38MAPK but not ERK1/2 activations (Fig. 8A and B). As shown in Fig. 8C, we confirmed that SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor), but not PD98059 (ERK inhibitor), sup- pressed iNOS, COX-2 and HO-1 induction in quercetin treated and LPS-stimulated cells. These results suggest that JNK and p38MAPK are involved both in the induction of anti-inflammatory (HO- 1) and proinflammatory mediators (iNOS and COX-2 enzyme) activations.

LPS-induced PI3K and Akt activations are suppressed by quercetin pretreatment

Since PI3K-Akt pathway is involved in LPS activation of signaling pathways and expression of inflammatory mediators in RAW 264.7 cells, we examined the effect of quercetin treatment in the indicated signaling pathways. As shown in Fig. 9A–C, quercetin markedly inhibited LPS-induced-PI3K, PDK1 and Akt phosphoryla- tion in a time- and concentration-dependent manner, respectively. A similar inhibition on JNK and p38 activations (Fig. 8A and B), and PI3k, PDK1 and Akt phosphorylations (Fig. 9A–C) suggest the existence of a possible cross-talk between PI3K/Akt and JNK/p38 signaling pathways.

Quercetin inhibits LPS-activated Src phosphorylaton

Quercetin is reported to be protein tyrosine kinase (PTK) inhibitor including Src tyrosine kinase (Houseman et al. 2002). Also, PTK activation is required for LPS-induced release of cytokines such as TNF-α, IL-6 and IL-1β from human blood monocytes (Geng et al. 1993). We, therefore, hypothesized that quercetin might contribute to augmented NF-nB translocation by LPS via the involvement of Src family kinases. To study whether Src tyrosine kinase is involved in quercetin mediated inhibition of PI3K/Akt activation, we examined the effect of quercetin treatment to Src phosphorylation. Expo- sure of RAW 264.7 cells to LPS led to a time-dependent increase in Src phosphorylation that peaked at 15 min after LPS stimulation (Fig. 10A). As depicted in Fig. 8A and B, quercetin pretreatment attenuated LPS-induced Src phosphorylation in a time- and dose- dependent manner. This observation was further confirmed by the use of PP2 (Src inhibitor) and LY274002 (PI3K inhibitor). LY274002 suppressed iNOS, PI3K and Atk activations (Fig. 10C). This effect was associated with inhibition of Src activation by its inhibitor, PP2 (Fig. 10C), suggesting that tyrosine kinases may be a target for quercetin action.

Quercetin suppresses LPS-induced kinase activities and Src, Syk and PI3K tyrosine phosphorylations

Since PI3K is activated by tyrosine phosphorylation (Cuevas et al. 2001) and since it has been reported to be activated by

Src kinase (Check et al. 2010) and Syk kinase (Zhang et al. 2009) up on TLR4 activation, we examined the effect of quercetin on the tyrosine phosphorylations of the indicated proteins. As shown in Fig. 11A, quercetin suppressed LPS-induced Src (upper panel), Syk (middle panel) and PI3K (p85) (lower panel) tyrosine phos- phorylations in a time dependent manner. Furthermore, kinase activity of Src and Syk were determined in quercetin treated and LPS-stimulated cells. Src and Syk were immunoprecipitated from RAW 264.7 cells stimulated with LPS in the presence and absence of quercetin and assayed for kinase activity in vitro. As shown in Fig. 11B and C, the kinase activities of Src and Syk, respec- tively, were inhibited by quercetin in a concentration-dependent manner.

Quercetin inhibits LPS-induced PI3kinase with MyD88 association and suppresses multiple components of TLR4 downstream signaling pathway activation

Since it has been reported that a putative PI 3-kinase binding site YXXM exists in the C-terminus of MyD88 (Ojaniemi et al. 2003), we studied whether LPS induced signaling complex formation between PI3k and MyD88 would be affected by quercetin treatment. MyD88 was immunoprecipitated from untreated cells and from cells treated with quercetin for various times, as indicated in Fig. 12A. The immunoprecipitates were separated on SDS-PAGE, and the membrane was blotted with an antibody that recognizes the p85 regulatory subunit of the PI 3-kinase. MyD88 precipitation brought down the P85, the amount of which was increased by the LPS chal- lenge and inhibited by quercetin (Fig. 12A, middle panel). We were able to detect TLR4 in the same immunoprecipitates with a weaker but similar pattern of expression to PI3K (Fig. 12A, upper panel). However, we did not detect co-immuno-precipitation between TLR4 and p85 subunit of PI3K (data not shown). Since the p85 sub- unit of PI 3-kinase interacts with tyrosine-phosphorylated motifs on other signaling proteins, the tyrosine phosphorylation status of MyD88 was studied. Anti-MyD88 immunoprecipitates from LPS- stimulated and quercetin treated cell lysates were analyzed by immunoblot with anti-phosphotyrosine antibody. Tyrosine phos- phorylation of MyD88 was increased by LPS challenge. However, the observed increased expression was restored to basal level by quercetin treatment (Fig. 12A, lower panel). In all immunoblots, the basal expression level of MyD88 was depleted by LPS chal- lenge, suggesting its activation. To confirm this, molecules that have been shown to lie downstream of the MYD88 were also examined by immunoblotting. Interestingly, LPS-stimulated acti- vation of IRAK1, TRAF6, p-TAK1, TAK1, p-IKKα/β and InBα were suppressed by quercetin treatment (Fig. 12B). While quercetin inhibited LPS-activated TAK1 and IKKα/β phosphorylations, it restored LPS-induced depletion of MyD88, IRAK1, TRAF6, TAK1 and InBα, suggesting its upstream inhibitory effect early in the signaling event through the inhibition of PI3K tyrosine phos- phorylation with a subsequent disruption of its association with MyD88.

Discussion

Both in vitro and in vivo data suggested that quercetin possesses immunomodulatory and anti-inflammatory properties. Although not fully understood, these health-promoting effects have been mainly related to their interactions with several key enzymes involving cytokines and regulatory transcription factors, and antioxidant systems (Gomes et al. 2008). Thus, inhibitory or sti- mulatory actions at these pathways are likely to profoundly affect cellular function by altering the phosphorylation state of target molecules and/or by modulating gene expressions. In line with our objective of identifying natural dietary products that are safe and have therapeutic potential as anti-inflammatory agents, we explore the molecular mechanism of quercetin action against LPS-induced inflammation in RAW 264.7 cells.

In this study, quercetin suppressed LPS-induced NO release, PGE synthesis, iNOS, COX-2 and cytokines (TNF-α, IL-1β, IL-6 and GM-CSF) gene and protein expressions. These findings suggest that a wide range of inflammatory mediators can be effectively attenuated by quercetin in LPS-activated RAW 264.7 cells. Even though quercetin mechanism of action against inflammation in LPS-activated RAW cell (suitable in vitro model) is limited, reports indicated that quercetin diminishes iNOS expression and InB degra- dation through the regulation of the IKK complex (Chen et al. 2005), and it inhibits NO and PGE2 production through suppression of iNOS and COX-2 protein expressions (Lin et al. 2003). In agree- ment to our study, quercetin has shown a decreased TNF-α protein, and IL-1β, IL-6, TNF-α, MIP-1α and iNOS mRNA levels (Boesch- Saadatmandi et al. 2010).

We here, observed that quercetin pretreatment suppressed InB phosphorylation, NF-nB and AP1 nuclear translocation, DNA-binding, and reporter transcriptional activation reflecting the ability of quercetin to inhibited NF-nB and AP-1 dependent inflam- matory mediators and pro-inflammatory cytokines transcriptions. The effect of quercetin on AP1 DNA binding in the present study was relatively low despite pronounced effect on reporter gene expres- sion. This was at least partially due to the inhibition of c-Jun or ATF2 phosphorylation, a process that does not affect DNA bind- ing, but influences transactivation instead (Karin et al. 1997). In line with this observation, studies have shown that flavonoids can modulate the NF-nB signaling pathway during inflammation, and this modulation can occur at early (IKK activation) as well as late (binding of NF-nB to DNA) stages (Dias et al. 2005). It has also been reported that quercetin prevents LPS-induced InB phosphorylation, and reduces InB-α and InBβ phosphorylations (Peet and Li 1999), that in turn inhibit the expression of NF-nB- and MAP kinase/AP1- dependent inflammatory mediators and cytokines (Pan et al. 2009; Russo et al. 2012), supporting the rationale of the present study to expand and add quercetin’s mechanism of action against inflam- mation on the exiting data.

Quercetin stimulated HO-1 induction and its expression level in the present study was dependent on p38 and JNK activities as SB20358 and SP600125 reversed quercetin-mediated HO-1 induc- tion, respectively. Inhibition of LPS-induced NO production by quercetin is reported to involve HO-1 (Lin et al. 2003) and IL-10 (Comalada et al. 2006) inductions via a p38MAPK-dependent path- way (Comalada et al. 2006, Otterbein et al. 2000). It is further reported that HO-1 and its product (carbon monoxide) inhibited the expression of LPS-induced TNF-α, IL-1β, and MIP-1β, but increased IL-10 expression in MAPK-dependent manner in vivo and in vitro (Otterbein et al. 2000). The potential clinical applications of enhanc- ing the HO-1 system (Soares and Bach 2009), and the mechanism underlying the cytoprotective effect of HO-1 are important roles of the enzyme (Gozzelino et al. 2010), suggesting that quercetin with HO-1 inducing capability can be an alternative anti-inflammatory agent.

In the present study, we have shown that activation of JNK and p38MAPK but not ERK1/2, were suppressed by quercetin. This was further confirmed as the respective inhibitors i.e. SB203580 and SP600125, but not PD98059 inhibited HO-1, iNOS and COX- 2 expressions, suggesting that the HO-1 induction by quercetin may be mediated by JNK and p38MAPK. The three MAPKs are activated by LPS- and cytokine-stimulations (Dong et al. 2002), and considered to be targets of the development of novel anti- inflammatory drugs (Karin 2004). Cho et al. (2003) reported that quercetin inhibits ERK and p38MAPK activation but not JNK activation in LPS-stimulated RAW 264.7 cells. The difference in quercetin preference to JNK over ERK in the present finding as opposed to the above indicated report may be attributed to the concentrations of quercetin used, the origin of macrophages and nature of stimulus. Quercetin concentrations used in the above indicated report is higher, which may have a nonspecific broad effects in protein kinases. This suggestion is supported by a report describing the contribution of ERK1/2 and p38 on TNF-α production depends on the dose, state of macrophages and the stimulus (Means et al. 2000). However, a similar result to our study has been reported elsewhere (Wadsworth et al. 2001), which states that quercetin inhibited iNOS and TNF-α- production by inhibiting p38MAPK and JNK in LPS-induced RAW cells, leading to the inhibition of AP-1-DNA binding. Santangelo et al. (2007) further indicated that the ERK route is frequently activated by mitogens and growth factors, while inflammation is a main trigger for JNK and p38. The present study did not rule out the possibility that LPS-induced TNF-α induction might be through stabilization of the TNF mRNA via p38-dependent post transcriptional mechanism and whether quercetin modulates this mechanism. The dominant regulatory mode for TNF-α induction is reported to be through stabilization of the TNF mRNA in p38-dependent post transcriptional mechanisms (Mahtani et al. 2001).

Our study demonstrated that quercetin significantly inhibited LPS-induced-PI3K, PDK1 and Akt phosphorylation. Quercetin- evoked inhibition of JNK and p38, and the above indicated kinases activation suggest the existence of a possible cross-talk between PI3K/Akt and p38/JNK signaling pathways. Interestingly, quercetin arrested Src and Syk kinase activations and tyrosine phosphorylations followed by inhibition in PI3K tyrosine phos- phorylation, confirming the involvement of the kinase activities and tyrosine phosphorylations of both kinases in quercetin- mediated inhibition of PI3K/Akt activation. This conclusion was further supported by LY274002 (PI3K inhibitor) and PP2 (Src inhibitor)-mediated suppression of iNOS, PI3K and Akt activation, suggesting that quercetin may inhibit tyrosine kinases. This is in line with previous reports that indicated PI3K activation is medi- ated by Src kinase (Check et al. 2010) and Syk kinase (Zhang et al. 2009) activations followed by PI3K tyrosine phosphoryla- tion (Cuevas et al. 2001) upon LPS-induced TLR4 stimulation. In agreement with the present study, reports indicated that quercetin inhibited tyrosine kinase activity and the downstream PI3K/Akt signaling pathway (Jeong et al. 2008), and a higher selectivity of quercetin and its derivatives as Src tyrosine kinase inhibitors is suggested (Huang et al. 2009). Quercetin mediated Src tyro- sine kinase inhibition has been reported within the dose range of this study (Houseman et al. 2002), and participated in a diminished LPS-induced TNF-α, IL-6 and IL-1β release (Geng et al. 1993).

Interestingly, LPS-induced PI3K activation and its p85 subunit association with the MyD88 was attenuated by quercetin. The increased tyrosine phosphorylation status of MyD88 by LPS was also correspondingly reversed by quercetin treatment, suggesting that the activation of MyD88 tyrosine phosphorylated motif, on which p85 interacts, is inhibited by quercetin. Although we did not detect co-immunoprecipitation between TLR4 and p85 subunit, LPS-induced TLR4 activation in MyD88 precipitated proteins were also observed to be inhibited by quercetin. It has been reported that a putative PI3K binding site YXXM exists in the C-terminus of MyD88 (Ojaniemi et al. 2003), and MyD88 association with the P85 subunit is increased by the LPS challenge. Thus, quercetin treat- ment may disrupt the association of PI3K to MyD88 and limit tyrosine phosphorylated TLR4/MyD88/PI3K complex formation in response to TLR4 LPS-stimulation. The inhibitory effect of quercetin on the above indicated complex formation is linked to suppression of LPS-induced activation of the molecules that have been shown to lie downstream of the MyD88, including IRAK1, TRAF6, p-TAK1, TAK1, p-IKKα/β and InBα. Quercetin restored the depletion of total MyD88, IRAK1, TRAF6, TAK1 and InBα expression, and inhibited LPS-stimulated TAK1 and IKKα/β phosphorylations, suggesting its upstream inhibitory effect early in the signaling event through the inhibition of PI3K tyrosine phosphorylation and disruption of its regulatory unit association with MyD88.

Direct interaction of PI3K with certain TLRs or TLR adapters has been reported, regulating downstream signaling to NF-nB trans- activation and/or MAPK activation (Ojaniemi et al. 2003; Li et al. 2003). Thus, TLR4/MyD88/PI3K interactions are reported to regu- late TLR4 signaling (Laird et al. 2009), and TLR4-mediated activation of PI3K functions either as a positive or negative regulator of TLR signaling. PI3K has also been reported to contribute to TLR4- mediated activation of NF-nB and COX-2, IL-6, MCP-1, IP-10, IL-12, and IL-10 (Li et al. 2003). Moreover, PI3K has positively regulated cytokine expression through formation of a complex between the p85 regulatory subunit, TLR4 and MyD88 (Ojaniemi et al. 2003). Thus, PI3K may act as a molecular switch to control TLR4 signaling depending on cellular needs and its state of activity. Here, we found that modulation of P13K activity by quercetin at early stage of the LPS-induced signaling event limits TLR4 activation pathway.

Our data in the present study shows that quercetin-mediated inhibition of LPS-activated TLR4/MyD88/PI3K association limits the IRAK-1, TRAF6, TAK1 and IKKα/β activation, which results in reduced InB phosphorylation and degradation followed by attenuation of NF-nB translocation and its target genes (inflam- matory mediators and cytokines) induction. Limited TAK1 activation in the present study also suggests diminished p38/JNK phosphorylations and reduced AP-1 activation and its target gene expressions, supporting the quercetin’s role in limiting LPS-induced inflammation. TAK1 activation is known to involve both IKK/NF-nB (Akira and Takeda 2004) and MAPKs/AP-1 pathways (Gay and Gangloff 2007).

In conclusion, we demonstrate that quercetin arrested Src and Syk mediated PI3K, PDK1 and Akt activations, and it disrupted LPS-induced p85 association to TLR4/MyD88 complex and limited IRAK1, TRAF6 and TAK1 activation with a subsequent suppression in IKKα/β mediated I-nB phosphorylation. In addition, it atten- uated TAK1 mediated JNK1/2 and p38MAPK phosphorylations but stimulated HO-1 induction. This led to inhibition in NF-nB and AP-1 regulated inflammatory mediators expressions. Thus, quercetin limits LPS-induced inflammation via inhibition of Src- and Syk- mediated PI3K (p85) tyrosine phosphorylation and subsequent TLR4/MyD88/PI3K complex formation that diminishes activation of downstream signaling pathways.