Targeting tumor necrosis factor (TNF)-α-mediated signal pathways may be a promising strategy for developing chemopreventive agents, because TNF-α-mediated cyclooxygenase (COX)-2 expression plays a key role in inflammation and carcinogenesis. Luteolin [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone] exerts anticarcinogenic effects, although little is known about the underlying molecular mechanisms and specific targets of this compound. In the present study, we found that luteolin inhibited TNF-α-induced COX-2 expression by down-regulating the transactivation of nuclear factor-κB and activator protein-1. Furthermore, luteolin inhibited TNF-α-induced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/ERK/p90RSK, mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase/c-Jun, and Akt/p70S6K. However, it had no effect on the phosphorylation of p38. These effects of luteolin on TNF-α-mediated signaling pathways and COX-2 expression are similar to those achieved by blocking tumor progression locus 2 serine/threonine kinase (TPL2) using pharmacologic inhibitors and small interfering RNAs. Luteolin inhibited TPL2 activity in vitro and in TPL2 immunoprecipitation kinase assays by binding directly in an ATP-competitive manner. Overall, these results indicate that luteolin exerts potent chemopreventive activities, which primarily target TPL2.
Tumor necrosis factor (TNF)-α is one of the most important mediators of inflammation and carcinogenesis (Balkwill and Mantovani, 2001). TNF-α was originally identified as an antitumor agent. However, multiple lines of evidence suggest that TNF-α is an endogenous tumor promoter (Balkwill, 2006). TNF-α can induce anchorage-independent growth of mouse epidermal P+ cells (Huang et al., 1999). TNF-α and TNF-α receptor (TNFR) 1-deficient mice are resistant to chemical-induced skin (Moore et al., 1999; Arnott et al., 2004) and liver carcinogenesis (Knight et al., 2000). TNF-α is usually produced by immune cells and binds to TNFR1 (p55 receptor) or TNFR2 (p75 receptor), and the subsequent association of adaptor proteins with TNFR leads to the activation of downstream signaling cascades, including the mitogen-activated protein kinase (MAPK) pathway. Transcription factors, such as nuclear factor κB (NF-κB) and activator protein-1 (AP-1), are activated by TNF-α and play key roles in TNF-α-induced carcinogenesis by up-regulating proinflammatory proteins, such as cyclooxygenase (COX)-2 (Pettus et al., 2003; Aggarwal et al., 2006).
COX is a rate-limiting enzyme for the production of prostaglandins. Two forms of COX have been identified: the constitutively expressed form, COX-1, and the inducible form, COX-2. COX-2 is expressed at low or negligible levels, although it can be dramatically induced in cells after activation by cytokines or growth factors. COX-2 plays crucial roles in inflammation, tumorigenesis, and angiogenesis (Harris, 2007), and it is reported to be overexpressed in many types of cancer (Harris, 2007). The development of intestinal tumors and skin papillomas was reduced in a cox-2 knockout mouse (Harris, 2007). Moreover, it has been demonstrated that pharmacologic inhibition of COX-2 impairs tumor formation in several animal models (Chun and Surh, 2004). Therefore, suppression of COX-2 expression seems to be a promising target for protecting against carcinogenesis.
Tumor progression locus 2 serine/threonine kinase (TPL2), also known as Cot, was initially cloned as a provirus insertion site in Moloney murine leukemia virus-induced T cell lymphomas in rats (Erny et al., 1996). TPL2 is a protooncogene that is overexpressed in various cancer types, including breast cancer, gastric cancer, colonic adenocarcinomas, Epstein-Barr virus-related Hodgkin lymphomas, and nasopharyngeal carcinomas (Sourvinos et al., 1999; Kikuchi et al., 2003; Christoforidou et al., 2004; Clark et al., 2004). Moreover, TPL2 is required for epidermal growth factor-induced neoplastic transformation, and TPL2 overexpression promotes the proliferation and transformation of mouse embryonic fibroblasts (Chiariello et al., 2000; Choi et al., 2008). Our recent study shows that TPL2 is required for arsenite-induced COX-2 expression (Lee et al., 2009).
TPL2 is an important mediator of TNF-α-induced signaling pathways. TPL2-deficient mice are resistant to TNF-α-induced, rapid, and slowly developing inflammatory syndrome and bowel disease (Kontoyiannis et al., 2002). TNF-α activates TPL2 through the TNFR1/TNF receptor-associated factor-2)/receptor-interacting kinase-1/Syk pathway (Eliopoulos et al., 2006). Activated TPL2 phosphorylates MEK and MKK4 as a mitogen-activated protein kinase kinase kinase (MAP3K). Downstream transcription factors, such as NF-κB and AP-1, are activated and subsequently induce the expression of inflammatory proteins, such as COX-2 (Vougioukalaki et al., 2011).
Luteolin [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone] (Fig. 1A) is a flavonoid that is found abundantly in green peppers, perilla, celery, and chamomile tea. Previous studies have demonstrated the cancer-chemopreventive effects of luteolin. Luteolin induces the apoptosis of many types of cancer cells. It impairs various types of stimuli induced by proinflammatory gene expression and chemical-induced breast, colon, and skin carcinogenesis (Lin et al., 2008). However, the underlying molecular mechanisms and target proteins involved in the suppression of carcinogenesis by luteolin are not fully understood. In the present study, we report that luteolin inhibits TNF-α-induced COX-2 expression by directly targeting TPL2 in JB6 P+ mouse skin epidermal cells. To our knowledge, the present study is the first to suggest that luteolin is an inhibitor of TPL2 activity and TPL2 is required for TNF-α-induced COX-2 expression.
Materials and Methods
Luteolin was purchased from Sigma-Aldrich (St. Louis, MO). Minimum essential medium (MEM), fetal bovine serum (FBS), Gentamicin, l-glutamine, and penicillin/streptomycin were obtained from Invitrogen (Carlsbad, CA). The TPL2 inhibitor [TPL2i; 4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine] was obtained from Calbiochem (San Diego, CA). The anti-COX-2 antibody was obtained from Cayman Chemical (Ann Arbor, MI), and the anti-β-actin antibody was purchased from Sigma-Aldrich. Antibodies against phosphorylated ERK1/2 (Thr202/Tyr204), total ERK1/2, and total JNK1 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies against total p38 and phosphorylated JNK (Thr183/Tyr185) were obtained from Cell Signaling Biotechnology (Danvers, MA). The antiphosphorylated p38 (Tyr180/Tyr182) antibody was purchased from BD Biosciences (San Jose, CA). The active TPL2 protein was obtained from Millipore (Billerica, MA). Anti-TPL2 and antiphosphorylated TPL2 were obtained from Abcam Inc. (Cambridge, MA). Cyanogen bromide-Sepharose 4B, [γ-32P]ATP, and the chemiluminescence detection kit were purchased from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK). The protein assay kit was obtained from Bio-Rad Laboratories (Hercules, CA). G418 [(2R,3S,4R,5R,6S)-5-amino-6-[(1R,2S,3S,4R,6S)-4,6-diamino-3-[(2R,3R,4R,5R)-3,5-dihydroxy-5-methyl-4-methylaminooxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-2-(1-hydroxyethyl)oxane-3,4-diol] and the luciferase assay substrate were purchased from Promega (Madison, WI). The murine TNF-α was purchased from ProSpec-Tany TechnoGene (Rehovot, Israel).
The JB6 P+ cell line was cultured in monolayers at 37°C in 5% CO2 in MEM that contained 5% FBS. The JB6 P+ cell lines that were stably transfected with the COX-2, AP-1, or NF-κB luciferase reporter plasmids were maintained in MEM that was supplemented with 5% FBS and contained 200 μg/ml of G418 to exclude nontransfected cells.
Western Blot Analysis.
Cells were cultured for 48 h and then incubated in MEM that contained 0.1% FBS for an additional 24 h. The cells were then treated with chemicals for 1 h before being exposed to TNF-α (4 ng/ml) and harvested at various time points. Cell lysates were prepared and treated with lysis buffer [10 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 10% glycerol, and a protease inhibitor cocktail tablet] for 40 min on ice, and then centrifuged at 16,000g for 10 min. The protein concentration of the supernatant fraction was measured using a dye-binding protein assay kit (Bio-Rad Laboratories), as described in the manufacturer's manual. Lysate protein samples (40 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore Corporation, Billerica, MA). After transfer, the membrane was blocked in 5% fat-free dry milk for 1 h, and then incubated with the specific primary antibody for 2 h at room temperature. After hybridization with the horseradish peroxidase-conjugated secondary antibody, protein bands were detected using an enhanced chemiluminescence detection kit (GE Healthcare).
Measurement of Cell Viability.
Cell viability was measured by 3-(4,5-dimethylthiazol-2-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 × 103) were cultured in 96 wells and incubated for 48 h. After incubation, the cells were pretreated with luteolin at the indicated concentrations for 1 h before incubation with TNF-α (4 ng/ml) for 18 h, followed by the addition of 20 μl of MTT stock solution (5 mg/ml; Sigma-Aldrich) to each well, and the plates were further incubated for 4 h at 37°C. The supernatant was removed, and 200 μl of DMSO was added to each well to solubilize the water-insoluble purple formazan crystals. The absorbency at a wavelength of 570 nm was measured with a microplate reader (Molecular Devices, Sunnyvale, CA). All measurements were performed in triplicate. Results are expressed as the percentage of proliferation with respect to untreated cells.
Luciferase Assays for COX-2 Promoter Activity and AP-1/NF-κB-Responsive Promoter Activity.
Confluent monolayers of JB6 P+ cells (5 × 103) that were stably transfected with a COX-2 promoter, AP-1-responsive promoter, or NF-κB-responsive promoter luciferase reporter plasmid were suspended in 200 μl of 5% FBS/MEM and added to each well of a 96-well plate. The plates were incubated at 37°C in a humidified atmosphere with 5% CO2. When the cells reached 80 to 90% confluence, they were cultured in 0.1% FBS/MEM for 24 h to reduce the background. The cells were then pretreated with chemicals for 1 h before being exposed to TNF-α (4 ng/ml) and harvested 3 h later. The cells were disrupted in 100 μl of lysis buffer (0.1 M potassium phosphate buffer, pH 7.8, 1% Triton X-100, 1 mM DTT, 2 mM EDTA), and luciferase activity was measured using a luminometer (Microlumat Plus LB 96V; Berthold Technologies, Bad Wildbach, Germany).
TPL2 activity was measured directly according to the instructions provided by Millipore. In brief, each reaction contained 2.5 μl of assay buffer (500 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM Na3VO4, 1% 2-mercaptoethanol, 0.1% Brij-35), to which was added 50 ng of active TPL2 protein. The TPL2 protein was incubated with various concentrations of luteolin (0, 1.25, 5, 20, and 80 μM) for 10 min at room temperature. Inactive MEK1 (355 ng), inactive MAPK2/Erk2 (3.4 μg), and myelin basic protein (5 μg) were added to 10 μl of [γ-32P]ATP in magnesium-acetate-ATP cocktail buffer (2.5 mM HEPES, pH 7.4, 50 mM magnesium acetate, 0.5 mM ATP) and incubated at 30°C for 10 min in the above assay buffer and substrate peptide. Then 15-μl aliquots were transferred onto p81 paper and washed three times with 0.75% phosphoric acid for 5 min and once with acetone for 5 min. Radioactive nuclide incorporation was measured using a scintillation counter (PerkinElmer Life and Analytical Sciences, Waltham, MA). Each experiment was performed three times.
TPL2 Immunoprecipitation and Kinase Assays for JB6 P+ Cells.
JB6 P+ cells were cultured to 80% confluence and then incubated with 0.1% FBS/MEM for 24 h at 37°C to reduce the background. The cells were treated with various concentrations of luteolin (5, 10, and 20 μM) or TPL2 inhibitor (20 μM) for 1 h before being exposed to TNF-α (4 ng/ml) for 15 min. The cells were then disrupted with lysis buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1 mM EGTA, 1% Triton X-100, 1 mM β-glycerophosphate, 1 mg/ml leupeptin, 1 mM Na3VO4, 1 mM PMSF) and centrifuged in a microcentrifuge at 14,000 rpm for 15 min. The lysates that contained 500 μg of protein were used for immunoprecipitation with the anti-TLP2 antibody and incubated at 4°C overnight with Protein A/G Sepharose beads. The beads were washed three times with kinase buffer (200 mM Tris-HCl, pH 7.5, 0.4 mM EGTA, 0.4 mM Na3VO4) and then resuspended in 2.5 μl of 10× kinase buffer. Inactive MEK1 (355 ng), inactive MAPK2/Erk2 (3.4 μg), and myelin basic protein (5 μg) were added to 10 μl of [γ-32P]ATP in magnesium-acetate-ATP cocktail buffer [2.5 mM HEPES, pH 7.4, 50 mM magnesium acetate, 0.5 mM ATP] and incubated at 30°C for 10 min in the above assay buffer and substrate peptide. Then 15-μl aliquots were transferred onto p81 paper and washed three times with 0.75% phosphoric acid for 5 min and once with acetone for 5 min. The incorporation of radioactivity was measured using a scintillation counter. Each experiment was performed three times.
Direct and Cell-Based Pull-Down Assays.
Active TPL2 protein (0.2 μg) or a JB6 P+ cellular supernatant fraction (500 μg) was incubated with luteolin-Sepharose 4B or Sepharose 4B (control) beads (100 μl, 50% slurry) in reaction buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% Nonidet P-40, 2 μg/ml bovine serum albumin, 0.02 mM PMSF, 1× protease inhibitor cocktail). After incubation with gentle rocking overnight at 4°C, the beads were washed five times with buffer (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 proteins bound to the beads were analyzed by Western blotting.
ATP and Luteolin Competition Assay.
Recombinant TPL2 (0.2 μg) was incubated with 100 μl of luteolin-Sepharose 4B or 100 μl of Sepharose 4B in reaction buffer (see Direct and Cell-Based Pull-Down Assays) for 12 h at 4°C, and ATP was added at either 10 or 100 μM to a final volume of 500 μl and incubated for 30 min. The samples were washed, and the proteins were detected by Western blotting.
Small Interfering RNA-Mediated Silencing of the TPL2 Gene.
Two small interfering RNAs (siRNAs) designed to knock down murine tpl2 gene expression and a control siRNA that contains a scrambled sequence that does not lead to the specific degradation of any known cellular mRNA were purchased from Bioneer (Daejun, Korea). JB6 P+ and JB6 P+ cells (5 × 103) that were stably transfected with the COX-2, AP-1, or NF-κB luciferase reporter plasmid were transfected with siRNA using INTERFERin (Polyplus-Transfection, Illkirch, France). The final siRNA concentration was 10 nM.
The homology model for TPL2 was generated by Geno3D (http://geno3d-pbil.ibcp.fr) using the coordinates of Mst1 (Protein Data Bank accession code 3COM) as a template. Insight II (Accelrys, San Diego, CA) was used for the docking study and structural analysis.
When necessary, data were expressed as mean ± S.D., and Student's t test was used for single statistical comparisons. A probability value of p < 0.05 was used as the criterion for statistical significance.
Luteolin Suppresses TNF-α-Induced COX-2 Expression in JB6 P+ Cells.
Because COX-2 is one of the most important inflammatory mediators of TNF-α-induced skin cancer (Yan et al., 2006), we first examined the effect of luteolin on TNF-α-induced COX-2 expression. COX-2 expression was dose-dependently inhibited by luteolin treatment (Fig. 1B). To determine whether the inhibitory effects of luteolin on COX-2 expression were mediated by transcriptional regulation, we investigated the effects of luteolin on TNF-α-induced COX-2 promoter activity. The luciferase assay data revealed that exposure to TNF-α (4 ng/ml) significantly induced COX-2 promoter activity, and that this was markedly inhibited by luteolin treatment (Fig. 1C). Luteolin had no effect on cell growth in cotreatment of TNF-α at dosages between 5 and 20 μM (Fig. 1D), which suggests that luteolin inhibits the up-regulation of COX-2 without affecting cell viability.
Luteolin Attenuates TNF-α-Induced Signaling in JB6 P+ Cells.
To elucidate the mechanism underlying the inhibitory effects of luteolin on TNF-α-induced COX-2 expression, we examined the effects of luteolin on TNF-α-induced MAP kinases and the Akt signaling pathway. Exposure of JB6 P+ cells to TNF-α (4 ng/ml) markedly induced the phosphorylation of ERK, JNK, p38, and Akt. Luteolin suppressed MEK/ERK/p90RSK (Fig. 2A), MKK4/JNK/c-Jun (Fig. 2B), and Akt/p70S6K (Fig. 2C). However, TNF-α-induced phosphorylation of p38 was not affected by luteolin treatment (Fig. 2D). We also measured AP-1- and NF-κB-responsive promoter activities using JB6 P+ cells that were stably transfected with an AP-1- or NF-κB-responsive promoter luciferase reporter plasmid. Luteolin significantly inhibited TNF-α-induced responsive promoter activity of AP-1 (Fig. 2E) and NF-κB (Fig. 2F).
Luteolin Directly Inhibits TPL2 Activity.
A previous study has shown that TPL2 regulates MEK and MKK4 without affecting p38 in the TNF-α-induced signaling pathway (Das et al., 2005). Thus, we examined the effect of luteolin on TPL2 activity. In an in vitro TPL2 assay luteolin strongly suppressed TPL2 activity in a dose-dependent manner (Fig. 3A). In a cellular system, phosphorylation of TPL2 was increased by luteolin treatment (Fig. 3B). However, luteolin suppressed TNF-α-induced TPL2 activity in JB6 P+ cells with the same potency as a commercial TPL2 inhibitor (Fig. 3C).
Luteolin Binds TPL2 Directly.
To identify the mechanism by which luteolin modulates TPL2 activity, we investigated whether luteolin binds directly to TPL2. The pull-down assay results revealed that luteolin bound directly to the active TPL2 protein (Fig. 3D, lane 3), but not to unconjugated Sepharose 4B beads (Fig. 3D, lane 2). The input lane (Fig. 3D, lane 1) shows the loading of 50 ng of the active TPL2 protein as a marker, verifying that the detected band represents the TPL2 protein. We also observed binding of luteolin to TPL2 in JB6 P+ cells (Fig. 3E).
TPL2 Is Involved in TNF-α-Induced COX-2 Up-Regulation.
We next examined whether inhibiting TPL2 activity could suppress TNF-α-induced COX-2 up-regulation in JB6 P+ cells. Treatment with either TPL2i or siTPL2 inhibited TNF-α-induced COX-2 expression and promoter activity (Fig. 4). We also examined the alteration of the TNF-α-induced signaling pathway using TPL2i (Fig. 5) and siTPL2 (Fig. 6). Inhibition of TPL2 using either TPL2i or siTPL2 was similar to the inhibitory effect of luteolin on the TNF-α-induced signaling pathway in JB6 P+ cells. The TPL2 inhibitor suppressed MEK/ERK/p90RSK (Fig. 5A), MKK4/JNK/c-Jun (Fig. 5B), and Akt/p70S6K (Fig. 5C), whereas the phosphorylation of p38 was unchanged, as observed for luteolin treatment (Fig. 5D). We also measured the levels of AP-1 and NF-κB transactivation in JB6 P+ cells that were stably transfected with the AP-1 or NF-κB luciferase reporter plasmid. The TPL2 inhibitor attenuated TNF-α-induced transactivation of AP-1 (Fig. 5E) and NF-κB (Fig. 5F) in a dose-dependent manner. Transfection of siTPL2 resulted in the down-regulation of TPL2 protein (Fig. 6G). The signaling pathway was generally similar to that observed after treatment with luteolin or TPL2 inhibitor (Fig. 6). Phosphorylation of p38 was increased in siTPL2-treated cells, compared with siMock-treated cells (Fig. 6D).
Binding Mode of Luteolin with TPL2 using Computer Modeling Study.
To investigate the molecular basis of TPL2 inhibition by luteolin, we carried out a docking study using a homology model of the structure of the TPL2 domain derived from the crystal structure of Mst1, which shares 50% amino acid sequence homology with TPL2. The binding ability of luteolin to TPL2 was reduced in the presence of ATP (Fig. 7A), indicating that luteolin binds to TPL2 in an ATP-competitive manner. Thus, we show that luteolin binds to TPL2 in an ATP-competitive manner. The kinase domain of TPL2 consists of an N-lobe and a C-lobe. These N- and C-lobes are linked through a loop, which is termed the “hinge region.” The backbone of this loop interacts with the adenine moiety of ATP by hydrogen bonding. Based on the experimental result that luteolin is an ATP-competitive inhibitor of TPL2, we docked the compound to the ATP binding site of TPL2. Luteolin was easily docked to the ATP binding site of TPL2, which is located between the N-lobe and C-lobe of the kinase domain (Fig. 7, B and C).
Flavonoids, which are chemicals that have a diphenylpropane (C6C3C6) skeleton, are generally secondary metabolites of plants. Flavonoids have been shown to exert health benefits for patients with cancer, cardiovascular diseases, and diabetes (Middleton et al., 2000). Because flavonoids possess antioxidant properties, many studies have suggested that the health benefits of flavonoids are related to their antioxidant effects (Di Carlo et al., 1999). However, antioxidant effects alone cannot explain all of the effects mediated by flavonoids, e.g., specific inhibition of signal transduction at low dosages. Our previous studies and other studies indicate that flavonoids act as kinase inhibitors (Kim et al., 2009, 2010; Kwon et al., 2009; Choi et al., 2010).
Luteolin, one of those abundant flavonoids that occur naturally, inhibits many types of inflammatory disease, such as cancer, cardiovascular disease, and neuronal disease (Seelinger et al., 2008). Luteolin controls the expression of inflammatory genes, such as those for TNF-α, interleukin-6, and COX-2, through inhibiting signaling molecules (Seelinger et al., 2008). Luteolin inhibits vascular epithermal growth factor-induced angiogenesis by inhibiting phosphatidylinositol 3-kinase enzyme activity (Agullo et al., 1997), and it inhibits protein kinase C activity (Ferriola et al., 1989).
In the present study, luteolin inhibited TNF-α-induced COX-2 expression in JB6 P+ cells. Luteolin also inhibited TNF-α-induced phosphorylation of MEK/ERK/p90RSK, MKK4/JNK/c-Jun, and Akt/p70S6K. However, it had no effect on the phosphorylation of p38. Luteolin inhibited AP-1 and NF-κB transactivation, which are transcription factors that regulate COX-2 expression (Kim et al., 2011). TPL2 plays crucial role in TNF-α induced activation of MEK/ERK and MKK4/JNK. However, phosphorylation of p38 is not affected by TPL2 knockout (Dasi et al., 2005). The same tendency was observed when treated with luteolin. Luteolin inhibited TPL2 activity in vitro and in TPL2 immunoprecipitation kinase assays. Although kinase activity was decreased, phosphorylation of Ser290 in TPL2, which is an important phosphorylation site for the activation of TPL2, was increased by luteolin treatment. In the case of inhibition of kinase activity by pharmacologic inhibitors, the phosphorylation status is usually unchanged. However, in some instances, phosphorylation is increased because of feedback effects (Harrington et al., 2005). Considering these results, we propose that TPL2 acts as a molecular target for luteolin-induced regulation of the TNF-α-induced signaling pathway in JB6 P+ cells.
Depending on our computer modeling study, we anticipated the binding mode of luteolin with TPL2. The hydroxyl groups at the 3′ and 4′ positions of luteolin can form hydrogen bonds with the backbone of Glu208 and Gly210 in the hinge region of TPL2. The hydroxyl group at the 7-position can form hydrogen bonds with the side chains of Gln173. In this scenario, the inhibitor would be sandwiched by the side chains of the hydrophobic residues in the ATP binding site, which includes Ala165, Met207, and Val152 from the N-lobe and Met262, Val260, and Val269 from the C-lobe. Given that staurosporine, which is a general kinase inhibitor, does not inhibit TPL2 and that the known TPL2 inhibitor has higher selectivity than other kinases, it seems that the ATP binding site of TPL2 has a unique structure and repels compounds that cannot accommodate this structural characteristic. However, the surface of the putative luteolin binding site of TPL2 accommodates luteolin without steric collision, owing to the small size of this compound, thereby conferring high-level inhibitory activity on the inhibitor for TPL2 (Fig. 7B). Further studies with X-ray crystallography to determine the inhibitor complex structure may elucidate the precise nature of luteolin binding to TPL2.
A pharmacologic inhibitor and siRNA were used to show that TPL2 is required for TNF-α-induced COX-2 expression in JB6 P+ cells. Although pharmacologic inhibition may lack selectivity, it inhibits signal transduction in a rapid and dose-dependent manner. Although siRNA systems have the advantage of selectivity, the long-term loss of gene function can lead to compensatory changes in the functions of other genes. Therefore, it is advisable to use both methods to study signal transduction (Jaeschke et al., 2006). TNF-α-induced COX-2 expression and transactivation were inhibited by both TPL2i and siRNA. To confirm siRNA blocking efficiency, we examined TPL2 protein levels. We could not detect TPL2 in the siTPL2-treated group. However, the level of TPL2 protein was diminished 4 h after treatment with siMock and TNF-α. There was no difference in the TPL2 protein level after 15 min of treatment with TNF-α. A previous study showed that TPL2 was degraded by TNF-α stimulation after the phosphorylation of downstream kinases (Cho and Tsichlis, 2005). In the present study, TPL2 protein degradation began 1 h after treatment with TNF-α (data not shown).
We used TPL2i and siRNA to elucidate the effects of suppressing TPL2 on the TNF-α-induced signaling pathway. Treatment with TPL2i or siTPL2 showed similar results to luteolin treatment with respect to the TNF-α induced signaling pathway. TNF-α-induced phosphorylation of p38 was inhibited in the macrophages, but not in the fibroblasts, of TPL2(−/−) mice (Dasi et al., 2005). This cell-specific phosphorylation of p38 by TPL2 abrogation may reflect the different roles of MKK4. MKK4 is required for the phosphorylation of p38 but only in cell types with low levels of MKK3/6 expression (Brancho et al., 2003). Our recent study shows that inhibition of MKK4 activity cannot impair phosphorylation of p38 (Kim et al., 2009). Therefore, MKK4 may not be required for the activation of p38 in TNF-α-stimulated JB6 P+ cells.
Our recent data and other studies show that natural small compounds present in foodstuffs or medicinal plants can directly target cellular proteins, primarily acting as kinase inhibitors. These compounds usually have multiple targets, because their specificities are generally lower than those of designed inhibitors. In recent times, multitargeting kinase inhibitors have received much attention. The fact that these inhibitors have broad reactivities means that they may be applicable to a number of conditions (Vogt and Kang, 2006). Thus, multitargeting kinase inhibitors may have broader applications than kinase inhibitors that have one specific target. Previous studies have suggested phosphatidylinositol 3-kinase and protein kinase C as targets of luteolin (Ferriola et al., 1989; Agullo et al., 1997). In the present study, we reveal TPL2 as a novel target of luteolin. Therefore, luteolin can confer multiple health benefits by targeting several kinases.
Participated in research design: K. W. Lee and H. J. Lee.
Conducted experiments: Kim, Son, Jang, D. E. Lee, Kang, Jung, and Heo.
Wrote or contributed to the writing of the manuscript: Kim, Heo, K. W. Lee, and H. J. Lee.
This work was supported in part by the World Class University Program [Grant R31-2008-00-10056-0] and the National Leap Research Program [Grant 2010-0029233] through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology, Republic of Korea.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- tumor necrosis factor
- TNF-α receptor
- tumor progression locus 2 serine/threonine kinase
- small interfering TLP2
- TPL2 inhibitor, 4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1,7]-naphthyridine
- activator protein-1
- nuclear factor-κB
- extracellular signal-regulated kinase
- fetal bovine serum
- c-Jun N-terminal kinase
- mitogen-activated protein kinase
- MAPK/ERK kinase 1
- mitogen-activated protein kinase kinase 4
- minimum essential medium
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- phenylmethylsulfonyl fluoride
- small interfering RNA
- small interfering Mock.
- Received January 13, 2011.
- Accepted June 23, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics