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Deoxynivalenol induces ectodomain shedding of TNF receptor 1 and thereby inhibits the TNF-α-induced NF-κB signaling pathway
European Journal of Pharmacology 701 (2013) 144–151
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Molecular and cellular pharmacology
Deoxynivalenol induces ectodomain shedding of TNF receptor 1 and thereby inhibits the TNF-a-induced NF-kB signaling pathway Seiya Hirano, Takao Kataoka n Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 September 2012 Received in revised form 27 November 2012 Accepted 9 January 2013 Available online 25 January 2013
Trichothecene mycotoxins are known to inhibit eukaryotic translation and to trigger the ribotoxic stress response, which regulates gene expression via the activation of the mitogen-activated protein (MAP) kinase superfamily. In this study, we found that deoxynivalenol induced the ectodomain shedding of tumor necrosis factor (TNF) receptor 1 (TNFRSF1A) and thereby inhibited the TNF-ainduced signaling pathway. In human lung carcinoma A549 cells, deoxynivalenol and 3-acetyldeoxynivalenol inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by TNF-a more strongly than that induced by interleukin 1a (IL-1a), whereas T-2 toxin and verrucarin A exerted nonselective inhibitory effects. Deoxynivalenol and 3-acetyldeoxynivalenol also inhibited the nuclear factor kB (NF-kB) signaling pathway induced by TNF-a, but not that induced by IL-1a. Consistent with these findings, deoxynivalenol and 3-acetyldeoxynivalenol induced the ectodomain shedding of TNF receptor 1 by TNF-a-converting enzyme (TACE), also known as a disintegrin and metalloproteinase 17 (ADAM17). In addition to the TACE inhibitor TAPI-2, the MAP kinase or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 and the p38 MAP kinase inhibitor SB203580, but not the c-Jun N-terminal kinase (JNK) inhibitor SP600125, suppressed the ectodomain shedding of TNF receptor 1 induced by deoxynivalenol and reversed its selective inhibition of TNF-a-induced ICAM-1 expression. Our results demonstrate that deoxynivalenol induces the TACEdependent ectodomain shedding of TNF receptor 1 via the activation of ERK and p38 MAP kinase, and thereby inhibits the TNF-a-induced NF-kB signaling pathway. & 2013 Elsevier B.V. All rights reserved.
Keywords: Deoxynivalenol ERK p38 MAP kinase TNF receptor 1 TNF-a-converting enzyme Trichothecene mycotoxin
1. Introduction Proinflammatory cytokines, such as tumor necrosis factor a (TNF-a) and interleukin-1 (IL-1), induce intracellular signaling pathways, one of which leads to the activation of the transcription factor nuclear factor kB (NF-kB) (Karin and Greten, 2005). When TNF-a and IL-1 engage with TNF receptor 1 (TNFRSF1A) and IL-1 receptor, respectively, they recruit distinct sets of adaptor proteins, this results in the activation of the inhibitor of kB (IkB) kinase as a common target (Hayden and Ghosh, 2008; Bhoj and Chen, 2009). Immediately after IkB kinase phosphorylates IkB, which prevents the nuclear translocation of the NF-kB subunits, phosphorylated IkB undergoes ubiquitination and proteolytic degradation by proteasomes (Perkins, 2006). The NF-kB subunits are released and translocated to the nucleus where they stimulate the transcription of diverse target genes that regulate inflammatory responses, including the gene encoding intercellular adhesion molecule-1 (ICAM-1) (Roebuck and Finnegan, 1999).
n
Corresponding author. Tel./fax: þ 81 75 724 7752. E-mail address: [email protected] (T. Kataoka).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.01.019
TNF-a-converting enzyme (TACE), also known as a disintegrin and metalloproteinase 17 (ADAM17), is a cell-surface metalloproteinase that mediates the ectodomain shedding of various ligands (e.g., TNF-a) and receptors (e.g., TNF receptor 1) (Scheller et al., 2011). TACE is ubiquitously expressed in many cell types and its activity is regulated by different mechanisms, including its posttranslational modification (Scheller et al., 2011). In response to various stimuli, extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase phosphorylate the cytoplasmic tail of TACE at threonine 735 and thereby TACEdependent ectodomain shedding is induced (Dı´az-Rodrı´guez et al., 2002; Soond et al., 2005; Liu et al., 2009; Xu and Derynck, 2010). Several translation inhibitors are known to induce the activation of the MAP kinase superfamily by interacting with ribosomes in an intracellular mechanism designated the ribotoxic stress response (Iordanov et al., 1997; Kataoka, 2012). Recently, we have shown that different translation inhibitors (acetoxycycloheximide and cytotrienin A) induce the ectodomain shedding of TNF receptor 1 via the activation of ERK and p38 MAP kinase (Ogura et al., 2008a, 2008b; Yamada et al., 2011a). Trichothecene mycotoxins are a large group of sesquiterpenoids produced by Fusarium and other fungi. They are often found
S. Hirano, T. Kataoka / European Journal of Pharmacology 701 (2013) 144–151
as contaminants in agricultural staples and are known to exert acute and chronic effects on animals and humans (Pestka et al., 2004; Pestka, 2010). Trichothecene mycotoxins interact with eukaryotic ribosomes and block the peptidyl transferase reaction. They also exhibit both immunosuppressive and immunostimulatory activities (Pestka et al., 2004; Pestka 2010). In addition to the induction of cell death in leukocytes, deoxynivalenol also upregulates the expression of proinflammatory cytokines (e.g., TNF-a) and chemokines in macrophages and monocytes via the ribotoxic stress response (Chung et al., 2003; Islam et al., 2006). However, it remains unclear whether deoxynivalenol modulates the intracellular signaling pathways induced by proinflammatory cytokines. In this study, we investigated the biological activities of deoxynivalenol and its structural analogs in the NF-kB signaling pathway and the gene expression in response to proinflammatory cytokines. Our results demonstrate for the first time that deoxynivalenol rapidly induces the ectodomain shedding of TNF receptor 1 and thereby inhibits the TNF-a-induced NF-kB signaling pathway.
2. Materials and methods 2.1. Cell culture Human lung carcinoma A549 cells (JCRB0076) and human hepatocellular carcinoma HepG2 cells (RCB1648) were provided by the Health Science Research Resources Bank (Tokyo, Japan) and RIKEN BRC through the National Bio-Resource Project of MEXT, Japan (Tsukuba, Japan), respectively. A549 cells and HepG2 cells were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) heat-inactivated fetal calf serum (JRH Biosciences, Lenexa, KS, USA) and a mixed penicillin–streptomycin solution (Nacalai Tesque Inc., Kyoto, Japan).
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TNF receptor 1 (H-5; Santa Cruz Biotechnology) were obtained commercially.
2.4. Assay for cell-surface expression of ICAM-1 A549 cells were washed twice with phosphate-buffered saline (PBS) and incubated with 1% paraformaldehyde–PBS for 15 min. The fixed cells were washed twice with PBS and incubated overnight in the presence of 1% bovine serum albumin (SigmaAldrich)–PBS for blocking. Then, the cells were incubated with mouse anti-human ICAM-1 IgG antibody (clone 15.2) for 60 min and thereafter washed three times with 0.02% Tween 20–PBS. The cells were further incubated with horseradish peroxidase (HRP)-linked anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 60 min and then washed three times with 0.02% Tween-20–PBS. The cells were incubated with the substrate solution (0.2 M sodium citrate (pH 5.3), 0.1% o-phenylenediamine dihydrochloride, 0.02% H2O2) at 37 1C for 20 min. The absorbance at 415 nm was measured with a Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
2.5. Assay for macromolecular synthesis A549 cells were pulse-labeled with [4,5-3H]L-leucine (41. 66 TBq/mmol; Moravek Biochemicals, Inc., Brea, CA, USA). The cells were washed three times with PBS and lysed with 0.25 M NaOH for 15 min. The proteins were precipitated by incubation on ice for 1 h in the presence of 5% trichloroacetic acid. Cell lysates were separated into supernatants and precipitates by centrifugation (10,000 g, 5 min). The precipitates were washed once with 5% trichloroacetic acid. Radioactivity of the supernatants and the precipitates was measured with a 1900CA TRI-CARBs liquid scintillation analyzer (Packard Instrument Co., Meriden, CT, USA).
2.2. Reagents Deoxynivalenol, 3-acetyldeoxynivalenol, verrucarin A, and 1,9-pyrazoloanthrone (SP600125) were purchased from SigmaAldrich Co. (St. Louis, MO, USA). T-2 toxin (Enzo Life Sciences International, Inc., Plymouth Meeting, PA, USA), N-(R)-(2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-L-t-butyl-glycylL-alanine 2-aminoethyl amide (TAPI-2; Peptide Institute, Inc., Osaka, Japan), 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126; Wako Pure Chemical Industries, Ltd., Osaka, Japan), and 4-(4-fluorophenyl)-2-(4-methysulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580; Cayman Chemical Co., Ann Arbor, MI, USA) were obtained commercially. Recombinant human TNF-a and human IL-1a were kindly provided by Dainippon Pharmaceutical (Osaka, Japan). 2.3. Antibodies Antibodies to b-actin (AC-15; Sigma-Aldrich), cyclooxygenase2 (Cox-2) (Clone 33; BD Biosciences, Franklin Lakes, NJ, USA), ERK1/ERK2 (#9102; Cell Signaling Technology, Inc., Danvers, MA, USA), cellular FLICE-inhibitor protein (c-FLIP) (Dave-2; Alexis Co., Lausen, Switzerland), ICAM-1 (clone 15.2; Leinco Technologies, Inc., St. Louis, MO, USA), ICAM-1 (clone 28; BD Biosciences), NFkB p50 (H-119; Santa Cruz Biotechnology, Santa Cruz, CA, USA), NF-kB p65 (C-20; Santa Cruz Biotechnology), p38 MAP kinase (#9212; Cell Signaling Technology), poly(ADP–ribose) polymerase (PARP) (C-2–10; Sigma-Aldrich), phospho-ERK1/ERK2 (Thr202/ Tyr204) (#9101; Cell Signaling Technology), phospho-p38 MAP kinase (Thr180/Tyr182) (#9211; Cell Signaling Technology), and
2.6. Preparation of cell lysates and western blotting A549 cells were washed once with PBS and lysed with Triton X-100 lysis buffer (50 mM Tris–HCl (pH 7.4), 1% Triton X-100, the protease inhibitor mixture CompleteTM (Roche Diagnostics, Mannheim, Germany), 2 mM DTT, 2 mM orthovanadate). Cell lysates were centrifuged (10,000 g, 5 min) and separated into supernatants as cytoplasmic fractions and pellets. The pellets were washed twice with Triton X-100 lysis buffer and then solubilized as nuclear fractions. The culture medium was centrifuged (10,000 g, 5 min) to remove cell debris and insoluble materials. The proteins were then precipitated with chloroform/ methanol. The protein samples (30 mg/lane) were separated by SDS–PAGE and transferred onto Hybond-ECL nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA). The membranes were incubated overnight with 4% skim milk in 0.5% Tween 20–PBS for blocking, and then incubated with the primary antibodies and HRP-linked secondary antibodies (Jackson ImmunoResearch). The protein bands were detected with ECL Western blotting detection reagents (GE Healthcare) and analyzed with the ImageQuant LAS 4000 mini (GE Healthcare).
2.7. Statistical analysis Statistical significance was assessed with one-way ANOVA followed by the Tukey test for multiple comparisons. Differences with P values of o0.05 were considered to be statistically significant.
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3. Results 3.1. Deoxynivalenol and 3-acetyldeoxynivalenol inhibit TNF-ainduced ICAM-1 expression more strongly than IL-1a-induced ICAM1 expression The trichothecene mycotoxins, deoxynivalenol, 3-acetyldeoxynivalenol, T-2 toxin, and verrucarin A (Supplementary Fig. 1), were investigated for their biological effects on the NF-kBresponsive gene expression induced by proinflammatory cytokines. Human lung carcinoma A549 cells were highly responsive to TNF-a or IL-1a, which induced cell-surface ICAM-1 expression in an NF-kB-dependent manner. Deoxynivalenol inhibited TNF-ainduced ICAM-1 expression in a dose-dependent manner and the inhibition was approximately 8-fold stronger than its inhibition of IL-1a-induced ICAM-1 expression (Fig. 1A and Supplementary Table 1). The inhibitory effects of 3-acetyldeoxynivalenol were at least 10-fold weaker than those of deoxynivalenol; nevertheless, 3-acetyldeoxynivalenol still inhibited the TNF-a-induced ICAM-1 expression in preference to the IL-1a-induced one (Fig. 1B and Supplementary Table 1). In contrast, T-2 toxin and verrucarin A inhibited both TNF-a- and IL-1a-induced ICAM-1 expression at almost equivalent concentrations with IC50 values less than 10 nM (Fig. 1C and D and Supplementary Table 1). Low to high concentrations of the trichothecene mycotoxins alone did not upregulate ICAM-1 expression in A549 cells (data not shown).
3.2. Deoxynivalenol inhibits TNF-a-induced expression of NF-kBresponsive genes In addition to the cell-surface expression of ICAM-1 measured by the Cell-ELISA assay, the total amount of ICAM-1 in the cell lysates was analyzed by Western blotting. Consistent with the results shown in Fig. 1, deoxynivalenol and 3acetyldeoxynivalenol inhibited the TNF-a-induced ICAM1expression more strongly than the IL-1a-induced one (Fig. 2A and B), whereas T-2 toxin and verrucarin A inhibited the TNF-aand IL-1a-induced ICAM-1 expression at similar concentrations (Fig. 2C and D). In human hepatocellular carcinoma HepG2 cells, ICAM-1was expressed constitutively in the absence of cytokines and ICAM-1 expression was increased to some extent by TNF-a or IL-1a (Fig. 2E). The constitutive expression of ICAM-1 was only weakly decreased by deoxynivalenol even at 10 mM in HepG2 cells (Fig. 2E). Thus, it is most likely that deoxynivalenol inhibits primarily the de novo synthesis of ICAM-1 protein, but barely affects the stability of ICAM-1 protein per se. In addition to ICAM-1, c-FLIP and Cox-2 are known to be NF-kB-responsive genes. As previously shown (Ogura et al., 2008a), TNF-a upregulated the cellular levels of c-FLIP (especially c-FLIPS) and Cox-2 in A549 cells (Fig. 2F). Deoxynivalenol inhibited the TNF-a-induced expression of c-FLIPS and Cox-2 (Fig. 2F). These results indicate that deoxynivalenol inhibits the TNF-a-induced expression of NF-kB-responsive genes.
Fig. 1. Deoxynivalenol and 3-acetyldeoxynivalenol inhibit TNF-a-induced ICAM-1 expression more strongly than IL-1a-induced ICAM-1 expression. (A–D) A549 cells were preincubated with various concentrations of deoxynivalenol (A), 3-acetyldeoxynivalenol (B), T-2 toxin (C), and verrucarin A (D) for 1 h and then incubated with TNF-a (2.5 ng/ml; filled circles) or IL-1a (0.25 ng/ml; open circles) for 6 h in the presence of the compounds. ICAM-1 expression (%) is shown as mean 7 S.D. (n¼ 3). nnP o 0.01 compared with control.
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Fig. 2. Deoxynivalenol inhibits TNF-a-induced expression of NF-kB-responsive genes. (A–D) A549 cells were preincubated with various concentrations of deoxynivalenol (A), 3-acetyldeoxynivalenol (B), T-2 toxin (C), and verrucarin A (D) for 1 h and then incubated with ( þ ) or without ( ) TNF-a (2.5 ng/ml) or IL-1a (0.25 ng/ml) for 6 h in the presence of the compounds. The cell lysates were analyzed by Western blotting. (E) HepG2 cells were incubated with ( þ ) or without ( ) TNF-a (2.5 ng/ml) or IL-1a (0.25 ng/ml) (left panel) and various concentrations of deoxynivalenol without cytokines (right panel) for 6 h. The cell lysates were analyzed by Western blotting. (F) A549 cells were preincubated with various concentrations of deoxynivalenol for 1 h and then incubated with ( þ ) or without (–) TNF-a (2.5 ng/ml) for 6 h in the presence of deoxynivalenol. The cell lysates were analyzed by Western blotting.
3.3. Trichothecene mycotoxins inhibit cellular protein synthesis Deoxynivalenol, 3-acetyldeoxynivalenol, T-2 toxin, and verrucarin A barely affected cell viability in the presence or absence of TNF-a or IL-1a (Supplementary Fig. 2), excluding the possibility that the inhibition of ICAM-1 expression by trichothecene mycotoxins is due to cytotoxic effects. To further evaluate the inhibitory effects of trichothecene mycotoxins on de novo protein synthesis, the incorporation of [3H]L-leucine into the acidinsoluble macromolecular fractions of A549 cells was measured. Deoxynivalenol and 3-acetyldeoxynivalenol inhibited cellular protein synthesis in a dose-dependent manner with IC50 values of 0.7 and 20 mM, respectively, whereas T-2 toxin and verrucarin A exerted much stronger inhibitory effects on protein synthesis with IC50 values of 8.6 and 2.1 nM, respectively (Supplementary Table 1). Deoxynivalenol, 3-acetyldeoxynivalenol, T-2 toxin, and verrucarin A also caused a marked increase in the radioactivity of the acidsoluble fractions, accompanied by a reduction in the radioactivity of the acid-insoluble fractions (Supplementary Fig. 3), confirming that these trichothecene mycotoxins did not affect the uptake of amino acids, but primarily inhibited the translation process. These results
indicate that the inhibitory effects of trichothecene mycotoxins on ICAM-1 expression are related to their inhibition of cellular protein synthesis.
3.4. Deoxynivalenol and 3-acetyldeoxynivalenol inhibit the NF-kB signaling pathway induced by TNF-a, but not that induced by IL-1a In response to TNF-a or IL-1a, NF-kB heterodimers composed of p65 and p50 were translocated from the cytoplasm to the nuclei of A549 cells. Deoxynivalenol inhibited the TNF-a-induced nuclear translocation of p65 and p50 at concentrations higher than 1 mM, whereas it barely affected the IL-1a-induced nuclear translocation of NF-kB (Fig. 3A). 3-Acetyldeoxynivalenol also markedly inhibited the TNF-a-induced nuclear translocation of p65 and p50 at 100 mM (Fig. 3B). In contrast, T-2 toxin and verrucarin A only weakly affected the TNF-a-induced nuclear translocation of NF-kB, even at much higher concentrations than those required for the inhibition of cellular protein synthesis (Fig. 3C and D). These results suggest that the selective inhibition of TNF-a-induced ICAM-1 expression by deoxynivalenol and
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Fig. 3. Deoxynivalenol and 3-acetyldeoxynivalenol inhibit TNF-a-induced nuclear translocation of the NF-kB subunits. (A–D) A549 cells were preincubated with various concentrations of deoxynivalenol (A), 3-acetyldeoxynivalenol (B), T-2 toxin (C), and verrucarin A (D) for 1 h and then incubated with ( þ) or without (–) TNF-a (2.5 ng/ml) or IL-1a (0.25 ng/ml) for 30 min in the presence of the compounds. The nuclear and cytoplasmic fractions were analyzed by Western blotting.
3-acetyldeoxynivalenol is largely attributable to the inhibition of the NF-kB signaling pathway. 3.5. Deoxynivalenol and 3-acetyldeoxynivalenol induce the ectodomain shedding of TNF receptor 1 We previously showed that translation inhibitors, such as acetoxycycloheximide and cytotrienin A, induced the ectodomain shedding of TNF receptor 1 (Ogura et al., 2008a; Yamada et al., 2011a). To determine whether deoxynivalenol and 3-acetyldeoxynivalenol also induced TNF receptor 1 shedding, A549 cells were exposed to these compounds for 1 h, and then culture media and cell lysates were analyzed by Western blotting using anti-TNF receptor 1 antibody. At concentrations of 1 and 10 mM, deoxynivalenol increased the amount of soluble TNF receptor 1 in the medium, and this was accompanied by a reduction of TNF receptor 1 expression in the cells (Fig. 4A). 3-Acetyldeoxynivalenol at concentrations higher than 10 mM also increased the amount of soluble TNF receptor 1 in the medium (Fig. 4B). The results indicate that deoxynivalenol and 3-acetyldeoxynivalenol induce the ectodomain shedding of TNF receptor 1. 3.6. Ectodomain shedding of TNF receptor 1 by deoxynivalenol causes the preferential inhibition of TNF-a-induced ICAM-1 expression TACE is known to mediate the proteolytic cleavage of TNF receptor 1 (Scheller et al., 2011). The TACE inhibitor TAPI-2 markedly suppressed the elevation of soluble TNF receptor 1 released by deoxynivalenol-treated A549 cells (Fig. 5A), indicating that deoxynivalenol induced the ectodomain shedding of TNF receptor 1 by TACE. We further investigated whether the ectodomain shedding of TNF receptor 1 was responsible for the
preferential inhibition of TNF-a-induced ICAM-1 expression by deoxynivalenol. In the presence of TAPI-2, relatively similar concentrations of deoxynivalenol inhibited both TNF-a- and IL-1a-induced ICAM-1 expression (Fig. 5B), corresponding to the IC50 values of 2.1 and 4.2 mM, respectively (calculated from three independent experiments, including that shown in Fig. 5B), in contrast to the IC50 values of 0.56 mM and 4.5 mM, respectively, in the absence of TAPI-2 (Supplementary Table 1).
3.7. Deoxynivalenol induces ectodomain shedding of TNF receptor 1 via ERK and p38 MAP kinase TACE activity is regulated by ERK and p38 MAP kinase in response to various stimuli (Scheller et al., 2011). Upon treatment with deoxynivalenol, ERK1/ERK2 phosphorylation was increased during the period of 5–30 min in A549 cells (Fig. 6A). In addition, p38 MAP kinase was phosphorylated within 5 min following exposure to deoxynivalenol and it remained phosphorylated for as long as 2 h (Fig. 6A). To investigate whether these members of the MAP kinase superfamily were necessary for the ectodomain shedding of TNF receptor 1 induced by deoxynivalenol, specific kinase inhibitors were used as blocking agents. The MAP kinase or ERK kinase (MEK) inhibitor U0126 and the p38 MAP kinase inhibitor SB203580 reduced the increase in the amount of soluble TNF receptor 1 released by deoxynivalenol-treated A549 cells, whereas the c-Jun N-terminal kinase (JNK) inhibitor SP600125 had no such effect (Fig. 6B). The ectodomain shedding of TNF receptor 1 induced by deoxynivalenol was completely suppressed by the combined treatment with U0126 and SB203580 (Fig. 6B). Consistent with these data, almost equivalent concentrations of deoxynivalenol inhibited the ICAM-1 expression induced by TNFa and that induced by IL-1a, when A549 cells were treated with a combination of U0126 and SB203580 (Fig. 6C).
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Fig. 4. Deoxynivalenol and 3-acetyldeoxynivalenol induce the ectodomain shedding of TNF receptor 1. (A and B) A549 cells were incubated with various concentrations of deoxynivalenol (A) or 3-acetyldeoxynivalenol (B) for 1 h. The cell lysates (cell) and the culture media (medium) were analyzed by Western blotting.
Fig. 5. The TACE-dependent ectodomain shedding of TNF receptor 1 induced by deoxynivalenol is responsible for its selective inhibition of TNF-a-induced ICAM-1 expression. (A) A549 cells were preincubated with ( þ ) or without (–) TAPI-2 (25 mM) for 1 h and then incubated with (þ ) or without (–) deoxynivalenol (1 mM) for 1 h in the presence or absence of TAPI-2. The cell lysates (Cell) and the culture media (Medium) were analyzed by Western blotting. (B) A549 cells were pretreated with or without TAPI-2 (25 mM) for 1 h. Then, the cells were treated with various concentrations of deoxynivalenol for 1 h and incubated with TNF-a (2.5 ng/ml; filled circles) or IL-1a (0.25 ng/ml; open circles) for 6 h in the presence of absence of TAPI-2 or deoxynivalenol. ICAM-1 expression (%) is shown as mean 7 S.D. (n¼3). nP o 0.05 and nn P o 0.01 compared with control.
4. Discussion Trichothecene mycotoxins are known translation inhibitors. In this study, we found that deoxynivalenol and 3-acetyldeoxynivalenol inhibited TNF-a-induced ICAM-1 expression more strongly than IL-1a-induced ICAM-1 expression, largely by suppressing the
TNF-a-induced NF-kB signaling pathway. The TACE inhibitor TAPI-2 or the MEK inhibitor U0126 plus the p38 MAP kinase inhibitor SB203580 markedly suppressed the ectodomain shedding of TNF receptor 1 induced by deoxynivalenol, and reversed the selective inhibition of TNF-a-induced ICAM-1 expression by deoxynivalenol. These results demonstrate for the first time that deoxynivalenol
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Fig. 6. Deoxynivalenol induces the ectodomain shedding of TNF receptor 1 via ERK and p38 MAP kinase. (A) A549 cells were incubated with deoxynivalenol (1 mM) for the indicated times. Phosphorylated and total amounts of ERK and p38 MAP kinase were analyzed by Western blotting. (B) A549 cells were pretreated with or without U0126 (10 mM), SB203580 (10 mM), SP600125 (10 mM), or U0126 (10 mM) plus SB203580 (10 mM) for 1 h, and then treated with (þ) or without (–) deoxynivalenol (1 mM) for 1 h in the presence or absence of protein kinase inhibitors. The cell lysates (Cell) and the culture media (Medium) were analyzed by Western blotting. (C) A549 cells were pretreated with or without U0126 (10 mM), SB203580 (10 mM), SP600125 (10 mM), or U0126 (10 mM) plus SB203580 (10 mM) for 1 h. Then, the cells were treated with various concentrations of deoxynivalenol for 1 h and incubated with TNF-a (2.5 ng/ml; filled circles) or IL-1a (0.25 ng/ml; open circles) for 6 h in the presence or absence of the protein kinase inhibitors or deoxynivalenol. ICAM-1 expression (%) is shown as mean 7 S.D. (n¼ 3). **P o0.01 compared with control.
induces the TACE-dependent ectodomain shedding of TNF receptor 1 by activating ERK and p38 MAP kinase, thereby inhibiting TNF-ainduced NF-kB activation. Trichothecene mycotoxins have a common tricyclic 12,13-epoxytrichothec-9-ene core structure and are classified into structural types based on the substitution pattern at the C-8 position (types A and B) and the additional ring linking the C-4 and C-15 positions (type D) (McCormick et al., 2011). In this study, we investigated the capacities of four trichothecene mycotoxins to inhibit the ICAM-1 expression induced by TNF-a or IL-1a. Deoxynivalenol (type B) and 3-acetyldeoxynivalenol (type B), but neither T-2 toxin (type A) nor verrucarin A (type D), inhibited TNF-a-induced ICAM-1 expression more strongly than IL-1a-induced ICAM-1 expression. Recently, we have shown that the selective inhibition of TNF-a-induced ICAM-1 expression by translation inhibitors is attributable to the ribotoxic stress response, which induces the downregulation of cell-surface TNF receptor 1 expression (Ogura et al., 2008a; Yamada et al., 2011a). Trichothecene mycotoxins are classified into strong (e.g., deoxynivalenol), intermediate (e.g., 3-acetyldeoxynivalenol), and weak (e.g., T-2 toxin, verrucarin A) inducers of the ribotoxic stress response at the concentration of 10 mM (Shifrin and Anderson, 1999). Those studies are in agreement with our present finding that
TNF-a-induced ICAM-1 expression is selectively inhibited by deoxynivalenol and 3-acetyldeoxynivalenol, but by neither T-2 toxin nor verrucarin A. It has also been shown that some type A trichothecene mycotoxins induce the ribotoxic stress response as efficiently as deoxynivalenol (Shifrin and Anderson, 1999). Unlike the type B trichothecene mycotoxins that have a carbonyl group at the C-8 position, only selected structural derivatives of the type A trichothecene mycotoxins, which have other groups at the C-8 position, are likely to induce the ribotoxic stress response. Several translation inhibitors can induce the ribotoxic stress response, which leads to the activation of the MAP kinase superfamily (Iordanov et al., 1997; Kataoka, 2012). The TACE-dependent ectodomain shedding of cell-surface proteins is upregulated by ERK and p38 MAP kinase, which phosphorylate threonine 735 in the cytoplasmic tail of TACE (Dı´az-Rodrı´guez et al., 2002; Soond et al., 2005; Liu et al., 2009; Xu and Derynck, 2010). Consistent with those studies, we have recently shown that different translation inhibitors (acetoxycycloheximide and cytotrienin A) induce the TACE-dependent ectodomain shedding of TNF receptor 1 by activating ERK and p38 MAP kinase (Ogura et al., 2008a; Ogura et al., 2008b; Yamada et al., 2011a). Acetoxycycloheximide induced the maximum activation of ERK and p38 MAP kinase within 15 min and subsequently stimulated the
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ectodomain shedding of TNF receptor 1 at 30–60 min (Kadohara et al., 2005; Ogura et al., 2008a; Ogura et al., 2008b). The kinetics of MAP kinase activation by acetoxycycloheximide is similar to the kinetics of its activation by deoxynivalenol (Zhou et al., 2005). Moreover, as previously observed in acetoxycycloheximide and cytotrienin A (Ogura et al., 2008b; Yamada et al., 2011a), the pharmacological inhibition of the ERK pathway or the p38 MAP kinase pathway suppressed the TACE-dependent ectodomain shedding of TNF receptor 1 induced by deoxynivalenol. Thus, it is highly likely that the rapid and strong activation of ERK and p38 MAP kinase by translation inhibitors is required for the TACE-dependent ectodomain shedding of TNF receptor 1. Deoxynivalenol exerts both immunostimulatory (e.g., activation of gene expression) and immunosuppressive (e.g., induction of cell death) effects depending on the experimental conditions, including the dose and duration of exposure (Pestka et al., 2004; Pestka, 2010). Among its immunostimulatory effects, deoxynivalenol upregulates the expression of proinflammatory cytokines and chemokines by activating their transcription and increasing the stability of their mRNAs (Chung et al., 2003; Islam et al., 2006; Gray and Pestka, 2007; Choi et al., 2009). In particular, it has been shown that TNF-a production is upregulated by deoxynivalenol in macrophages (Chung et al., 2003; Zhou et al., 2005). In this study, we showed that deoxynivalenol induced the ectodomain shedding of TNF receptor 1 by TACE and thereby diminished the activity of the TNF-a-induced NF-kB signaling pathway. Therefore, in contrast to other proinflammatory cytokines, deoxynivalenol may exert two opposite effects in regulating TNF-a-dependent cellular responses: the upregulation of TNF-a production in selected cells, including macrophages, and the downregulation of TNF-a responsiveness in many cells ubiquitously expressing TNF receptor 1 and TACE. In conclusion, our data demonstrated that deoxynivalenol induced the ectodomain shedding of TNF receptor 1 via the activation of ERK and p38 MAP kinase, a novel biological effect of trichothecene mycotoxins. We have previously evaluated the biological effects of translation inhibitors on ICAM-1 expression and cellular protein synthesis (Yamada et al., 2011b). The present and previous results indicate that deoxynivalenol is likely to induce the ribotoxic stress response as efficiently as other translation inhibitors. Trichothecene mycotoxins are a family of over 200 derivatives, which may include active inducers of the ribotoxic stress response with different efficacies as well as inactive analogs. Therefore, as representative translation inhibitors, trichothecene mycotoxins may be useful tools in determining the molecular basis of the ribotoxic stress response.
Acknowledgment This work was supported in part by JSPS KAKENHI Grant number 22380060.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2013. 01.019.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Deoxynivalenol induces ectodomain shedding of TNF receptor 1 and thereby inhibits the TNF-a-induced NF-kB signaling pathway Seiya Hirano, Takao Kataoka n Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 September 2012 Received in revised form 27 November 2012 Accepted 9 January 2013 Available online 25 January 2013
Trichothecene mycotoxins are known to inhibit eukaryotic translation and to trigger the ribotoxic stress response, which regulates gene expression via the activation of the mitogen-activated protein (MAP) kinase superfamily. In this study, we found that deoxynivalenol induced the ectodomain shedding of tumor necrosis factor (TNF) receptor 1 (TNFRSF1A) and thereby inhibited the TNF-ainduced signaling pathway. In human lung carcinoma A549 cells, deoxynivalenol and 3-acetyldeoxynivalenol inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by TNF-a more strongly than that induced by interleukin 1a (IL-1a), whereas T-2 toxin and verrucarin A exerted nonselective inhibitory effects. Deoxynivalenol and 3-acetyldeoxynivalenol also inhibited the nuclear factor kB (NF-kB) signaling pathway induced by TNF-a, but not that induced by IL-1a. Consistent with these findings, deoxynivalenol and 3-acetyldeoxynivalenol induced the ectodomain shedding of TNF receptor 1 by TNF-a-converting enzyme (TACE), also known as a disintegrin and metalloproteinase 17 (ADAM17). In addition to the TACE inhibitor TAPI-2, the MAP kinase or extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 and the p38 MAP kinase inhibitor SB203580, but not the c-Jun N-terminal kinase (JNK) inhibitor SP600125, suppressed the ectodomain shedding of TNF receptor 1 induced by deoxynivalenol and reversed its selective inhibition of TNF-a-induced ICAM-1 expression. Our results demonstrate that deoxynivalenol induces the TACEdependent ectodomain shedding of TNF receptor 1 via the activation of ERK and p38 MAP kinase, and thereby inhibits the TNF-a-induced NF-kB signaling pathway. & 2013 Elsevier B.V. All rights reserved.
Keywords: Deoxynivalenol ERK p38 MAP kinase TNF receptor 1 TNF-a-converting enzyme Trichothecene mycotoxin
1. Introduction Proinflammatory cytokines, such as tumor necrosis factor a (TNF-a) and interleukin-1 (IL-1), induce intracellular signaling pathways, one of which leads to the activation of the transcription factor nuclear factor kB (NF-kB) (Karin and Greten, 2005). When TNF-a and IL-1 engage with TNF receptor 1 (TNFRSF1A) and IL-1 receptor, respectively, they recruit distinct sets of adaptor proteins, this results in the activation of the inhibitor of kB (IkB) kinase as a common target (Hayden and Ghosh, 2008; Bhoj and Chen, 2009). Immediately after IkB kinase phosphorylates IkB, which prevents the nuclear translocation of the NF-kB subunits, phosphorylated IkB undergoes ubiquitination and proteolytic degradation by proteasomes (Perkins, 2006). The NF-kB subunits are released and translocated to the nucleus where they stimulate the transcription of diverse target genes that regulate inflammatory responses, including the gene encoding intercellular adhesion molecule-1 (ICAM-1) (Roebuck and Finnegan, 1999).
n
Corresponding author. Tel./fax: þ 81 75 724 7752. E-mail address: [email protected] (T. Kataoka).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.01.019
TNF-a-converting enzyme (TACE), also known as a disintegrin and metalloproteinase 17 (ADAM17), is a cell-surface metalloproteinase that mediates the ectodomain shedding of various ligands (e.g., TNF-a) and receptors (e.g., TNF receptor 1) (Scheller et al., 2011). TACE is ubiquitously expressed in many cell types and its activity is regulated by different mechanisms, including its posttranslational modification (Scheller et al., 2011). In response to various stimuli, extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase phosphorylate the cytoplasmic tail of TACE at threonine 735 and thereby TACEdependent ectodomain shedding is induced (Dı´az-Rodrı´guez et al., 2002; Soond et al., 2005; Liu et al., 2009; Xu and Derynck, 2010). Several translation inhibitors are known to induce the activation of the MAP kinase superfamily by interacting with ribosomes in an intracellular mechanism designated the ribotoxic stress response (Iordanov et al., 1997; Kataoka, 2012). Recently, we have shown that different translation inhibitors (acetoxycycloheximide and cytotrienin A) induce the ectodomain shedding of TNF receptor 1 via the activation of ERK and p38 MAP kinase (Ogura et al., 2008a, 2008b; Yamada et al., 2011a). Trichothecene mycotoxins are a large group of sesquiterpenoids produced by Fusarium and other fungi. They are often found
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as contaminants in agricultural staples and are known to exert acute and chronic effects on animals and humans (Pestka et al., 2004; Pestka, 2010). Trichothecene mycotoxins interact with eukaryotic ribosomes and block the peptidyl transferase reaction. They also exhibit both immunosuppressive and immunostimulatory activities (Pestka et al., 2004; Pestka 2010). In addition to the induction of cell death in leukocytes, deoxynivalenol also upregulates the expression of proinflammatory cytokines (e.g., TNF-a) and chemokines in macrophages and monocytes via the ribotoxic stress response (Chung et al., 2003; Islam et al., 2006). However, it remains unclear whether deoxynivalenol modulates the intracellular signaling pathways induced by proinflammatory cytokines. In this study, we investigated the biological activities of deoxynivalenol and its structural analogs in the NF-kB signaling pathway and the gene expression in response to proinflammatory cytokines. Our results demonstrate for the first time that deoxynivalenol rapidly induces the ectodomain shedding of TNF receptor 1 and thereby inhibits the TNF-a-induced NF-kB signaling pathway.
2. Materials and methods 2.1. Cell culture Human lung carcinoma A549 cells (JCRB0076) and human hepatocellular carcinoma HepG2 cells (RCB1648) were provided by the Health Science Research Resources Bank (Tokyo, Japan) and RIKEN BRC through the National Bio-Resource Project of MEXT, Japan (Tsukuba, Japan), respectively. A549 cells and HepG2 cells were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) heat-inactivated fetal calf serum (JRH Biosciences, Lenexa, KS, USA) and a mixed penicillin–streptomycin solution (Nacalai Tesque Inc., Kyoto, Japan).
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TNF receptor 1 (H-5; Santa Cruz Biotechnology) were obtained commercially.
2.4. Assay for cell-surface expression of ICAM-1 A549 cells were washed twice with phosphate-buffered saline (PBS) and incubated with 1% paraformaldehyde–PBS for 15 min. The fixed cells were washed twice with PBS and incubated overnight in the presence of 1% bovine serum albumin (SigmaAldrich)–PBS for blocking. Then, the cells were incubated with mouse anti-human ICAM-1 IgG antibody (clone 15.2) for 60 min and thereafter washed three times with 0.02% Tween 20–PBS. The cells were further incubated with horseradish peroxidase (HRP)-linked anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 60 min and then washed three times with 0.02% Tween-20–PBS. The cells were incubated with the substrate solution (0.2 M sodium citrate (pH 5.3), 0.1% o-phenylenediamine dihydrochloride, 0.02% H2O2) at 37 1C for 20 min. The absorbance at 415 nm was measured with a Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
2.5. Assay for macromolecular synthesis A549 cells were pulse-labeled with [4,5-3H]L-leucine (41. 66 TBq/mmol; Moravek Biochemicals, Inc., Brea, CA, USA). The cells were washed three times with PBS and lysed with 0.25 M NaOH for 15 min. The proteins were precipitated by incubation on ice for 1 h in the presence of 5% trichloroacetic acid. Cell lysates were separated into supernatants and precipitates by centrifugation (10,000 g, 5 min). The precipitates were washed once with 5% trichloroacetic acid. Radioactivity of the supernatants and the precipitates was measured with a 1900CA TRI-CARBs liquid scintillation analyzer (Packard Instrument Co., Meriden, CT, USA).
2.2. Reagents Deoxynivalenol, 3-acetyldeoxynivalenol, verrucarin A, and 1,9-pyrazoloanthrone (SP600125) were purchased from SigmaAldrich Co. (St. Louis, MO, USA). T-2 toxin (Enzo Life Sciences International, Inc., Plymouth Meeting, PA, USA), N-(R)-(2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-L-t-butyl-glycylL-alanine 2-aminoethyl amide (TAPI-2; Peptide Institute, Inc., Osaka, Japan), 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126; Wako Pure Chemical Industries, Ltd., Osaka, Japan), and 4-(4-fluorophenyl)-2-(4-methysulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580; Cayman Chemical Co., Ann Arbor, MI, USA) were obtained commercially. Recombinant human TNF-a and human IL-1a were kindly provided by Dainippon Pharmaceutical (Osaka, Japan). 2.3. Antibodies Antibodies to b-actin (AC-15; Sigma-Aldrich), cyclooxygenase2 (Cox-2) (Clone 33; BD Biosciences, Franklin Lakes, NJ, USA), ERK1/ERK2 (#9102; Cell Signaling Technology, Inc., Danvers, MA, USA), cellular FLICE-inhibitor protein (c-FLIP) (Dave-2; Alexis Co., Lausen, Switzerland), ICAM-1 (clone 15.2; Leinco Technologies, Inc., St. Louis, MO, USA), ICAM-1 (clone 28; BD Biosciences), NFkB p50 (H-119; Santa Cruz Biotechnology, Santa Cruz, CA, USA), NF-kB p65 (C-20; Santa Cruz Biotechnology), p38 MAP kinase (#9212; Cell Signaling Technology), poly(ADP–ribose) polymerase (PARP) (C-2–10; Sigma-Aldrich), phospho-ERK1/ERK2 (Thr202/ Tyr204) (#9101; Cell Signaling Technology), phospho-p38 MAP kinase (Thr180/Tyr182) (#9211; Cell Signaling Technology), and
2.6. Preparation of cell lysates and western blotting A549 cells were washed once with PBS and lysed with Triton X-100 lysis buffer (50 mM Tris–HCl (pH 7.4), 1% Triton X-100, the protease inhibitor mixture CompleteTM (Roche Diagnostics, Mannheim, Germany), 2 mM DTT, 2 mM orthovanadate). Cell lysates were centrifuged (10,000 g, 5 min) and separated into supernatants as cytoplasmic fractions and pellets. The pellets were washed twice with Triton X-100 lysis buffer and then solubilized as nuclear fractions. The culture medium was centrifuged (10,000 g, 5 min) to remove cell debris and insoluble materials. The proteins were then precipitated with chloroform/ methanol. The protein samples (30 mg/lane) were separated by SDS–PAGE and transferred onto Hybond-ECL nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA). The membranes were incubated overnight with 4% skim milk in 0.5% Tween 20–PBS for blocking, and then incubated with the primary antibodies and HRP-linked secondary antibodies (Jackson ImmunoResearch). The protein bands were detected with ECL Western blotting detection reagents (GE Healthcare) and analyzed with the ImageQuant LAS 4000 mini (GE Healthcare).
2.7. Statistical analysis Statistical significance was assessed with one-way ANOVA followed by the Tukey test for multiple comparisons. Differences with P values of o0.05 were considered to be statistically significant.
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3. Results 3.1. Deoxynivalenol and 3-acetyldeoxynivalenol inhibit TNF-ainduced ICAM-1 expression more strongly than IL-1a-induced ICAM1 expression The trichothecene mycotoxins, deoxynivalenol, 3-acetyldeoxynivalenol, T-2 toxin, and verrucarin A (Supplementary Fig. 1), were investigated for their biological effects on the NF-kBresponsive gene expression induced by proinflammatory cytokines. Human lung carcinoma A549 cells were highly responsive to TNF-a or IL-1a, which induced cell-surface ICAM-1 expression in an NF-kB-dependent manner. Deoxynivalenol inhibited TNF-ainduced ICAM-1 expression in a dose-dependent manner and the inhibition was approximately 8-fold stronger than its inhibition of IL-1a-induced ICAM-1 expression (Fig. 1A and Supplementary Table 1). The inhibitory effects of 3-acetyldeoxynivalenol were at least 10-fold weaker than those of deoxynivalenol; nevertheless, 3-acetyldeoxynivalenol still inhibited the TNF-a-induced ICAM-1 expression in preference to the IL-1a-induced one (Fig. 1B and Supplementary Table 1). In contrast, T-2 toxin and verrucarin A inhibited both TNF-a- and IL-1a-induced ICAM-1 expression at almost equivalent concentrations with IC50 values less than 10 nM (Fig. 1C and D and Supplementary Table 1). Low to high concentrations of the trichothecene mycotoxins alone did not upregulate ICAM-1 expression in A549 cells (data not shown).
3.2. Deoxynivalenol inhibits TNF-a-induced expression of NF-kBresponsive genes In addition to the cell-surface expression of ICAM-1 measured by the Cell-ELISA assay, the total amount of ICAM-1 in the cell lysates was analyzed by Western blotting. Consistent with the results shown in Fig. 1, deoxynivalenol and 3acetyldeoxynivalenol inhibited the TNF-a-induced ICAM1expression more strongly than the IL-1a-induced one (Fig. 2A and B), whereas T-2 toxin and verrucarin A inhibited the TNF-aand IL-1a-induced ICAM-1 expression at similar concentrations (Fig. 2C and D). In human hepatocellular carcinoma HepG2 cells, ICAM-1was expressed constitutively in the absence of cytokines and ICAM-1 expression was increased to some extent by TNF-a or IL-1a (Fig. 2E). The constitutive expression of ICAM-1 was only weakly decreased by deoxynivalenol even at 10 mM in HepG2 cells (Fig. 2E). Thus, it is most likely that deoxynivalenol inhibits primarily the de novo synthesis of ICAM-1 protein, but barely affects the stability of ICAM-1 protein per se. In addition to ICAM-1, c-FLIP and Cox-2 are known to be NF-kB-responsive genes. As previously shown (Ogura et al., 2008a), TNF-a upregulated the cellular levels of c-FLIP (especially c-FLIPS) and Cox-2 in A549 cells (Fig. 2F). Deoxynivalenol inhibited the TNF-a-induced expression of c-FLIPS and Cox-2 (Fig. 2F). These results indicate that deoxynivalenol inhibits the TNF-a-induced expression of NF-kB-responsive genes.
Fig. 1. Deoxynivalenol and 3-acetyldeoxynivalenol inhibit TNF-a-induced ICAM-1 expression more strongly than IL-1a-induced ICAM-1 expression. (A–D) A549 cells were preincubated with various concentrations of deoxynivalenol (A), 3-acetyldeoxynivalenol (B), T-2 toxin (C), and verrucarin A (D) for 1 h and then incubated with TNF-a (2.5 ng/ml; filled circles) or IL-1a (0.25 ng/ml; open circles) for 6 h in the presence of the compounds. ICAM-1 expression (%) is shown as mean 7 S.D. (n¼ 3). nnP o 0.01 compared with control.
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Fig. 2. Deoxynivalenol inhibits TNF-a-induced expression of NF-kB-responsive genes. (A–D) A549 cells were preincubated with various concentrations of deoxynivalenol (A), 3-acetyldeoxynivalenol (B), T-2 toxin (C), and verrucarin A (D) for 1 h and then incubated with ( þ ) or without ( ) TNF-a (2.5 ng/ml) or IL-1a (0.25 ng/ml) for 6 h in the presence of the compounds. The cell lysates were analyzed by Western blotting. (E) HepG2 cells were incubated with ( þ ) or without ( ) TNF-a (2.5 ng/ml) or IL-1a (0.25 ng/ml) (left panel) and various concentrations of deoxynivalenol without cytokines (right panel) for 6 h. The cell lysates were analyzed by Western blotting. (F) A549 cells were preincubated with various concentrations of deoxynivalenol for 1 h and then incubated with ( þ ) or without (–) TNF-a (2.5 ng/ml) for 6 h in the presence of deoxynivalenol. The cell lysates were analyzed by Western blotting.
3.3. Trichothecene mycotoxins inhibit cellular protein synthesis Deoxynivalenol, 3-acetyldeoxynivalenol, T-2 toxin, and verrucarin A barely affected cell viability in the presence or absence of TNF-a or IL-1a (Supplementary Fig. 2), excluding the possibility that the inhibition of ICAM-1 expression by trichothecene mycotoxins is due to cytotoxic effects. To further evaluate the inhibitory effects of trichothecene mycotoxins on de novo protein synthesis, the incorporation of [3H]L-leucine into the acidinsoluble macromolecular fractions of A549 cells was measured. Deoxynivalenol and 3-acetyldeoxynivalenol inhibited cellular protein synthesis in a dose-dependent manner with IC50 values of 0.7 and 20 mM, respectively, whereas T-2 toxin and verrucarin A exerted much stronger inhibitory effects on protein synthesis with IC50 values of 8.6 and 2.1 nM, respectively (Supplementary Table 1). Deoxynivalenol, 3-acetyldeoxynivalenol, T-2 toxin, and verrucarin A also caused a marked increase in the radioactivity of the acidsoluble fractions, accompanied by a reduction in the radioactivity of the acid-insoluble fractions (Supplementary Fig. 3), confirming that these trichothecene mycotoxins did not affect the uptake of amino acids, but primarily inhibited the translation process. These results
indicate that the inhibitory effects of trichothecene mycotoxins on ICAM-1 expression are related to their inhibition of cellular protein synthesis.
3.4. Deoxynivalenol and 3-acetyldeoxynivalenol inhibit the NF-kB signaling pathway induced by TNF-a, but not that induced by IL-1a In response to TNF-a or IL-1a, NF-kB heterodimers composed of p65 and p50 were translocated from the cytoplasm to the nuclei of A549 cells. Deoxynivalenol inhibited the TNF-a-induced nuclear translocation of p65 and p50 at concentrations higher than 1 mM, whereas it barely affected the IL-1a-induced nuclear translocation of NF-kB (Fig. 3A). 3-Acetyldeoxynivalenol also markedly inhibited the TNF-a-induced nuclear translocation of p65 and p50 at 100 mM (Fig. 3B). In contrast, T-2 toxin and verrucarin A only weakly affected the TNF-a-induced nuclear translocation of NF-kB, even at much higher concentrations than those required for the inhibition of cellular protein synthesis (Fig. 3C and D). These results suggest that the selective inhibition of TNF-a-induced ICAM-1 expression by deoxynivalenol and
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Fig. 3. Deoxynivalenol and 3-acetyldeoxynivalenol inhibit TNF-a-induced nuclear translocation of the NF-kB subunits. (A–D) A549 cells were preincubated with various concentrations of deoxynivalenol (A), 3-acetyldeoxynivalenol (B), T-2 toxin (C), and verrucarin A (D) for 1 h and then incubated with ( þ) or without (–) TNF-a (2.5 ng/ml) or IL-1a (0.25 ng/ml) for 30 min in the presence of the compounds. The nuclear and cytoplasmic fractions were analyzed by Western blotting.
3-acetyldeoxynivalenol is largely attributable to the inhibition of the NF-kB signaling pathway. 3.5. Deoxynivalenol and 3-acetyldeoxynivalenol induce the ectodomain shedding of TNF receptor 1 We previously showed that translation inhibitors, such as acetoxycycloheximide and cytotrienin A, induced the ectodomain shedding of TNF receptor 1 (Ogura et al., 2008a; Yamada et al., 2011a). To determine whether deoxynivalenol and 3-acetyldeoxynivalenol also induced TNF receptor 1 shedding, A549 cells were exposed to these compounds for 1 h, and then culture media and cell lysates were analyzed by Western blotting using anti-TNF receptor 1 antibody. At concentrations of 1 and 10 mM, deoxynivalenol increased the amount of soluble TNF receptor 1 in the medium, and this was accompanied by a reduction of TNF receptor 1 expression in the cells (Fig. 4A). 3-Acetyldeoxynivalenol at concentrations higher than 10 mM also increased the amount of soluble TNF receptor 1 in the medium (Fig. 4B). The results indicate that deoxynivalenol and 3-acetyldeoxynivalenol induce the ectodomain shedding of TNF receptor 1. 3.6. Ectodomain shedding of TNF receptor 1 by deoxynivalenol causes the preferential inhibition of TNF-a-induced ICAM-1 expression TACE is known to mediate the proteolytic cleavage of TNF receptor 1 (Scheller et al., 2011). The TACE inhibitor TAPI-2 markedly suppressed the elevation of soluble TNF receptor 1 released by deoxynivalenol-treated A549 cells (Fig. 5A), indicating that deoxynivalenol induced the ectodomain shedding of TNF receptor 1 by TACE. We further investigated whether the ectodomain shedding of TNF receptor 1 was responsible for the
preferential inhibition of TNF-a-induced ICAM-1 expression by deoxynivalenol. In the presence of TAPI-2, relatively similar concentrations of deoxynivalenol inhibited both TNF-a- and IL-1a-induced ICAM-1 expression (Fig. 5B), corresponding to the IC50 values of 2.1 and 4.2 mM, respectively (calculated from three independent experiments, including that shown in Fig. 5B), in contrast to the IC50 values of 0.56 mM and 4.5 mM, respectively, in the absence of TAPI-2 (Supplementary Table 1).
3.7. Deoxynivalenol induces ectodomain shedding of TNF receptor 1 via ERK and p38 MAP kinase TACE activity is regulated by ERK and p38 MAP kinase in response to various stimuli (Scheller et al., 2011). Upon treatment with deoxynivalenol, ERK1/ERK2 phosphorylation was increased during the period of 5–30 min in A549 cells (Fig. 6A). In addition, p38 MAP kinase was phosphorylated within 5 min following exposure to deoxynivalenol and it remained phosphorylated for as long as 2 h (Fig. 6A). To investigate whether these members of the MAP kinase superfamily were necessary for the ectodomain shedding of TNF receptor 1 induced by deoxynivalenol, specific kinase inhibitors were used as blocking agents. The MAP kinase or ERK kinase (MEK) inhibitor U0126 and the p38 MAP kinase inhibitor SB203580 reduced the increase in the amount of soluble TNF receptor 1 released by deoxynivalenol-treated A549 cells, whereas the c-Jun N-terminal kinase (JNK) inhibitor SP600125 had no such effect (Fig. 6B). The ectodomain shedding of TNF receptor 1 induced by deoxynivalenol was completely suppressed by the combined treatment with U0126 and SB203580 (Fig. 6B). Consistent with these data, almost equivalent concentrations of deoxynivalenol inhibited the ICAM-1 expression induced by TNFa and that induced by IL-1a, when A549 cells were treated with a combination of U0126 and SB203580 (Fig. 6C).
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Fig. 4. Deoxynivalenol and 3-acetyldeoxynivalenol induce the ectodomain shedding of TNF receptor 1. (A and B) A549 cells were incubated with various concentrations of deoxynivalenol (A) or 3-acetyldeoxynivalenol (B) for 1 h. The cell lysates (cell) and the culture media (medium) were analyzed by Western blotting.
Fig. 5. The TACE-dependent ectodomain shedding of TNF receptor 1 induced by deoxynivalenol is responsible for its selective inhibition of TNF-a-induced ICAM-1 expression. (A) A549 cells were preincubated with ( þ ) or without (–) TAPI-2 (25 mM) for 1 h and then incubated with (þ ) or without (–) deoxynivalenol (1 mM) for 1 h in the presence or absence of TAPI-2. The cell lysates (Cell) and the culture media (Medium) were analyzed by Western blotting. (B) A549 cells were pretreated with or without TAPI-2 (25 mM) for 1 h. Then, the cells were treated with various concentrations of deoxynivalenol for 1 h and incubated with TNF-a (2.5 ng/ml; filled circles) or IL-1a (0.25 ng/ml; open circles) for 6 h in the presence of absence of TAPI-2 or deoxynivalenol. ICAM-1 expression (%) is shown as mean 7 S.D. (n¼3). nP o 0.05 and nn P o 0.01 compared with control.
4. Discussion Trichothecene mycotoxins are known translation inhibitors. In this study, we found that deoxynivalenol and 3-acetyldeoxynivalenol inhibited TNF-a-induced ICAM-1 expression more strongly than IL-1a-induced ICAM-1 expression, largely by suppressing the
TNF-a-induced NF-kB signaling pathway. The TACE inhibitor TAPI-2 or the MEK inhibitor U0126 plus the p38 MAP kinase inhibitor SB203580 markedly suppressed the ectodomain shedding of TNF receptor 1 induced by deoxynivalenol, and reversed the selective inhibition of TNF-a-induced ICAM-1 expression by deoxynivalenol. These results demonstrate for the first time that deoxynivalenol
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Fig. 6. Deoxynivalenol induces the ectodomain shedding of TNF receptor 1 via ERK and p38 MAP kinase. (A) A549 cells were incubated with deoxynivalenol (1 mM) for the indicated times. Phosphorylated and total amounts of ERK and p38 MAP kinase were analyzed by Western blotting. (B) A549 cells were pretreated with or without U0126 (10 mM), SB203580 (10 mM), SP600125 (10 mM), or U0126 (10 mM) plus SB203580 (10 mM) for 1 h, and then treated with (þ) or without (–) deoxynivalenol (1 mM) for 1 h in the presence or absence of protein kinase inhibitors. The cell lysates (Cell) and the culture media (Medium) were analyzed by Western blotting. (C) A549 cells were pretreated with or without U0126 (10 mM), SB203580 (10 mM), SP600125 (10 mM), or U0126 (10 mM) plus SB203580 (10 mM) for 1 h. Then, the cells were treated with various concentrations of deoxynivalenol for 1 h and incubated with TNF-a (2.5 ng/ml; filled circles) or IL-1a (0.25 ng/ml; open circles) for 6 h in the presence or absence of the protein kinase inhibitors or deoxynivalenol. ICAM-1 expression (%) is shown as mean 7 S.D. (n¼ 3). **P o0.01 compared with control.
induces the TACE-dependent ectodomain shedding of TNF receptor 1 by activating ERK and p38 MAP kinase, thereby inhibiting TNF-ainduced NF-kB activation. Trichothecene mycotoxins have a common tricyclic 12,13-epoxytrichothec-9-ene core structure and are classified into structural types based on the substitution pattern at the C-8 position (types A and B) and the additional ring linking the C-4 and C-15 positions (type D) (McCormick et al., 2011). In this study, we investigated the capacities of four trichothecene mycotoxins to inhibit the ICAM-1 expression induced by TNF-a or IL-1a. Deoxynivalenol (type B) and 3-acetyldeoxynivalenol (type B), but neither T-2 toxin (type A) nor verrucarin A (type D), inhibited TNF-a-induced ICAM-1 expression more strongly than IL-1a-induced ICAM-1 expression. Recently, we have shown that the selective inhibition of TNF-a-induced ICAM-1 expression by translation inhibitors is attributable to the ribotoxic stress response, which induces the downregulation of cell-surface TNF receptor 1 expression (Ogura et al., 2008a; Yamada et al., 2011a). Trichothecene mycotoxins are classified into strong (e.g., deoxynivalenol), intermediate (e.g., 3-acetyldeoxynivalenol), and weak (e.g., T-2 toxin, verrucarin A) inducers of the ribotoxic stress response at the concentration of 10 mM (Shifrin and Anderson, 1999). Those studies are in agreement with our present finding that
TNF-a-induced ICAM-1 expression is selectively inhibited by deoxynivalenol and 3-acetyldeoxynivalenol, but by neither T-2 toxin nor verrucarin A. It has also been shown that some type A trichothecene mycotoxins induce the ribotoxic stress response as efficiently as deoxynivalenol (Shifrin and Anderson, 1999). Unlike the type B trichothecene mycotoxins that have a carbonyl group at the C-8 position, only selected structural derivatives of the type A trichothecene mycotoxins, which have other groups at the C-8 position, are likely to induce the ribotoxic stress response. Several translation inhibitors can induce the ribotoxic stress response, which leads to the activation of the MAP kinase superfamily (Iordanov et al., 1997; Kataoka, 2012). The TACE-dependent ectodomain shedding of cell-surface proteins is upregulated by ERK and p38 MAP kinase, which phosphorylate threonine 735 in the cytoplasmic tail of TACE (Dı´az-Rodrı´guez et al., 2002; Soond et al., 2005; Liu et al., 2009; Xu and Derynck, 2010). Consistent with those studies, we have recently shown that different translation inhibitors (acetoxycycloheximide and cytotrienin A) induce the TACE-dependent ectodomain shedding of TNF receptor 1 by activating ERK and p38 MAP kinase (Ogura et al., 2008a; Ogura et al., 2008b; Yamada et al., 2011a). Acetoxycycloheximide induced the maximum activation of ERK and p38 MAP kinase within 15 min and subsequently stimulated the
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ectodomain shedding of TNF receptor 1 at 30–60 min (Kadohara et al., 2005; Ogura et al., 2008a; Ogura et al., 2008b). The kinetics of MAP kinase activation by acetoxycycloheximide is similar to the kinetics of its activation by deoxynivalenol (Zhou et al., 2005). Moreover, as previously observed in acetoxycycloheximide and cytotrienin A (Ogura et al., 2008b; Yamada et al., 2011a), the pharmacological inhibition of the ERK pathway or the p38 MAP kinase pathway suppressed the TACE-dependent ectodomain shedding of TNF receptor 1 induced by deoxynivalenol. Thus, it is highly likely that the rapid and strong activation of ERK and p38 MAP kinase by translation inhibitors is required for the TACE-dependent ectodomain shedding of TNF receptor 1. Deoxynivalenol exerts both immunostimulatory (e.g., activation of gene expression) and immunosuppressive (e.g., induction of cell death) effects depending on the experimental conditions, including the dose and duration of exposure (Pestka et al., 2004; Pestka, 2010). Among its immunostimulatory effects, deoxynivalenol upregulates the expression of proinflammatory cytokines and chemokines by activating their transcription and increasing the stability of their mRNAs (Chung et al., 2003; Islam et al., 2006; Gray and Pestka, 2007; Choi et al., 2009). In particular, it has been shown that TNF-a production is upregulated by deoxynivalenol in macrophages (Chung et al., 2003; Zhou et al., 2005). In this study, we showed that deoxynivalenol induced the ectodomain shedding of TNF receptor 1 by TACE and thereby diminished the activity of the TNF-a-induced NF-kB signaling pathway. Therefore, in contrast to other proinflammatory cytokines, deoxynivalenol may exert two opposite effects in regulating TNF-a-dependent cellular responses: the upregulation of TNF-a production in selected cells, including macrophages, and the downregulation of TNF-a responsiveness in many cells ubiquitously expressing TNF receptor 1 and TACE. In conclusion, our data demonstrated that deoxynivalenol induced the ectodomain shedding of TNF receptor 1 via the activation of ERK and p38 MAP kinase, a novel biological effect of trichothecene mycotoxins. We have previously evaluated the biological effects of translation inhibitors on ICAM-1 expression and cellular protein synthesis (Yamada et al., 2011b). The present and previous results indicate that deoxynivalenol is likely to induce the ribotoxic stress response as efficiently as other translation inhibitors. Trichothecene mycotoxins are a family of over 200 derivatives, which may include active inducers of the ribotoxic stress response with different efficacies as well as inactive analogs. Therefore, as representative translation inhibitors, trichothecene mycotoxins may be useful tools in determining the molecular basis of the ribotoxic stress response.
Acknowledgment This work was supported in part by JSPS KAKENHI Grant number 22380060.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2013. 01.019.
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