MKK4 is a novel target for the inhibition of tumor necrosis factor-α-induced vascular endothelial growth factor expression by myricetin

biochemical pharmacology 77 (2009) 412–421

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MKK4 is a novel target for the inhibition of tumor necrosis factor-a-induced vascular endothelial growth factor expression by myricetin Jong-Eun Kim a, Jung Yeon Kwon b, Dong Eun Lee a, Nam Joo Kang a,b, Yong-Seok Heo c, Ki Won Lee b,*, Hyong Joo Lee a,** a Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, San 56-1, Shillim-dong, Gwanak-gu, Seoul 151-921, Republic of Korea b Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea c Department of Chemistry, Konkuk University, Seoul 143-701, Republic of Korea

article info

abstract

Article history:

Tumor necrosis factor-a (TNF-a) is a mediator of multiple inflammatory diseases. Vascular

Received 28 August 2008

endothelial growth factor (VEGF) plays a critical role in TNF-a-mediated diseases. We

Accepted 24 October 2008

investigated the inhibitory effects of 3,30 ,40 ,5,50 ,7-hexahydroxyflavone (myricetin), an abundant natural flavonoid, on TNF-a-induced VEGF upregulation and the underlying molecular mechanism. Myricetin is a direct inhibitor of mitogen-activated protein kinase (MAPK)/

Keywords:

extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) and inhibits neoplastic cell

Mitogen-activated protein kinase

transformation. We found that myricetin inhibited TNF-a-induced VEGF expression in

kinase 4

JB6 P+ mouse epidermal cells by targeting MAPK kinase 4 (MKK4), as well as MEK1. The

Myricetin

activation of activator protein-1 by TNF-a was inhibited by myricetin in a dose-dependent

Vascular endothelial growth factor

manner. The phosphorylation of c-Jun N-terminal kinase (JNK) and ERK was inhibited by myricetin, but not the phosphorylation of their upstream kinases MKK4 and MEK1. TNF-ainduced VEGF expression was inhibited by SP600125 and U0126, which are inhibitors of JNK and MEK, respectively. Myricetin inhibited TNF-a-induced MKK4 activity and bound glutathione S-transferase-MKK4 directly by competing with ATP. Computer modeling suggested that myricetin docks onto the ATP-binding site in MKK4, which is located between the N- and C-lobes of the kinase domain. Overall, our results indicate that myricetin has potent chemopreventive effects against TNF-a-related disease, mainly by targeting MKK4 and MEK1. # 2008 Elsevier Inc. All rights reserved.

* Corresponding author. Tel.: +82 2 2049 6178; fax: +82 2 3436 6178. ** Corresponding author. Tel.: +82 2 880 4853; fax: +82 2 873 5095. E-mail addresses: [email protected] (K.W. Lee), [email protected] (H.J. Lee). Abbreviations: AP, activator protein; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HIF-1, hypoxia inducible factor; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK1, MAPK/ extracellular signal-regulated kinase ERK kinase 1; MEM, Eagle’s minimum essential medium; MKK4, MAPK kinase 4; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Myricetin, 3,30 ,40 ,5,50 ,7-hexahydroxyflavone; NF, nuclear factor; Sp-1, specificity protein-1; TNF-a, tumor necrosis factor-a; VEGF, vascular endothelial growth factor. 0006-2952/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2008.10.027

biochemical pharmacology 77 (2009) 412–421

1.

Introduction

Tumor necrosis factor-a (TNF-a) is a cytokine that had critical roles in multiple inflammatory diseases, including cancer, sepsis, diabetes, osteoporosis, multiple sclerosis, rheumatoid arthritis, and psoriasis [1–3]. Elevated serum or tissue levels of TNF-a are commonly observed during inflammation or infection. The targeting of TNF-a is recognized as a fundamental approach for the treatment of inflammatory disease. Monoclonal antibodies against TNF-a such as infliximab and adalimumab have been used clinically to treat rheumatoid arthritis and psoriasis [4–7]. Receptor binding of TNF-a activates a number of signaling pathways, including the nuclear factor (NF)-kB, activator protein (AP)-1, mitogen-activated protein kinase (MAPK), and phosphatidylinositol (PI) 3-kinase pathways. Several agents known to inhibit the signaling pathways triggered by TNF-a have been suggested as chemopreventive agents for TNF-amediated diseases [4–7]. Vascular endothelial growth factor (VEGF), cylooxygenase-2, and inducible nitric oxide synthase, which are closely related to inflammation and carcinogenesis, are overexpressed mainly through TNF-a-activated pathways [8]. VEGF, which has a highly conserved receptor-binding cysteine-knot structure, is expressed in various cell types in response to TNF-a and is an important regulator of pathogenesis in a variety of disorders, including cancer, rheumatoid arthritis, and psoriasis [9]. AP-1 is a well-known transcription factor that regulates the expression of various inflammatory genes, including VEGF [10]. Signal cascades, including the MAPK and PI3 kinase pathways, are involved in controlling VEGF expression via the regulation of AP-1 [11,12]. The modulation of the TNF-a-triggered VEGF signaling pathway by targeting specific signaling molecules is potentially useful for the treatment of inflammatory disease. The compound 3,30 ,40 ,5,50 ,7-hexahydroxyflavone (myricetin) is an abundant natural flavonoid found in fruits, vegetables, and common drinks such as red wine or garlic, guava, and onion juice [13]. Of the various flavonoids tested, myricetin exerts relatively strong antioxidant effects [14]. Several reports have suggested that myricetin also has anti-inflammatory activity [5,15]. Myricetin inhibits the TNF-a-induced upregulation of intercellular adhesion molecule-1, a transmembrane glycoprotein related to inflammatory disease [5], and an IgE-mediated proinflammatory factor released from human mast cells [15]. Myricetin also exerts protective effects against skin tumorigenesis [16] and inhibits the growth of A549 lung cancer cells by suppressing thioredoxin reductase activity [17]. The development of small kinase inhibitors has been suggested as a useful approach to modulate the effects of flavonoids like myricetin [18]. Recent studies have also suggested that PI3-kinase and PIM1 kinase are targets of myricetin [19,20]. Myricetin directly inhibits MEK1, suppressing neoplastic transformation [21]. Here, we uncovered the mechanism underlying the inhibitory effect of myricetin on TNF-a-induced VEGF upregulation and its molecular target.

2.

Materials and methods

2.1.

Chemicals

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Myricetin was purchased from Sigma (St. Louis, MO). U0126 and SP600125 were purchased from Tocris Bioscience (Ellisville, MO). Eagle’s minimum essential medium (MEM), gentamicin, fetal bovine serum (FBS), and L-glutamine were purchased from Gibco BRL (Carlsbad, CA). Mouse TNF-a was purchased from ProSpec-Tany TechnoGene (Rehovot, Israel). An enzyme-linked immunosorbent assay (ELISA) kit for the measurement of VEGF was purchased from R&D Systems (Minneapolis, MN). Antibodies against phosphorylated (p) MEK1 (Ser217/221), pMKK4 (Ser257/Thr261), total ERK, pc-Jun (ser 63), total c-Jun, pp90-kDa ribosomal S6 kinase (p90RSK, Thr359/Ser363), total p90RSK, and total Akt were obtained from Cell Signaling Biotechnology (Beverly, MA). Antibodies against pAkt (ser 473), pERK (Thr202/Tyr204), total ERK, total MEK1, and total MKK4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). pp38 (Tyr180/Tyr182) antibody was purchased from BD bioscience (San Jose, CA). The MEK1/MKK4 kinase assay kit was purchased from Upstate Biotechnology (Lake Placid, NY). CNBr–Sepharose 4B, glutathione–Sepharose 4B, and [g-32P] ATP were obtained from GE Healthcare (Piscataway, NJ), and a protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA). G418 and the luciferase assay substrate were obtained from Promega (Madison, WI).

2.2.

Cell culture

JB6 P+ mouse epidermal cells were cultured in monolayers in 5% FBS/MEM, 2 mM L-glutamine, and 25 mg/ml gentamicin at 37 8C under 5% CO2. JB6 P+ cells stably transfected with the AP1 luciferase reporter plasmid luciferase reporter plasmid was a gift from Dr. Zigang Dong [22] and were maintained in 5% FBS/ MEM and 200 mg/ml G418.

2.3.

Determination of VEGF production

JB6 P+ cells (5  105) were cultured in 96-well plates and incubated for 48 h. The cells were then pretreated with chemicals at the indicated concentrations for 1 h before incubation with 4 ng/ml TNF-a for 18 h. The culture medium was then harvested, and the amount of VEGF was measured according to the manufacturer’s instructions. The data were normalized by the protein concentration in each sample.

2.4.

Measurement of cell viability

Cell viability was measured using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which is based on the ability of live cells to convert tetrazolium salt into purple formazan. JB6 P+ cells (5  105) were cultured in 96-well plates and incubated for 48 h before pretreatment with myricetin at the indicated concentrations for 1 h. The cells were then incubated with 4 ng/ml TNF-a for 18 h. Next, 20 ml of MTT stock solution (5 mg/ml, Sigma, St. Louis, MO) was added to each well, and the plates were further incubated for 4 h at 37 8C. The supernatant was then removed, and 200 ml of DMSO

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was added to each well to solubilize the water-insoluble purple formazan crystals. The absorbance at 570 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, CA). All measurements were performed in triplicate. The results are expressed as the percent proliferation with respect to untreated cells.

2.5.

continuously for another 3 h at 4 8C. The beads were then washed three times with kinase buffer (20 mM MOPS [pH 7.2], 25 mM b-glycerol phosphate, 5 mM EGTA, 1 mM Na3VO4, and 1 mM DTT). For MEK1, the mixture was supplemented with 1 mg of inactive ERK2 and incubated for an additional 30 min at 30 8C. Subsequently, 20 mg of MBP and 10 ml of a diluted

Luciferase assay for AP-1 transcriptional activity

Confluent monolayers of JB6 P+ cells stably transfected with an AP-1 luciferase reporter plasmid were trypsinized, and 8  103 viable cells suspended in 100 ml of 5% FBS/MEM were added to each well of a 96-well plate. The plates were then incubated at 37 8C in a humidified atmosphere of 5% CO2. When the cells reached 80–90% confluence, they were starved by culturing them in 0.1% FBS/MEM for another 24 h. The cells were then treated with myricetin for 15 min and exposed to 4 ng/ml TNF-a for an additional 3 h. After treatment, the cells were disrupted with 100 ml of lysis buffer (0.1 M potassium phosphate buffer [pH 7.8], 1% Triton X-100, 1 mM dithiothreitol [DTT], and 2 mM EDTA), and luciferase activity was measured using a Microlumat Plus LB 96 V luminometer (Berthold Technologies, Bad Wildbad, Germany).

2.6.

Western blotting

JB6 P+ cells (1  106) were cultured in 10 cm diameter dishes for 48 h and then starved in serum-free medium for another 24 h to eliminate the influence of FBS on kinase activation. The cells were treated with 0–10 mM myricetin for 1 h before exposure to 4 ng/ml TNF-a. After centrifugation, cell lysis was performed at 4 8C for 30 min in cell lysis buffer (20 mM Tris– HCl [pH 7.5], 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 mg/ml leupeptin, and 1 mM PMSF). The lysates were then centrifuged at 23,000  g for 15 min, and the resulting supernatants were stored at 70 8C prior to Western blotting. The protein concentration in each sample was measured by subjecting 30 mg of protein to 10% SDS-PAGE, followed by electrophoretic transfer to nitrocellulose membranes (Whatman Inc., Clifton, NJ). The protein bands were visualized using a chemiluminescence detection kit (GE Healthcare) after hybridization with horseradish peroxidaseconjugated secondary antibodies.

2.7. MKK4 and MEK1 immunoprecipitation and kinase assays JB6 P+ cells were cultured to 80% confluence and then serumstarved in 0.1% FBS/MEM for 24 h at 37 8C. The cells were then either treated or not treated with 0–10 mg/ml myricetin for 1 h before being exposed to 4 ng/ml TNF-a for 15 min, disrupted with lysis buffer (20 mM Tris–HCl [pH 7.4], 1 mM EDTA, 150 mM NaCl, 1 mM EGTA, 1% Nonidet P-40, 1 mM bglycerophosphate, 1 mg/ml leupeptin, 1 mM Na3VO4, and 1 mM PMSF), and centrifuged at 14,000 rpm for 10 min in a microcentrifuge. Lysates containing 500 mg of protein were used for immunoprecipitation with antibodies against MEK1 or MKK4 and incubated at 4 8C overnight. Protein A/G Plus agarose beads were then added, and the mixture was rotated

Fig. 1 – Structure of myricetin and the TNF-a-induced upregulation of VEGF in JB6 P+ cells. (A) Chemical structure of myricetin. (B and C) Dose- and time-dependent upregulation of VEGF in TNF-a-treated JB6 P+ cells. The cells were seeded into 96-well plates, cultured to 70–80% confluence with 5% FBS/MEM, and then starved by replacing the medium with 0.1% FBS/MEM for 24 h. The cells were then exposed to various doses of TNF-a (B) for various time periods (C). The conditioned medium was then collected and analyzed for VEGF expression using ELISA as described in Section 2. VEGF is expressed as the percentage relative to that in untreated controls. The data are presented as the mean W S.D. as determined from three independent experiments.

biochemical pharmacology 77 (2009) 412–421

415

[g-32P]ATP solution were added, and the mixture was incubated for 10 min at 30 8C. For MKK4, the mixture was supplemented with 2 mg of inactive JNK 1a1. A 10-ml aliquot was removed following 30 min of incubation at 30 8C in the presence of 25 mg of ATF-2 and 10 ml of a diluted [g-32P]ATP solution. A 20-ml aliquot was transferred to p81 paper and washed three times with 0.75% phosphoric acid for 5 min each and once with acetone for 2 min. Radioactive incorporation was determined using a scintillation counter.

2.8.

Pull-down assay

Myricetin–Sepharose 4B beads were prepared as described previously [21]. Recombinant MKK4 (2 mg) or a JB6 P+ supernatant fraction (500 mg) was incubated with myricetin– Sepharose 4B (or Sepharose 4B alone as a control) beads (100 ml, 50% slurry) in a reaction buffer containing 50 mM Tris (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% Nonidet P-40, 2 mg/ml bovine serum albumin, 0.02 mM PMSF, and 1 mg of protease inhibitor mixture. After incubation with gentle rocking overnight at 4 8C, the beads were washed five times with 50 mM Tris (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% Nonidet P-40, and 0.02 mM PMSF, and the proteins bound to the beads were analyzed by immunoblotting.

2.9.

ATP and myricetin competition assay with MKK4

Active MKK4 (2 mg) was incubated with 100 ml of myricetin– Sepharose 4B or Sepharose 4B beads in the reaction buffer described in the previous section for 12 h at 4 8C; subsequently, ATP was added at 0, 100, or 1 mM in a final volume of 500 ml, and the mixture was incubated for 30 min. The samples were washed, and the proteins were detected by Western blotting.

2.10.

Molecular modeling

The homology model structure of MKK4 was generated by Geno3D (http://geno3d-pbil.ibcp.fr) using the coordinates for MKK7 (PDB accession code 2DYL) as a template. Insight II (Accelrys Inc., San Diego, CA) was used for the docking study and structural analysis.

2.11. Fig. 2 – Effects of myricetin on TNF-a-induced VEGF upregulation and AP-1 activation in JB6 P+ cells. (A and B) Myricetin inhibits TNF-a-induced VEGF upregulation in JB6 P+ cells. JB6 P+ cells were seeded into 96-well plates, cultured to 70–80% confluence with 5% FBS/MEM, and then starved by replacing the medium with 0.1% FBS/MEM for 24 h. The cells were then treated with the indicated concentration of myricetin (A) for 1 h before treatment with 4 ng/ml TNF-a for 18 h. The conditioned medium was then collected and analyzed for VEGF expression using ELISA as described in Section 2. (B) The effect of myricetin on JB6 P+ cell viability. JB6 P+ cells were treated with 2.5, 5, or 10 mM myricetin for 1 h before treatment with 4 ng/ml TNF-a for 18 h. Cell viability was measured using the MTT assay as described in Section 2. (C) Myricetin inhibits TNFa-induced AP-1 transactivation. For the luciferase assay,

Statistical analysis

The data are expressed as the mean  S.D. Student’s t-test was used for single statistical comparisons, with a probability of p < 0.05 as the criterion for statistical significance.

JB6 P+ cells stably transfected with an AP-1 luciferase reporter plasmid were cultured as described in Section 2. The cells were then starved in MEM containing 0.1% FBS and treated or not treated with 2.5, 5, or 10 mM myricetin for 1 h before treatment with 4 ng/ml TNF-a for 3 h. The luciferase activity was then measured. AP-1 activity is expressed relative to the level in control cells (without TNFa treatment). The data are presented as the mean W S.D. as determined from three independent experiments. Asterisks indicate a significant difference compared to cells treated with TNF-a only (*p < 0.05, **p < 0.01).

416 3.

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Results

3.1. Dose- and time-dependent VEGF upregulation by TNF-a in JB6 P+ cells To optimize the concentration and time conditions for the TNFa-induced upregulation of VEGF in JB6 P+ cells, we examined VEGF expression at various times and doses of TNF-a. The stimulatory effect of TNF-a on VEGF expression was measured between 1 and 8 ng/ml (Fig. 1B) and for 6–24 h (Fig. 1C). The greatest effect was observed following exposure to 4 ng/ml TNFa for 18 h (Fig. 1C) although it is not statistically significance.

3.2. Inhibition of the TNF-a-induced upregulation of VEGF in JB6 P+ cells by myricetin We examined the effect of myricetin on TNF-a-induced VEGF expression in JB6 P+ cells using the conditions described above. Myricetin inhibited the TNF-a-induced expression of VEGF in a dose-dependent manner (Fig. 2A). The results of an MTT assay indicated that myricetin had no effect on cell viability between 2.5 and 10 mM (Fig. 2B), suggesting that myricetin inhibits the upregulation of VEGF without affecting cell viability.

3.3.

Effects of myricetin on AP-1 transactivation

Several transcription factors, including AP-1, nuclear factor kB (NF-kB), specificity protein-1 (Sp-1), and hypoxia inducible factor (HIF)-1, are reportedly influenced by VEGF expression [10]. To evaluate the effects of myricetin on these transcription factors, we examined the protein expression and DNA-binding activity of HIF-1a and Sp-1. Neither transcription factor was activated by TNF-a in JB6 P+ cells (data not shown). We measured the effects of myricetin on the transactivation of AP-1 and NF-kB in TNF-a-treated JB6 P+ cells using a luciferase reporter gene assay. AP-1 and NF-kB was activated by TNF-a. TNF-a-induced AP-1 activation was inhibited by myricetin (Fig. 2C). However, myricetin had no effect on TNF-a-induced NF-kB activation (data not shown).

3.4.

Effects of myricetin on MAP kinases and Akt

We examined the effects of myricetin on various MAP kinases and Akt, which are representative upstream regulators of AP1. Myricetin inhibited the TNF-a-induced phosphorylation of JNK and ERK in a dose-dependent manner. It also inhibited the phosphorylation of the c-Jun and p90RSK, which are substrates of JNK and ERK, respectively. However, the phosphorylation of MKK4, an upstream kinase of JNK, was increased by myricetin, and the phosphorylation of MEK, which acts upstream of ERK, was slightly inhibited in the presence of 10 mM myricetin (Fig. 3A and B). Myricetin cannot inhibit TNF-a-induced phosphorylation of p38 or Akt (Fig. 3C).

3.5. JNK and ERK mediate the induction of VEGF by TNF-a in JB6 P+ cells We next examined the involvement of JNK and ERK signaling in the TNF-a-induced upregulation of VEGF in JB6 P+ cells.

Fig. 3 – The effects of myricetin on the TNF-a-induced phosphorylation of ERK and JNK. (A) Myricetin inhibited the TNF-a-induced phosphorylation of JNK and c-Jun, but not MKK4. (B) Myricetin inhibited the TNF-a-induced phosphorylation of ERK and p90RSK, but not MEK. (C) Myricetin did not inhibit the TNF-a-induced phosphorylation of Akt and p38. The cells were pretreated with 2.5, 5, or 10 mM myricetin for 1 h, then stimulated with 4 ng/ml TNF-a and harvested after 15 min (pMKK4, pJNK, pMEK, pERK, pp90RSK, and pp38), 1 h (pAkt), or 4 h (pc-Jun). The phosphorylated and total protein levels were determined by Western blotting using antibodies against the corresponding phosphorylated and total proteins as described in Section 2.

biochemical pharmacology 77 (2009) 412–421

Fig. 4 – JNK and ERK activation is required for TNF-ainduced VEGF expression. (A and B) JB6 P+ cells were seeded into 96-well plates, cultured to 70–80% confluence with 5% FBS/MEM, and then starved by replacing the medium with 0.1% FBS/MEM for 24 h. The cells were then treated with the indicated doses of the JNK inhibitor SP600125 (A) or the MEK1 inhibitor U0126 (B) for 1 h before treatment with 4 ng/ml TNF-a for 18 h. The data are presented as the mean W SD as determined from three independent experiments. Asterisks indicate a significant difference compared to cells treated with TNF-a only (*p < 0.05, **p < 0.01).

SP600125, a JNK inhibitor, and U0126, a MEK inhibitor, effectively inhibited TNF-a-induced VEGF upregulation in JB6 P+ cells (Fig. 4A and B), suggesting the critical role of JNK and MEK signaling in TNF-a-induced VEGF upregulation.

3.6. Myricetin inhibits TNF-a-induced MKK4 and MEK1 activity MKK4 plays an important role in TNF-a-induced JNK activation [23]. Because our results indicated that myricetin inhibits JNK phosphorylation but increases MKK4 phosphorylation, we predicted that myricetin would inhibit MKK4 activity. Myricetin inhibited TNF-a-stimulated MKK4 activity in JB6 P+ cells in a dose-dependent manner (Fig. 5A). It also suppressed the TNF-a-induced phosphorylation of ERK, but not MEK1. We investigated whether the myricetin-induced suppression of

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Fig. 5 – Myricetin inhibits TNF-a-induced MKK4 and MEK1 activity. (A and B) JB6 P+ cells were pretreated with myricetin at the indicated concentrations for 1 h, stimulated with 4 ng/ml TNF-a for 15 min, and harvested. Immunoprecipitation for MKK4 (A) or MEK1 (B) was performed as described in Section 2. MKK4 and MEK1 activity is expressed as the percentage relative to that in untreated controls. The data are presented as the mean W S.D. as determined from three independent experiments. Asterisks indicate significant differences compared to cells treated with TNF-a only (*p < 0.05, **p < 0.01).

VEGF involves the direct inhibition of MEK1 activity. Myricetin dose-dependently suppressed TNF-a-induced MEK1 activity in JB6 P+ cells (Fig. 5B).

3.7.

Myricetin binds MKK4 in competition with ATP

To determine whether myricetin binds directly to MKK4, we performed in vitro and ex vivo pull-down assays. Active MKK4 was pulled down by myricetin–Sepharose 4B beads, but not by Sepharose 4B beads alone (Fig. 6A). We also observed the ex vivo binding of myricetin and MKK4 in JB6 P+ cell lysates (Fig. 6B). Furthermore, ATP competed with myricetin for binding with MKK4 (Fig. 6C). These results suggest that myricetin inhibits MKK4 activity competitively with ATP. Direct binding between myricetin and MEK in vitro and ex vivo was shown previously [21]. Thus, MKK4 and MEK are molecular targets of myricetin.

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Fig. 6 – Myricetin binds MKK4 directly. (A) Myricetin specifically binds MKK4 in vitro. The in vitro binding of MKK4 with myricetin was confirmed by immunoblotting using antibodies against MKK4. Lane 1, MKK4 standard (input control); lane 2, Sepharose 4B was used to pull down MKK4 as described in Section 2 (negative control); lane 3, MKK4 was pulled down using myricetin–Sepharose 4B beads. (B) Myricetin specifically binds MEK4 ex vivo. The ex vivo binding of MKK4 with myricetin was confirmed by immunoblotting using antibodies against MKK4. Lane 1, whole-cell lysate from JB6 P+ cells (input control); lane 2, a JB6 P+ cell lysate precipitated with Sepharose 4B beads as described in Section 2 (negative control); lane 3, a wholecell lysate from JB6 P+ cells precipitated with myricetin– Sepharose 4B beads as described in Section 2. (C) Myricetin competes with ATP for binding to MKK4. Active MEK4 (0.25 mg) was incubated with 0, 1, or 10 mM ATP and 50 ml of myricetin–Sepharose 4B or Sepharose 4B (negative control) beads in reaction buffer (final volume of 500 ml). The mixture was incubated at 4 8C overnight with shaking. After washing, the proteins were detected by Western blotting. Lane 2, negative control (MKK4 could not bind Sepharose 4B); lane 3, positive control (MKK4 successfully bound myricetin-Sepharose 4B); lanes 4–6 (myricetin–MEK4 binding was unaffected by the concentration of ATP).

4.

Discussion

Increasing evidence suggests that VEGF plays an important role in TNF-a-mediated inflammatory disease. The inhibition of TNF-a-induced VEGF upregulation and its related signaling is a potential target for the treatment of such diseases. Myricetin, an abundant dietary flavonoid, inhibited the TNF-ainduced upregulation of VEGF via the direct inhibition of MEK and MKK4 activity. AP-1 is a dimeric transcription factor composed of proteins from the c-Fos, c-Jun, ATF, and JDP families [25,26]. AP-1 activation is required for VEGF production in response to a variety of extracellular stimuli [10]. Myricetin inhibited TNF-a-induced AP-1 transactivation in JB6

P+ cells, thereby downregulating VEGF. MAPK signaling is critical for AP-1 regulation [25]. JNK is a key regulator of c-Jun and is essential for AP-1 activation [27]. Phosphorylation by MKK4 of the Thr-Pro-Tyr motif located in the activation loop activates JNK [28]. Although MKK4 activates both JNK and p38 MAPK, the deletion of MKK4 affects TNF-a-induced JNK, but not p38, activation [29]. Therefore, we investigated the effects of myricetin on the MKK4-JNK-c-Jun pathway. The phosphorylation of JNK and c-Jun was suppressed by myricetin; in contrast, the phosphorylation of MKK4 was increased in a dose-dependent manner. Because pharmacological inhibitors sometimes increase the phosphorylation of their target kinases via a feedback loop [30], we assumed that MKK4 was a possible target of myricetin. Myricetin inhibited MKK4 activity through direct binding in an ATP-competitive manner. Using computer modeling, we studied the mode of binding of myricetin to MKK4. To investigate the molecular basis of MKK4 inhibition by myricetin, we performed a docking study using a homology model structure of the MKK4 kinase domain derived from the crystal structure of MKK7, which has 66% homology with MKK4 in terms of its amino acid sequence. The kinase domain of MKK4 consists of an N-lobe and a C-lobe, which are linked through a loop known as the hinge region. The backbone of this loop interacts with the adenine moiety in ATP via hydrogen bonds. Considering the finding that myricetin is an ATP-competitive inhibitor of MKK4, we docked the compound onto the ATP-binding site of MKK4. Myricetin fit easily onto the ATP-binding site of MKK4, located between the N- and C-lobes of the kinase domain (Fig. 7). Myricetin can form hydrogen bonds with the backbone of the hinge region in MKK4, as do other ATP-competitive kinase inhibitors. The hydroxyl group at the 30 position acts as a hydrogen bond donor in the interaction with the carbonyl of Glu179. The hydroxyl group at the 40 position functions as a hydrogen bond donor in the interaction with the carbonyl of Met181; at the same time, it can also function as a hydrogen bond acceptor in the interaction with the amide group of Met181. The carbonyl group at position 4 and the hydroxyl group at position 5 for hydrogen bonds with the side chains of Ser233 and Lys187, respectively. In addition, the inhibitor is sandwiched by the side chains of the hydrophobic residues in the ATP-binding site (e.g., Ala120, Met178, Ile108, Val116, Cys156, Leu236, and Met181). The surface of the putative myricetin-binding site in MKK4 accommodates the compound without any steric collision, leading to the high level of activity of the inhibitor against MKK4 (Fig. 7). MKK7 also regulates JNK; however, we were unable to study MKK7 because we were unable to detect phosphorylation of MKK7 by Western blotting. The c-Fos, a component of the AP-1 dimer, is a substrate of ERK and p90RSK. The blocking of ERK activity using dominantnegative ERK2 or the MEK1 inhibitor U0126 repressed AP-1 transactivation in response to extracellular stimuli [31] and inhibited the TNF-a-induced upregulation of VEGF in JB6 P+ cells. Myricetin inhibits the TPA- and EGF-induced phosphorylation of ERK, but not MEK [21]. In an in vitro kinase assay, myricetin at 1 or 5 mM suppressed MEK1 activity by 29 and 88%, respectively, and ERK2 activity to a lesser degree. Furthermore, myricetin inhibits TPA- and EGF-induced MEK1 activity ex vivo in a dose-dependent manner non-competitively with ATP [21]. Here, we found that myricetin suppressed TNF-a-induced

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Fig. 7 – Hypothetical model of the MKK4 kinase domain/myricetin complex. (A) Myricetin (atomic color) binds the ATPbinding site in the kinase domain of MKK4. Hydrogen bonds are indicated by white lines; hydrophobic contacts are indicated by small curves. (B) Myricetin binding to the ATP-binding cleft represented as an electrostatic potential surface. (C) Simplified depiction of the effects of myricetin on TNF-a-induced VEGF expression.

MEK1 activity. Therefore, myricetin can act as an MEK inhibitor against various stimuli. Using computer modeling, we analyzed the mode of binding with MEK1 of quercetin, which has one less hydroxyl group at the 50 position than myricetin [24]. Because of the rotation of the B ring, the hydroxyl groups at the 50 and 30 positions are not distinct. Moreover, the opposing hydroxyl group does not hinder the

binding of myricetin to MEK1. Therefore, myricetin binds with MEK1 in a fashion similar to quercetin. Further studies using X-ray crystallography are needed to determine the inhibitorcomplex structure and elucidate the exact mode of binding of myricetin to MKK4 and MEK1. Given the clinical success of Gleevec, multitarget kinase inhibitors have received much attention. The broad reactivity

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of these inhibitors means that they may be applicable for a number of conditions. For example, Gleevec, which was originally developed for the treatment of chronic myelogenous leukemia by targeting BCR-ABL kinase, can also be used to treat gastrointestinal stromal tumors by inhibiting KIT kinase. The combinatorial inhibition provided by multitarget kinase inhibitors may synergistically increase their effects. For instance, the dual PI3K-mTOR inhibitor PI-103 is more effective than the inhibitors of either kinase alone [32]. Thus, multitarget kinase inhibitors may have broader applications than kinase inhibitors that have one specific target. Myricetin is generally regarded as safe because of its longtime use in food. Therefore, the potential applications and efficacy of myricetin are of great interest. Moreover, additional novel targets of myricetin may be found in the future. In summary, the interaction of myricetin with MKK4 and MEK1 suppresses their activity and inhibits downstream JNK and ERK signaling, leading to the suppression of TNF-a-induced VEGF expression (Fig. 7C). Our results indicate that myricetin may have potent inhibitory effects against TNF-a-mediated diseases.

[7]

[8] [9] [10]

[11]

[12]

[13]

Acknowledgements [14]

This work was supported by research grants from the BioGreen 21 Program (no. 20070301-034-027), Rural Development Administration, and the Korea Science and Engineering Foundation (no. R01-2008-000-12200-0), Ministry of Science and Technology, Republic of Korea.

Appendix A. Supplementary data

[15]

[16]

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bcp.2008.10.027.

references [17]

[1] Tweedie D, Sambamurti K, Greig NH. TNF-inhibition as a treatment strategy for neurodegenerative disorders: new drug candidates and targets. Current Alzheimer Research 2007;4:378–85. [2] Clark IA. How TNF was recognized as a key mechanism of disease. Cytokine & Growth Factor Reviews 2007;18:335–43. [3] Feldmann M, Maini RN. Anti-TNFa therapy of rheumatoid arthritis: what have we learned? Annual Review of Immunology 2001;163–96. [4] Takada Y, Aggarwal BB. Flavopiridol inhibits NF-kB activation induced by various carcinogens and inflammatory agents through inhibition of IkBa kinase and p65 phosphorylation. Abrogation of cyclin D1, cyclooxygenase-2, and matrix metalloprotease-9. Journal of Biological Chemistry 2004;279:4750–9. [5] Chen CC, Chow MP, Huang WC, Lin YC, Chang YJ. Flavonoids inhibit tumor necrosis factor-a-induced upregulation of intercellular adhesion molecule-1 (ICAM-1) in respiratory epithelial cells through activator protein-1 and nuclear factor-kB: Structure-activity relationships. Molecular Pharmacology 2004;66:683–93. [6] Manna SK, Mukhopadhyay A, Van NT, Aggarwal BB. Silymarin suppresses TNF-induced activation of NF-kB, c-

[18]

[19]

[20]

[21]

[22]

[23]

Jun N-terminal kinase, and apoptosis. Journal of Immunology 1999;163:6800–9. Paneas J, Gerritsen ME, Anderson DC, Miyasaka M, Granger DN. Apigenin inhibits tumor necrosis factor-induced intercellular adhesion molecule-1 upregulation in vivo. Microcirculation 1996;3:279–86. Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death and Differentiation 2003;10:45–65. Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocrine Reviews 2004;25:581–611. Joseko J, Mazurek M. Transcription factors having impact on vascular endothelial growth factor (VEGF) gene expression in angiogenesis. Medical Science Monitor 2004;10. Huang C, Li J, Song L, Zhang D, Tong Q, Ding M, et al. Black raspberry extracts inhibit benzo(a)pyrene diol-epoxideinduced activator protein 1 activation and VEGF transcription by targeting the phosphotidylinositol 3kinase/Akt pathway. Cancer Research 2006;66:581–7. Lee CC, Chen SC, Tsai SC, Wang BW, Liu YC, Lee HM, et al. Hyperbaric oxygen induces VEGF expression through ERK, JNK and c-Jun/AP-1 activation in human umbilical vein endothelial cells. Journal of Biomedical Science 2006;13:143–56. Harnly JM, Doherty RF, Beecher GR, Holden JM, Haytowitz DB, Bhagwat S, et al. Flavonoid content of U.S. fruits, vegetables, and nuts. Journal of Agricultural and Food Chemistry 2006;54:9966–77. Pietta PG. Flavonoids as antioxidants. Journal of Natural Products 2000;63:1035–42. Kempuraj D, Madhappan B, Christodoulou S, Boucher W, Cao J, Papadopoulou N, et al. Flavonols inhibit proinflammatory mediator release, intracellular calcium ion levels and protein kinase C theta phosphorylation in human mast cells. British Journal of Pharmacology 2005;145:934–44. Mukhtar H, Das M, Khan WA, Wang ZY, Bik DP, Bickers DR. Exceptional activity of tannic acid among naturally occurring plant phenols in protecting against 7,12dimethylbenz(a)anthracene-, benzo(a)pyrene-, 3methylcholanthrene-, and N-methyl-N-nitrosoureainduced skin tumorigenesis in mice. Cancer Research 1988;48:2361–5. Lu J, Papp LV, Fang J, Rodriguez-Nieto S, Zhivotovsky B, Holmgren A. Inhibition of mammalian thioredoxin reductase by some flavonoids: implications for myricetin and quercetin anticancer activity. Cancer Research 2006;66:4410–8. Williams RJ, Spencer JPE, Rice-Evans C. Flavonoids: antioxidants or signalling molecules? Free Radical Biology and Medicine 2004;36:838–49. Walker EH, Pacold ME, Perisic O, Stephens L, Hawkins PT, Wymann MP, et al. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Molecular Cell 2000;6:909–19. Holder S, Zemskova M, Zhang C, Tabrizizad M, Bremer R, Neidigh JW, et al. Characterization of a potent and selective small-molecule inhibitor of the PIM1 kinase. Molecular Cancer Therapeutics 2007;6:163–72. Lee KW, Kang NJ, Rogozin EA, Kim H-G, Cho YY, Bode AM, et al. Myricetin is a novel natural inhibitor of neoplastic cell transformation and MEK1. Carcinogenesis 2007;28:1918–27. Huang C, Li J, Ma WY, Dong Z. JNK activation is required for JB6 cell transformation induced by tumor necrosis factor-a but not by 12-O-tetradecanoylphorbol-13-acetate. Journal of Biological Chemistry 1999;274:29672–6. Whitmarsh AJ, Davis RJ. Role of mitogen-activated protein kinase kinase 4 in cancer. Oncogene 2007;26:3172–84.

biochemical pharmacology 77 (2009) 412–421

[24] Lee KW, Kang NJ, Heo Y-S, Rogozin EA, Pugliese A, Hwang MK, et al. Raf and MEK protein kinases are direct molecular targets for the chemopreventive effect of quercetin, a major flavonol in red wine. Cancer Research 2008;68:946–55. [25] Eferl R, Wagner EF. AP-1: a double-edged sword in tumorigenesis. Nature Reviews Cancer 2003;3:859–68. [26] Shaulian E, Karin M. AP-1 as a regulator of cell life and death. Nature Cell Biology 2002;4. [27] Jaeschke A, Karasarides M, Ventura J-J, Ehrhardt A, Zhang C, Flavell RA, et al. JNK2 is a positive regulator of the cJun transcription factor. Molecular Cell 2006;23:899–911. [28] Weston CR, Davis RJ. The JNK signal transduction pathway. Current Opinion in Genetics and Development 2002;12:14–21.

421

[29] Brancho D, Tanaka N, Jaeschke A, Ventura JJ, Kelkar N, Tanaka Y, et al. Mechanism of p38 MAP kinase activation in vivo. Genes and Development 2003;17:1969–78. [30] Harrington LS, Findlay GM, Lamb RF. Restraining PI3K: mTOR signalling goes back to the membrane. Trends in Biochemical Sciences 2005;30:35–42. [31] Watts RG, Huang C, Young MR, Li JJ, Dong Z, Pennie WD, et al. Expression of dominant negative Erk2 inhibits AP-1 transactivation and neoplastic transformation. Oncogene 1998;17:3493–8. [32] Vogt PK, Kang S. Kinase inhibitors: vice becomes virtue. Cancer Cell 2006;9:327–8.