Fisetin is a selective adenosine triphosphate-competitive inhibitor for mitogen-activated protein kinase kinase 4 to inhibit lipopolysaccharide-stimulated inflammation

Abstract

The mitogen-activated protein kinase kinase 4 (MKK4), a member of the MAP kinase kinase family, directly phosphorylates and activates the c-Jun NH2-terminal kinases (JNK), in response to proinflammatory cytokines and cellular stresses. Regulation of the MKK4 activity is considered to be a novel approach for the prevention and treatment of inflammation. The aim of this study was to identify whether fisetin, a potential anti-inflammatory compound, targets MKK4-JNK cascade to inhibit lipopolysaccharide (LPS)-stimulated inflammatory response. RAW264 macrophage pretreated with fisetin following LPS stimulation was used as a cell model to investigate the transactivation and expression of related-inflammatory genes by transient transfection assay, electrophoretic mobility shift assay (EMSA), or enzyme-linked immunosorbent assay (ELISA), and cellular signaling as well as binding of related-signal proteins by Western blot, pull-down assay and kinase assay, and molecular modeling. The transactivation and expression of cyclooxygenase-2 (COX-2) gene as well as prostaglandin E₂ (PGE₂) secretion induced by LPS were inhibited by fisetin in a dose-dependent manner. Signaling transduction analysis demonstrated that fisetin selectively inhibited MKK4-JNK1/2 signaling to suppress the phosphorylation of transcription factor AP-1 without affecting the NF-κB and Jak2-Stat3 signaling as well as the phosphorylation of Src, Syk, and TAK1. Furthermore, in vitro and ex vivo pull-down assay using cell lysate or purified protein demonstrated that fisetin could bind directly to MKK4. Molecular modeling using the Molecular Operating Environment™ software indicated that fisetin docked into the ATP-binding pocket of MKK4 with a binding energy of −71.75 kcal/mol and formed a 1.70 Å hydrogen bound with Asp247 residue of MKK4. The IC₅₀ of fisetin against MKK4 was estimated as 2.899 μM in the kinase assay, and the ATP-competitive effect was confirmed by ATP titration. Taken together, our data revealed that fisetin is a potent selective ATP-competitive MKK4 inhibitor to suppress MKK4-JNK1/2-AP-1 cascade for inhibiting LPS-induced inflammation.

1 INTRODUCTION

Macrophages are considered as the first-line defense against infection and are essential in innate immunity.¹ Macrophages display highly phenotypic plasticity, and the aberrant polarized macrophages participate in the pathogenesis of various inflammation-related human diseases including inflammatory bowel disease, nonalcoholic fatty liver disease, as well as cancer.^2-4 Lipopolysaccharide (LPS), a component from the outer membrane of Gram-negative bacteria, belongs to pathogen-associated molecular patterns (PAMPs) and acts as the agonist for toll-like receptor 4 (TLR4).⁵ Using LPS to stimulate macrophages into a proinflammatory phenotype is a common model for mimicking in vivo bacterial infection as well as studying immune response. In general, LPS activates canonical pathways including nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathway.^{6, 7} Whether Jak–Stat pathway directly activated by LPS is controversial in previous works.^{8, 9} Other kinases including Src and Syk may also respond to LPS stimulation, and they may have a crosstalk with NF-κB or MAPK pathway.^10-12 Signal pathways activated by LPS mediate the further transcription of proinflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-6 as well as proinflammatory enzymes including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).

Among inflammation-related pathways, MAPK pathway is one of the most important pathway in the regulation of proinflammatory mediators.⁶ Kinases in MAPK pathway play critical role in inflammatory signal transduction, and they are believed as the potential targets in controlling inflammation.¹³ In our previous works, some flavonoids were found to act as protein kinase inhibitors to regulate certain biological progress.¹⁴ For example, 8-prenyl quercetin directly binds MKK4-JNK1/2 and MEK1-ERK1/2,¹⁵ cyanidin 3-glucoside, and (−)-epicatechin (EC) binds TAK1,¹⁶ while quercetagetin targets and inhibits JNK1.¹⁷ Therefore, kinases in MAPK family may be the important targets for fisetin to inhibit inflammation.

Dietary flavonoids consumption has been reported to modulate host immune system for preventing excessive immune response.^{15, 18} Fisetin is a typical flavonoid belonging to the flavonol group and wildly exists in natural products including strawberries, green tea, and lime blossom.¹⁹ Fisetin was composed of two benzene rings that linked by a three-carbon chain, forming a C6-C3-C6 skeleton, with four free hydroxyl groups at C-3, C-3′, C-4′, and C-7, and a carbonyl group at C-4. Previous studies have reported that fisetin had potent anti-inflammatory capability in both animal and cell models. In animal model, fisetin administration downregulated LPS or high fat diet (HFD)-induced serum proinflammatory cytokines,^{20, 21} inflammatory mediators in kidney,²² and colonic inflammation level in colitis model.²³ In cell model, fisetin treatment downregulated the expression of LPS-stimulated proinflammatory mediators including iNOS, COX-2, IL-6, and TNF-α in macrophages.²³ The major potential mechanisms are associated with the inhibition of NF-κB²³ and MAPK²⁴ pathways. Other mechanisms might include the inhibition of Src and Syk kinases,²⁵ the prevention of β-catenin degradation,²⁶ and the modulation of PI3K/Akt/mTOR axis.²⁷ However, the results reported from several lines of studies are inconsistent on intracellular signaling and molecular targets of fisetin although all revealed anti-inflammatory effects. The major reason is possible due to different experimental models (cell or animal), dose, and treatment time of LPS and fisetin. After reviewing these reports, we optimized the cell model and challenged to identify the potential molecular targets of fisetin in LPS-stimulated RAW264 macrophages by multiple biochemical methods in this study.

First of all, the inhibitory effect of fisetin on LPS-stimulated inflammatory mediators was confirmed by detecting the level of intracellular COX-2 and prostaglandin E2 (PGE₂) secreted in culture medium. Next, the effect of fisetin on transactivation and signaling of these inflammatory mediators was investigated by electrophoretic mobility shift assay (EMSA) and phosphorylation status of relative protein kinases by Western blot. These data revealed that MKK4 acted as a potential molecular target for fisetin anti-inflammatory effect. To clarify this point, a pull-down assay was used to examine the direct binding of fisetin to MKK4 in ex vivo and then supported using in silico molecular docking analysis. Furthermore, kinase assay was used to confirm the inhibitory effects of fisetin on MKK4. Our data demonstrated that fisetin selectively targeted the ATP pocket of MKK4, a key kinase in MAPK pathway, to inhibit LPS-induced inflammation.

2 MATERIALS AND METHODS

2.1 Reagents and cell culture

Fisetin (≥99%) was purchased from EXTRASYNTHESE (Genay, Rhône, France), Takinib was from Cayman Chemical Co. (Ann Arbor, MI, USA), BSJ-04-122 (BSJ) was from Sigma-Aldrich (St. Louis, MO, USA), and staurosporine was from Tokyo Chemical Industry Co., Ltd (Tokyo, JP), and they were all dissolved in DMSO. For the experiment, the final concentration of DMSO with or without the above samples was 0.2% in cell culture medium. LPS (Escherichia coli Serotype O55:B5) was from Sigma-Aldrich (St. Louis, MO, USA), and IFN-γ was from PeproTech, Inc. (Cranbury, NJ, USA). LipofectAMINE2000 was from Life Technologies, Inc. (Grand Island, NY, USA). The COX-2 promoter-luciferase constructs (−1432/+59) were described previously.^{28, 29} Antibodies against COX-2, IκBα, p65, Jak2, phospho-Stat3 (Y705), MKP-1, and α-tubulin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against iNOS, phospho-IκBα (S32), phospho-p65 (S536), phospho-Jak2 (Y1007/1008), Stat3, phospho-Src (Y416), phospho-Syk (Y525/526), phospho-TAK1 (T184/187), phospho-MKK4 (T261), MKK4, phospho-JNK1/2 (T183/Y185), JNK1/2, phospho-c-Jun (S73), c-Jun, phospho-MEK1/2 (S217/221), phospho-ERK1/2 (T202/Y204), ERK1/2, phospho-MKK3 (S189)/6 (S207), phospho-p38 (T180/Y182), and p38 were from Cell Signaling Technology (Beverly, MA, USA). Antibody against phospho-MKK7 (S271/T275) and recombinant human MEK4/MKK4 protein were from Abcam (Cambridge, UK). Human JNK1 recombinant protein was from Thermo Fisher Scientific Inc. (Rockford, IL, USA). CNBr-activated Sepharose 4B was purchased from GE Healthcare (Uppsala, Sweden). Mouse macrophage-like RAW264 cell line (Cell No. RCB0535) was from RIKEN Bio-Resource Center Cell Bank (Tsukuba, Japan) and was cultured in DMEM containing 10% FBS and 2 mM L-glutamine at 37°C in a 5% CO₂ atmosphere.

2.2 PGE₂ measurement

PGE₂ in culture medium was measured with a PGE₂ enzyme immunoassay kit (Cayman Co, Ann Arbor, MI, USA) according to manufacturer's manual.³⁰ Briefly, RAW264 cells (5 × 10⁵ cells) were seeded into each well of 6-well plates. After incubation for 24 h, the cells were starved by being cultured in serum-free medium for another 2.5 h to eliminate the influence of FBS. The cells were then treated with 0 ~ 20 μM fisetin for 30 min before exposure to 40 ng/mL LPS for 12 h. The level of PGE₂ released into culture medium was determined by measuring absorbance at 405 nm with a microplate reader.

2.3 Transient transfection assay

Transient transfection was performed according to the modified method in our previous study.³¹ RAW264 cells (1 × 10⁵) were plated into each well of 12-well plates and cultured for 24 h. The cells were then transfected with 0.5 μg COX-2 promoter-luciferase plasmids using LipofectAMINE2000. After 5 h incubation, the medium was replaced and cultured for another 20.5 h. The cells were treated with 0 ~ 20 μM fisetin for 30 min before exposure to 40 ng/mL LPS for 6 h. The luciferase activity in cell lysate was measured by a luminometer (Berthold) according to the supplier's recommendations. The transactivation activity of COX-2 promoter was expressed as fold induction relative to the control cells without LPS treatment.

2.4 Electrophoretic mobility shift assay (EMSA)

Nuclear extracts for EMSA were prepared basing on our previous study.³² Briefly, RAW264 cells were precultured for 24 h and then starved by being cultured in serum-free medium for another 2.5 h to eliminate the influence of FBS. The cells were treated with 20 μM fisetin for 30 min before exposure to 40 ng/mL LPS for 4 h. Harvested cells were lysed by incubation in buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, and 0.2 mM PMSF] on ice for 15 min and then centrifuged at 13,500×g for 10 min at 4°C. The nuclear pellets were resuspended in Buffer B [20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF] for 15 min at 4°C and then centrifuged at 13,500×g for 15 min at 4°C. The supernatants containing nuclear extracts were stored at −80°C until use. Oligonucleotide probes were synthesized by Genenet Co., Ltd. (Fukuoka, Japan) and then annealed in TE buffer. The sequence of oligonucleotides was reported in our previous paper.³³ The 5′-end labeling of oligonucleotides was performed by T4 polynucleotide kinase (Takara Bio Inc., Shiga, Japan) with 10 pmol of double-stranded oligonucleotide and 50 μCi of [γ-³²P] ATP (5000 Ci/mmol; Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). The labeled oligonucleotides were purified using a Sephadex G-25 spin column (Amersham Biosciences, U.K.). Five μg nuclear extract was incubated at 25°C for 30 min with labeled or unlabeled competitor oligonucleotides in binding buffer [25 mM Tris–HCl, pH 7.5, 75 mM KCl, 0.3% Nonidet-40, 7.5% glycerol, 2.5 mM dithiothreitol, 1 mg/mL bovine serum albumin and 1 μg of poly(dI)⋅ poly(dC)]. The products were electrophoresed at 4°C on a 5% nondenaturing polyacrylamide gel in 0.5 × Tris borate/EDTA buffer, and the gel was then dried on 3MM chromatography paper. The radioactivity on paper was detected with FLA-2000 machine (Fuji Photo Film, Tokyo, Japan).

2.5 Nuclear protein extraction

Nuclear extracts for Western blot assay were prepared by using the Nuclear Extract Kit from Active Motif (Carlsbad, CA).³² Briefly, RAW264 cells were precultured for 24 h and then starved by being cultured in serum-free medium for another 2.5 h to eliminate the influence of FBS. The cells were treated with or without 20 μM fisetin for 30 min before exposure to 40 ng/mL LPS for 30 min. The cell pellet was suspended in 1 × hypotonic buffer supplemented with complete protease inhibitor cocktail, incubated for 15 min at 4°C, vortexed in presence of detergents, and centrifuged briefly. The nuclear pellet was washed twice with the cytoplasmic buffer followed by resuspension in the lysis buffer supplemented with 1 mM DTT and protease inhibitors. The suspension was incubated on rocking platform at 4°C for 30 min. The suspension was then vortexed briefly and centrifuged for 10 min at 14,000×g at 4°C. The supernatant (nuclear fraction) was collected, and nuclear extracts were analyzed by Western blotting assay.

2.6 Western blot assay

Western blot assay was performed as described previously.³⁴ RAW264 cells were precultured for 24 h and then starved in serum-free medium for 2.5 h to eliminate the influence from FBS before experiment. The cells were then pretreated with 0–20 μM fisetin for 30 min following LPS or IFN-γ stimulation. After a defined time, the cells were lysed in a lysis buffer (62.5 mM Tris–HCl [pH 6.8], 4% SDS, 100 mM DTT, 10% Glycerol), and boiled for 6–8 min. Equal amounts of protein (10–40 μg) were run on 8%–12% SDS-PAGE and then transferred to a PVDF membrane. After blocking, the membrane was incubated with specific primary antibody (4°C, overnight) and further incubated with corresponding HRP-conjugated secondary antibody (room temperature, 1 h). The bound antibodies were finally detected by the ECL system, and the relative amounts of proteins were quantified by Lumi Vision Imager software (TAITEC Co., Saitama, Japan).

2.7 Molecular modeling

The modeling of fisetin bound with MKK4 kinase (PDB codes: 3ALO) was performed by Molecular Operating Environment™ software (MOE, Version 2016.08, Chemical Computing Group Inc.) according to our previous report.³⁵ Hydrogen atoms were added first and then force field (MMFF94x) atomic charges were assigned. Docking of fisetin to kinase structures was done by MOE-ASEDock 2013 software.³⁵ The modeling of fisetin with MKK4 structure was repeated for 30 times. For all the possible interactions from each modeling, the interaction form with lowest Udock (binding energy) was recorded as the most possible interaction result in this single modeling. It is generally considered that the lower the Udock value is, the more stable the complex structure is formed.

2.8 Pull-down assay

Pull-down assay was performed according to our previous study.³⁶ First, fisetin (5 mg) was coupled to CNBr-activated Sepharose 4B beads (75 mg) in a coupling buffer [25% DMSO, 0.5 M NaCl, and 0.1 M NaHCO₃ (pH 8.3)] at 4°C overnight. After centrifugation (4°C, 1000 rpm, 3 min), the beads were washed with 5 volumes of coupling buffer and then suspended in 5 volumes of 0.1 M Tris–HCl buffer (pH 8.0) with rotation for 2 h at room temperature. After a three-cycle washing of acetate buffer (0.1 M acetic acid [pH 4.0] and 0.5 M NaCl) and wash buffer (0.1 M Tris–HCl [pH 8.0] and 0.5 M NaCl), 500 μg RAW264 lysates (for ex vivo assay) or 100 ng recombinant active His-MKK4 or His-JNK1 (for in vitro assay) were added and incubated with control beads or fisetin-conjugated beads in a reaction buffer (150 mM NaCl, 50 mM Tris–HCl [pH 8.5], 5 mM EDTA, 1 mM DTT, 0.01% Nonidet P-40, 2 μg/mL BSA, 0.02 mM PMSF and 0.1% [v/v] protease inhibitor cocktail) and rotated overnight at 4°C. The complexes were washed with wash buffer (200 mM NaCl, 50 mM Tris–HCl [pH 7.5], 5 mM EDTA, 1 mM DTT, 0.02% Nonidet P-40 and 0.02 mM PMSF) for five times and finally boiled for 5 min. The proteins that bound to the beads were further detected through Western blot assay.

2.9 Kinase assay

Inhibitory effect of fisetin on MKK4 was measured by ADP-Glo kinase assay kit (Promega Co., Madison, WI, USA). First, kinase reaction was performed using MEK4 (MAP2K4) Kinase Enzyme System (VA7219, Promega Co., Madison, WI, USA). Unactive JNK1 (0.1 μg), active MKK4 (7 ng), ATP (3 μM, equal to the ATP K_M value for MKK4),³⁷ and different concentrations of samples were added in kinase reaction buffer (40 mM Tris [pH: 7.5], 20 mM MgCl₂, 0.1 mg/mL BSA, 50 μM DTT) and reacted at room temperature for 2 h. ADP-Glo reagent was then added to deplete the ATP. After a 40-min incubation, kinase detection reagent was mixed followed by a final incubation for 30 min. The luminescence (RLU) was recorded using Infinite 200 PRO MPlex (TECAN Co., Zürich, Switzerland) with an integration time of 0.8 s for each well. The percent MKK4 activity was calculated according to the RLU of 0% kinase activity group (neither compound nor kinase) and 100% kinase activity group (no compound). For ATP competition study, the ATP concentration was set at 10, 50, or 250 μM in the kinase reaction. Curve fitting was done, and IC₅₀ was calculated using sigmoidal dose–response (variable slope) in GraphPad Prism®.

2.10 Statistical analysis

Results are presented as means ± SD. Significant differences were analyzed by one-way ANOVA followed by Tukey's multiple range test or analyzed by independent samples t-test (SPSS19, IBM Corp., Armonk, NY, USA). p < 0.05 was considered as statistically significant.

3 RESULTS

3.1 Fisetin suppresses COX-2 induction and PGE₂ production

COX-2 enzyme and its synthesized product PGE₂ were reported to be induced in LPS-stimulated macrophage.³⁰ Therefore, COX-2 was chosen as the inflammation marker in the present study. First, a promoter activity assay of COX-2 gene with a full COX-2 promoter-luciferase plasmid (−1432/+59) was performed by transient transfection assay. The data revealed that fisetin suppressed LPS-induced COX-2 promoter activity in a dose-dependent manner (Figure 1A). Subsequently, a dose-dependent inhibition of LPS-induced COX-2 expression, but not COX-1, a constitutive protein (Figure 1B) was also observed. Moreover, fisetin suppressed the production of PGE₂, which is synthesized by COX-2, in the same concentration ranges (Figure 1C). These results indicated that fisetin may be a potential inhibitor for COX-2.

FIGURE 1

Fisetin reduced LPS-mediated transactivation and expression of COX-2 gene to suppress PGE₂ secretion. (A) Fisetin suppressed LPS-induced COX-2 transcription activity in a dose-dependent manner. The transient transfection assay was performed as described in Materials and Methods. (B) Fisetin inhibited LPS-induced COX-2 expression in a dose-dependent manner. RAW264 cells were precultured for 21 h and starved in serum-free medium for 2.5 h. The cells were then treated with the indicated concentration of fisetin for 30 min and exposed to 40 ng/mL LPS for 12 h. COX-2 and COX-1 were detected by Western blot assay with the corresponding antibodies. The blots presented are the examples from at least three independent experiments. The relative density of COX-2 was calculated as the intensity of the treatment relative to that of LPS-treated group by densitometry. (C) Fisetin downregulated LPS-induced PGE₂ secretion. RAW264 cells were treated as the same as above, and the amount of PGE₂ in cell cultural medium were measured with ELISA kit according to the manufacturer's instruction. Each value represents the mean ± SD of three to four separate experiments. *p < 0.05 and **p < 0.01 versus LPS-treated group.

3.2 Effect of fisetin on LPS-stimulated signaling pathways

NF-κB pathway is one of the key cascades in LPS-stimulated immune response.³⁸ Therefore, we first investigated the effect of fisetin in the modulation of NF-κB pathway. EMSA result revealed that fisetin did not reduce the bound complex of NF-κB-DNA induced by LPS (Figure 2A, line 3). Western blot results showed that fisetin did not inhibit the phosphorylation and degradation of the inhibitory protein IκBα (Figure 2C) induced by LPS. Consequentially, the phosphorylation and nuclear translocation of transcription factor NF-κB/p65 (Figure 2B) were not suppressed by fisetin. These results demonstrated that fisetin did not suppress LPS-provoked NF-κB pathway.

FIGURE 2

Fisetin did not modulate NF-κB pathway. (A) Fisetin did not reduce the binding complex of NF-κB-DNA. RAW264 cells were precultured for 24 h and then starved in serum-free medium for another 2.5 h to eliminate the influence of FBS. The cells were treated with vehicle or 20 μM fisetin for 30 min before exposure to 40 ng/mL LPS for 4 h. Nuclear extracts were harvested and then incubated with labeled wild NF-kB probe (lane 1–4), 10-fold molar excess of cold NF-kB oligonucleotides (lane C) or labeled mutant NF-kB probe (lane MT). After separation in 5% nondenaturing polyacrylamide gel, the gel was dried on 3 MM chromatography paper and exposed to a radioactive imaging plate. (B) Fisetin did not inhibit LPS-induced phosphorylation and nuclear translocation of NF-κB/p65. (C) Fisetin did not prohibit the phosphorylation and degradation of IκBα. RAW264 cells were precultured as described above. The cells were then treated with 20 μM fisetin for 30 min and exposed to 40 ng/mL LPS for 30 min. Whole cell lysates or nuclear protein were harvested and analyzed by Western blot assay. The proteins were detected by their corresponding antibodies. The relative density was calculated as the intensity of the treatment relative to that of control normalized to α-tubulin, respective total proteins, or p65 in whole cell lysates (for nuclear p65) by densitometry. The blots presented are the examples from at least three independent experiments. Each value represents the mean ± SD of at least three separate experiments.

Next, we investigated the effect of fisetin on MAPK pathway, another vital pathway in controlling proinflammatory mediators. EMSA data indicated that fisetin markedly decreased the binding complex of AP-1-DNA (Figure 3A, lane 3). The specificity of AP-1-binding complexes was further demonstrated by competition experiment with cold and mutant AP-1 probe. Ten-fold molar excess of the unlabeled AP-1 oligonucleotides completely blocked the formation of this DNA–protein complex (lane C), and 10-fold molar excess of the labeled mutant AP-1 oligonucleotides did not block this complex (lane MT). These results indicated that fisetin inhibited AP-1 binding activity induced by LPS. Consistent with the EMSA result, fisetin also significantly inhibited the phosphorylation of transcription factor AP-1/c-Jun (Figure 3B). Pathway analysis revealed that fisetin significantly suppressed the phosphorylation of upstream kinases JNK1/2 and MKK4, but not MEK1/2-ERK1/2 (Figure 3C) and MKK3/6-p38 cascades (Figure 3D) in MAPK pathway. TAK1 is generally considered as an important upstream kinase to regulate both NF-κB and MAPK pathway,³⁹ our data showed that fisetin did not inhibit TAK1 phosphorylation (Figure 3E). Takinib, a TAK1-specific inhibitor as positive control, suppressed LPS-triggered phosphorylation of both TAK1 and MKK4 in a dose-dependent manner (Figure 4A,B). The phosphorylation of IκB-α and p65 in NF-κB pathway was also inhibited by Takinib (Figure 4C). These facts indicated that fisetin might inhibit LPS-induced inflammation in macrophages by selectively targeting MKK4-JNK1/2-AP-1 cascade. Additionally, we also observed that LPS did not activate Jak2-Stat3 cascade (Figure S1) and Src-Syk kinases (Figure S2).

FIGURE 3

Fisetin inhibited MKK4-JNK1/2-AP-1 cascade. (A) Fisetin reduced the binding complex of AP-1-DNA. Cell treatment and the EMSA experiment was performed as described in Figure 2A. (B) Fisetin inhibited the phosphorylation of c-Jun. (C) Fisetin selectively inhibited the phosphorylation of JNK1/2 in MAPKs. (D) Fisetin selectively inhibited the phosphorylation of MKK4 in MAP2Ks. (E) Fisetin did not suppress the phosphorylation of upstream kinase TAK1. RAW264 cells were pre-cultured as described above. The cells were then treated with 20 μM fisetin for 30 min and exposed to 1 μg/mL (for p-TAK1) or 40 ng/mL LPS for 10 (for p-TAK1) or 30 min. Whole cell lysates were harvested and analyzed by Western blot assay. The proteins were detected by their corresponding antibodies. The relative density was calculated as the intensity of the treatment relative to that of control normalized to α-tubulin or respective total proteins by densitometry. The blots presented are the examples from at least three independent experiments. Each value represents the mean ± SD of at least three separate experiments. *p < 0.05 and **p < 0.01 versus LPS-treated group.

FIGURE 4

Takinib inhibited both TAK1-MKK4-JNK1/2 and NF-κB pathway. Takinib dose-dependently inhibited the phosphorylation of TAK1 (A) and MKK4 (B). (C) Takinib inhibited the phosphorylation of JNK1/2-c-Jun and IκBα-p65. RAW264 cells were precultured as described above. The cells were then treated with Takinib at the indicated concentration for 30 min and exposed to 1 μg/mL (for p-TAK1) or 40 ng/mL LPS for 10 (for p-TAK1) or 30 min. Whole cell lysates were harvested and analyzed by Western blot assay. The proteins were detected by their corresponding antibodies. The relative density was calculated as the intensity of the treatment relative to that of control normalized to α-tubulin by densitometry. The blots presented are the examples from at least three independent experiments. Each value represents the mean ± SD of at least three separate experiments. *p < 0.05 and **p < 0.01 versus LPS-treated group.

3.3 Fisetin inhibits MKP-1 expression

Activated MAPKs can be dephosphorylated by MAPK phosphatases (MKPs).⁶ To clarify whether fisetin also influence the MKPs, the expression of MKP-1 and MKP-5 involved in dephosphorylating JNK^{40, 41} were investigated. First, a time-course study showed that LPS significantly induced JNK phosphorylation from 0.25 h, reaching peak at 0.5 h and continuing at least for 4 h. On the other hand, MKP-1 expression was observed from 1 to 12 h after LPS induction, and MKP-5 expression was not changed (Figure 5A). Treatment with 20 μM of fisetin completely inhibited LPS-induced MKP-1 expression even at 1–4 h (Figure 5B). A dose-dependent inhibition by fisetin was further observed in the concentration range of 2.5–20 μM (Figure 5C). These results suggested that the inhibitory effects of fisetin on MKK4-JNK1/2 cascade were independent of MKPs.

FIGURE 5

Fisetin inhibited MKP-1 expression. (A) Time-course experiment of LPS-induced JNK1/2 phosphorylation and MKP-1 expression. LPS did not further induce the expression of MKP-5. (B) Fisetin inhibited LPS-induced MKP-1 expression at different time points. (C) Fisetin dose-dependently suppressed LPS-induced MKP-1 expression. RAW264 cells were precultured as described above. The cells were then treated with vehicle or different doses of fisetin for 30 min and exposed to 40 ng/mL LPS. Whole cell lysates were harvested after a certain stimulation time and then analyzed by Western blot assay. The proteins were detected by their corresponding antibodies. The relative density was calculated as the intensity of the treatment relative to that of control normalized to α-tubulin by densitometry. The blots presented are the examples from at least three independent experiments. Each value represents the mean ± SD of at least three separate experiments. **p < 0.01 versus control group (A). *p < 0.05 and **p < 0.01 (B and C).

3.4 Fisetin directly binds MKK4 ex vivo and in vitro

To clarify whether fisetin specifically targets MKK4, the binding ability of fisetin to MKK4 was investigated by pull-down assay. Ex vivo pull-down assay using whole cell lysates from RAW264 cells revealed a higher binding ability of fisetin to MKK4 than α-tubulin, a negative control protein. The binding ability to JNK1 and c-Jun was almost the same as α-tubulin (Figure 6A). To further confirm the binding specificity of fisetin to MKK4, we performed in vitro pull-down assay with purified recombinant protein. As shown in Figure 6B, fisetin showed a higher binding rate to MKK4 (65.7%) than that to JNK1 (19.1%), which is consistent with the result from ex vivo pull-down assay. These data indicated that fisetin might directly bind MKK4 to suppress its phosphorylation.

FIGURE 6

Fisetin selectively bound MKK4. (A) Binding effect of fisetin in MKK4-JNK1/2-AP-1 cascade. Ex vivo pull-down assay was performed as described in Materials and Methods. Whole cell lysate (line 1, input control), lysate incubated with Sepharose 4B beads (line 2, negative control), and lysate incubated with fisetin-coupled Sepharose 4B beads (line 3) were applied to SDS-PAGE, and the proteins were detected by their corresponding antibodies. The binding rate of fisetin to α-tubulin was regarded as nonspecific binding. (B) Binding of fisetin to MKK4 or JNK1. In vitro pull-down assay was performed as described in Materials and Methods. Active kinase (line 1, input control), kinase incubated with Sepharose 4B beads (line 2, negative control), kinase incubated with fisetin-coupled Sepharose 4B beads (line 3) were applied to SDS-PAGE, and the MKK4 or JNK1 were detected by their corresponding antibodies. The relative density was calculated as the intensity of the treatment relative to that of input control by densitometry. The blots presented are the examples from at least three independent experiments. Each value represents the mean ± SD of at least three separate experiments. **p < 0.01 versus the relative binding rate of fisetin to MKK4.

3.5 Fisetin binds MKK4 competitively with ATP

To further elucidate the properties of fisetin binding to MKK4, we performed computational modeling based on the structure of MKK4 and fisetin. As shown in Figure 7A, fisetin docked to the ATP-binding pocket of MKK4, which is located in the middle of the hinge connecting the N and C lobes. Hydrogen bond is regarded as one of the most important direct noncovalent forces in protein–ligand interaction, and binding affinity of the ligand with target protein may increase one order of magnitude after forming each hydrogen bond.⁴² One hydrogen bond length 1.70 Å was formed between the 3′ positions of fisetin and Asp247 residues of MKK4, which configured the binding pocket. When fisetin superposed on MKK4, the hydrophobic surface was formed at the pocket of ATP-binding site, which allows fisetin to bind MKK4 more effectively (Figure 7A). The average binding energy is −71.75 kcal/mol. These docking data further supported our binding data ex vivo and in vitro between fisetin and MKK4.

FIGURE 7

Fisetin inhibited MKK4 in an ATP-competitive way. (A) The model of fisetin docking into the ATP-binding pocket. The probability of occurrence is 46.7% (14 out of total 30 times), with an average Udock of −71.75 kcal/mol. Molecular modeling was performed as described in Materials and Methods. Orange ribbon: MKK4, red ribbon: ATP-binding site, yellow: Fisetin. The formed hydrogen bond is showed in upper right panel, and is presented by green line in bottom right panel. Distance between atoms was measured in MOE software. (B) IC₅₀ measurement of fisetin, staurosporine, and BSJ to MKK4. (C) ATP titration suggests the ATP-competitive manner of fisetin toward MKK4. kinase reaction condition and kinase assay were performed as described in Materials and Methods. Curve was generated, and IC₅₀ was calculated using sigmoidal dose–response (variable slope) in GraphPad Prism®.

Next, we performed kinase assay to determine the inhibitory capacity of fisetin on MKK4. The ATP-competitive kinase inhibitor of MKK4, BSJ-04-122, and nonselective ATP-competitive kinase inhibitor, staurosporine, were used as the positive controls, respectively. The MKK4 was incubated with 3 μM ATP (a K_M value of ATP for MKK4) and then competed with fisetin and controls. As shown in Figure 7B, fisetin and BSJ showed a dose-dependent inhibition on MKK4 activity and the IC₅₀ against MKK4 was estimated as 2.899 μM and 21.9 nM, respectively. Furthermore, the inhibitory potency of fisetin was lost with the increased ATP concentration in the kinase reaction (Figure 7C). These data suggested that fisetin is an ATP-competitive inhibitor against MKK4.

3.6 Combination effect of fisetin and MKK4-specific inhibitor on MKK4-JNK1/2 cascade

To verify the potency of fisetin as a MKK4 inhibitor, the combination effect of fisetin and MKK4-specific inhibitor, BSJ, on MKK4-JNK1/2 signaling was investigated. As shown in Figure 8A, both fisetin and BSJ inhibited LPS-induced phosphorylation of MKK4 and JNK1/2, respectively, at the concentration range of 5–20 μM and 0.5–8 μM (Figure 8A) in a dose-dependent manner. As expected, combination treatment of fisetin and BSJ at each half of effective concentration inhibited LPS-induced phosphorylation of both MKK4 and JNK1/2 (Figure 8B). These data confirmed that fisetin specifically inhibited MKK4 phosphorylation.

FIGURE 8

Fisetin and MKK4-specific inhibitor BSJ complementarily suppressed LPS-activated MKK4-JNK1/2 cascade. (A) Fisetin or BSJ dose-dependently suppressed LPS-triggered MKK4 and JNK1/2 phosphorylation. (B) Combination effect of fisetin and BSJ on MKK4-JNK1/2 cascade. RAW264 cells were precultured as described above. The cells were then treated with vehicle or different doses of fisetin or BSJ for 30 min and exposed to LPS for 30 min (for p-MKK4 and p-JNK1/2). Whole-cell lysates were harvested and analyzed by Western blot assay. The proteins were detected by their corresponding antibodies. The relative density was calculated as the intensity of the treatment relative to that of control normalized to α-tubulin by densitometry. The blots presented are the examples from at least three independent experiments. Each value represents the mean ± SD of at least three separate experiments. *p < 0.05 and **p < 0.01 versus LPS-treated group.

4 DISCUSSION

As a typical flavonoid, fisetin occurs naturally in the plant products including strawberry, ceylon tea, and camomile.¹⁹ The anti-inflammatory effect of fisetin was reported in different models, but the molecular targets is still controversial and remained to be defined. In the present study, we found that fisetin reduced LPS-triggered proinflammatory mediators in RAW264 macrophages by selectively inhibiting MKK4-JNK1/2-AP-1 cascade.

In mechanism study, we comprehensively screened the modulation effect of fisetin on inflammation-related signal pathways. First, we investigated NF-κB pathway and found that fisetin failed to inhibit LPS-induced phosphorylation and further degradation of IκB-α, following no effect on phosphorylation and nuclear translocation of p65. EMSA data further demonstrated that fisetin did not inhibit LPS-stimulated NF-κB-DNA binding complex. The effect of fisetin on the inhibition of NF-κB pathway is controversial in previous studies. Kim, et al.²⁵ reported that fisetin did not prevent LPS-induced phosphorylation and degradation of IκBα and the nuclear translocation of p65/p50 in RAW264.7 macrophages. While immunostaining data from other studies showed that fisetin inhibited the nuclear translocation of p65 after LPS stimulation.^{24, 26} Sahu, et al.²³ reported that fisetin suppressed LPS-activated NF-κB pathway at 18 h in peritoneal macrophages. Although the contradiction is hard to be fully explained, the differences in cell culture, concentration of LPS for polarizing macrophages, especially the detection time after LPS stimulation may lead to a different outcome. Due to these reasons, we optimized the cell model in this study after reviewing these reports and challenged to identify the potential molecular targets of fisetin in RAW264 macrophages by multiple biochemical methods. Conformation of the present results in mouse primary macrophages are also under planning and will be performed in next step. MAPK pathway is another vital cascade involved in LPS-triggered immune response.⁶ We found that fisetin inhibited the binding of AP-1 to DNA. Signaling analysis demonstrated that fisetin selectively inhibited MKK4-JNK1/2 phosphorylation and suppressed the activation of transcription factor AP-1 (reflected by p-c-Jun), but did not affect other members in MAPK pathway including MKK7, MEK1/2-ERK1/2, and MKK3/6-p38. In addition, fisetin did not suppress the activation of TAK1, an upstream kinase leading to the activation of both MKK4-JNK cascade and NF-κB pathway (Figure 4). These data suggested that MKK4 might be a selective target kinase for fisetin.

Under normal physiology, there exist negative feedback mechanisms in limiting excessive MAPK activation for preventing inflammation-induced tissue damage.⁶ Activated MAPKs can be dephosphorylated by MKPs.⁶ Although murine macrophages express six MKPs, MKP-1, and MKP-5 prefer to dephosphorylate phospho-JNK.^{40, 41} Some natural products, including ursolic acid and obacunone, have been reported to induce MKP-1 expression under stress or proinflammatory stimulation, which may partly explain their anti-inflammatory ability.^{43, 44} However, we found that fisetin almost completely inhibited LPS-induced MKP-1 expression. As the feedback regulation for MAPKs, the transcription of MKP-1 depends on the MAPKs activity themselves,⁴⁵ and normal JNK1 activation is required for MKP-1 induction in LPS-stimulated macrophages.⁴⁶ Therefore, downregulation of MKP-1 by fisetin may be due to its suppression in JNK1/2-AP-1 in advance, which also supported the direct inhibition of fisetin on MKK4-JNK1/2-AP-1 cascade.

To further clarify whether fisetin specifically targets MKK4, the binding ability of fisetin to MKK4 was investigated by pull-down assay. Fisetin exhibited a higher binding ability to MKK4, compared to other proteins. Moreover, kinase assay revealed that fisetin directly inhibited MKK4 activity, although the inhibitory ability is lower than non-selective ATP-competitive kinase inhibitor staurosporine, or covalent MKK4 inhibitor BSJ. In kinase-mediated catalytic process, ATP binds in the cleft between the N-lobe and C-lobe in kinase structure, and kinases further catalyze the transfer of γ-phosphate from ATP to substrates.⁴⁷ Our molecular modeling result indicated that fisetin enters the ATP-binding pocket of MKK4 (Figure 6B). Further ATP titration validated this ATP-competitive effect because fisetin lost its inhibitory effect to MKK4 with the increased ATP concentration. Therefore, our results revealed that fisetin directly bound to MKK4 in an ATP-competitive manner.

MKK4 inhibitors play important role on controlling inflammation process. Beyond this, recent study further revealed the dominant role of MKK4 inhibitor in the regulation of liver regeneration,⁴⁸ which enhances liver regeneration and prevents liver failure. However, the selective MKK4 inhibitors are still lacking nowadays.⁴⁸ In this study, fisetin was demonstrated to be a potential selective MKK4 inhibitor. These data will not only provide new information for understanding the anti-inflammatory effects of fisetin but also provide inspiration to design new drugs using fisetin as molecular probe.

In the present study, fisetin concentration range (0 ~ 20 μM) was set up according to the previous studies in macrophages. Interestedly, a pharmacokinetics study has reported that the maximum plasma concentration (C_max) and half-life of fisetin were 0.5 ± 0.2 μg/mL (1.75 ± 0.70 μM) and 67.9 ± 24.5 min, respectively, at oral administration of 100 mg/kg fisetin, and were 10.6 ± 1.5 μg/mL (37.03 ± 5.24 μM) as well as 57.9 ± 16.1 min, respectively, at oral administration of 200 mg/kg fisetin.⁴⁹ These pharmacokinetics data suggested that the effective concentrations observed in our cell model can be achieved through the oral intake although the bioavailability of fisetin was relatively low. It is noticed that some methods for increasing the physiological levels of flavonoids are developing nowadays using drug delivery ssystems⁵⁰ or nanomaterials conjugation,⁵¹ which could enhance the physiological levels of fisetin through the oral intake.

Current studies also revealed that other natural products including procyanidin B2,⁵² dehydroglyasperin C,⁵³ or 7,3′,4′-trihydroxyisoflavone,⁵⁴ an isoflavone metabolite of the daidzein, also directly binds to MKK4 to inhibit its activation. On the other hand, fisetin also has been reported to directly inhibit PI3-kinase,⁵⁵ mTOR/p70S6K,⁵⁶ RSK (ERK effector),⁵⁷ casein kinase 2,⁵⁸ as well as c-Kit/CDK2⁵⁹ activity basing on kinase assay. Therefore, fisetin may have multiple targets of kinases. It is interesting to compare the effects of fisetin on these signal modulation effect by some omics approaches including kinome or phosphoproteomics in the future.

5 CONCLUSION

In conclusion, our study revealed that fisetin is a promising anti-inflammatory phytochemical that selectively targets and inhibits MKK4 in an ATP-competitive way to suppress MKK4-JNK1/2-AP-1 cascade-mediated inflammation.

AUTHOR CONTRIBUTIONS

Z.H., T.U., S.T., and D.H.: experimental design, experimental operation, investigation, and data analysis. Z.H: writing—original manuscript. T.K., K.X., and X.P.: methodology. K.S., S.W., Y.Y., M.K., and D.H.: supervision. D.H.: writing—manuscript review and revision. All authors read and approved the submission of final manuscript. This research was part of the dissertation submitted by the first author in partial fulfillment of a Ph.D. degree. All authors have provided consent.

FUNDING INFORMATION

This work was supported by the fund of Scholar Research of Kagoshima University (70030117) to De-Xing Hou.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

Open Research

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author.