* Department of Food Science and Human Nutrition;
Institute for Environmental Toxicology; and
Department of Microbiology and Molecular Genetics, Michigan State University, 234 G.M. Trout Bldg., East Lansing, Michigan 488241224
Received April 4, 2002; accepted June 4, 2002
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ABSTRACT |
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Key Words: tricothecenes; mycotoxins; vomitoxin; immunotoxins; COX-2 expression.
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INTRODUCTION |
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Animals exhibit feed refusal and weight loss upon chronic exposure to low dietary VT concentrations, whereas acute high-level exposure to VT can cause nausea, vomiting, and leukocytosis in experimental animals (Rotter et al., 1996). The immune system is particularly susceptible to VT with macrophages, B cells, and T cells being highly sensitive to the toxin (Bondy et al., 2000
). Notably, mice exposed to VT develop clinical signs that mimic human IgA nephropathy (Dong et al.; 1991
). The broad spectrum of toxicity found for VT and other trichothecenes is likely to relate, in part, to their capacity to evoke production of proinflammatory mediators (Azcona-Olivera et al., 1995a
,b
; Ji et al., 1998
; Rizzo et al., 1992
, 1994
; Wong et al., 1998
; Zhou et al., 1997
, 1998
).
Metabolites of arachidonic acid are known to play key roles in the proinflammatory responses (Smith et al., 2000). Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the oxygenation of arachidonic acid to prostaglandin endoperoxides. These metabolites are converted enzymatically into prostaglandins and thromboxane A2, which play both physiologic and pathologic roles in a diverse array of inflammatory sequelae (Smith et al., 2000
; Vane et al., 1998
). Two distinct isoforms of COX have been identified. COX-1 is constitutively expressed at low levels in most tissues and may be related to housekeeping function. In contrast, COX-2, a 70-kD protein, is strongly induced by mitogenic and proinflammatory stimuli, superinduced by protein synthesis inhibitors, and can be regulated at both transcriptional and post-transcriptional levels (Dixon et al., 2000
; Fletcher et al., 1992
; Newton et al., 1997a
,b
Newton et al., 1998; Wadleigh et al., 2000
).
Several inflammatory stimuli that induce COX-2 gene expression also activate the mitogen-activated protein kinases (MAPKs). Among the MAPKs, c-Jun N-terminal kinases 1 and 2 (JNK1/2), extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), and p38 MAPK have been extensively studied relative to their regulation of COX-2 gene expression (Guan et al., 1998; Scherle et al., 1998
; Ridley et al., 1998
; Xie et al., 1995
). Upon exposure to the prototypic inflammagen lipopolysaccharide (LPS), transcriptional regulation of COX-2 gene expression is redundantly modulated by the 3 MAPK families (Mestre et al., 2001
; Wadleigh et al., 2000
), whereas p38 plays an important role in the signaling pathway for the stability of COX-2 mRNA (Dean et al., 1999
; Lasa et al. 2000
).
Several investigations have suggested that trichothecenes can increase arachidonic acid metabolism and upregulate prostaglandin production (Naseem et al., 1989; Shohami and Feuerstein, 1986
). Recently, we have determined that a single acute exposure to VT induces COX-2 gene expression in the murine spleen (Islam et al., 2002
). Furthermore, trichothecenes have been suggested to activate MAPKs via a ribotoxic stress response (Shifrin and Anderson, 1999
; Yan et al., 2000
). Based on these observations, we hypothesized that VT induces COX-2 expression in macrophages, and that this was regulated at the level of MAPKs. The aim of this study was two-fold: first, to assess the effects of VT on COX-2 mRNA and protein expression in the RAW 264.7 macrophage model, and second, to relate VT-induced MAPK activation to transcriptional and post-transcriptional regulation of COX-2 gene expression. The results indicate that VT induces COX-2 expression and that MAPKs play critical roles in both transcriptional and post-transcriptional regulation of this gene response.
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MATERIALS AND METHODS |
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Prostaglandin E2 (PGE2) assay.
PGE2 was measured using an EIA kit (Cayman Chemical Co., Ann Arbor, MI). Cell culture supernatants were collected 18 h after VT treatment. The aliquots were diluted 10-fold in fresh culture medium and the assays were conducted according to the instructions of the supplier.
Western blot analysis.
At the time of harvest, cells were washed with ice-cold phosphate buffer, lysed in boiling lysis buffer (1% [w/v] SDS, 1.0 mM sodium ortho-vanadate, and 10 mM Tris pH 7.4), and sonicated for 5 s. Protein was measured by the Bradford assay (Bio-Rad, Cambridge, MA). Extracts (10 µg) were mixed with Laemmli sample buffer (Bio-Rad) and boiled for 5 min before resolving on a 10% (w/v) acrylamide gel. Resolved proteins were transferred to PVDF membrane and blocked with Tris-buffered saline (10 mM TrisHCl pH 7.5, 100 mM NaCl) containing 0.1 % (v/v) Tween-20 and 1% (w/v) BSA (TBST-BSA). The membrane was incubated for 1 h with MAPK antibodies (rabbit IgG, New England Biolabs, Beverly, MA) at a 1:1000 dilution or COX-2 antibody (mouse IgG1; Transduction Laboratories, Lexington, KY) at a 1:250 dilution in TBST-BSA, and then was washed 3 times with TBST. The membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling, Beverly, MA) at a 1:5000 dilution in TBST-BSA for MAPK detection or with HRP-conjugated anti-mouse IgG (Sigma, St. Louis, MO) at a 1:10,000 dilution for COX-2 detection. After washing 3 times with TBST, bound HRP-conjugated antibody was detected with the Enhanced Chemiluminescence (ECL) kit (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturers instructions.
Reverse transcription-competitive polymerase chain reaction (RT-cPCR).
RNA was extracted with Trizol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturers instructions. RNA (100 ng) from each sample was transcribed to cDNA by reverse transcriptase, according to Riedy et al.(1995). COX-2 cDNA was amplified competitively with a truncated COX-2 cDNA internal standard constructed by the bridging-deletion method (Hall et al., 1998). The amplification was performed in a 9600 Perkin Elmer Cycler (Perkin-Elmer Corp., Norwalk, CT) using the following parameters: 30 cycles of reactions of denaturation at 94°C for 30 s, annealing at 56°C for 45 s, and elongation at 72°C for 45 s. An aliquot of each PCR product was subjected to 1.5% (w/v) agarose gel electrophoresis and visualized by staining with ethidium bromide. Primers were synthesized at Michigan State Universitys Molecular Structure facility. The 5 forward and 3 reverse-complement PCR primers for amplification of mouse COX-2 cDNA were ACACTCTATCACTGGCATCC and GAAGGGACACCCTTTCACAT, respectively. Sizes of amplified COX-2 cDNA internal standard cDNA were 584 and 500 base pairs (bp), respectively. The densitometric ratio of COX-2 cDNA/COX-2 internal standard was used to construct a standard curve to calculate COX-2 cDNA concentrations in RT reaction products.
Plasmids and transfections.
The 5 upstream segment (-724/+7) of the mouse COX-2 gene from spleen chromosomal DNA was cloned into pXP2 (ATCC, Manassas, VA) at Hind III/Xho I restriction sites to construct a mouse COX-2 promoter-luciferase plasmid (pXP-5COX2). An expression vector for the dominant negative JNK1 (dnJNK) was kindly provided by Roger Davis (University of Massachusetts Medical School, Worcester, MA). All plasmids were purified with Endofree Plasmid Prep Kit (Qiagen, Valencia, CA).
For transfections, RAW 264.7 cells (4 x 105/ml) were washed twice in serum-free DMEM and then incubated for 3 h with a premixed complex of plasmid and lipopectamine (Life Technologies, Gaithersburg, MD) according to the manufacturers instructions. The cells were then replaced with fresh serum-containing DMEM and incubated for 24 h prior to VT exposure. pCMV-ß-Gal (BD Biosciences Clontech, Palo Alto, CA) was also co-transfected with 1 µg promoter-luciferase construct to standardize transfection efficiency.
JNK activity assay.
For determination of JNK activity, cells (5 x 105) were washed once with ice-cold PBS and incubated for 5 min with 0.5 ml ice-cold lysis buffer (20 mM Tris [pH 7.4] containing 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na3 VO4, 1 µg/ml leupeptin, and 1 mM PMSF). After incubation, cells were harvested, sonicated, centrifuged at 14,000 rpm in a microcentrifuge for 10 min, and the supernatant was collected for the assay. c-Jun fusion protein beads (20 µg; Cell Signaling, Beverly, MA) were added to the supernatant (250 µl), and the mixture was incubated with gentle rocking overnight at 4EC. The bead complex was collected, suspended, and incubated with 100 µM ATP in kinase buffer (25 mM Tris (pH 7.5), 5 mM ß-glycerolphosphate, 2 mM DTT, 0.1 mM Na3 VO4, 10 mM MgCl2) for 30 min at 30°C. The reaction was terminated by Laemmli sample buffer and centrifuged. The supernatant was analyzed by Western immunoblotting with phospho-c-Jun rabbit antibody (Cell Signaling, Beverly, MA).
Luciferase and ß-galactosidase assays.
Cells were washed with cold PBS, lysed with lysis buffer (25 mM Tris-H3PO4: pH 7.8, 2 mM EDTA, 2 mM DTT, 10% [v/v] glycerol, 1% [v/v] Triton X-100) and then centrifuged at 12,000 x g for 2 min. Resultant supernatant was stored at 80°C until assessment of luciferase activity and ß-galactosidase. Luciferase activity was measured with a luminometer (Model 20e, Turner Designs Co., Sunnyvale, CA) after briefly mixing the supernatant with an equal-volume Luciferase Assay Systems substrate solution (Promega, Madison, WI). ß-Galactosidase activity was measured with ß-galactosidase Enzyme Assay Kit (Promega, Madison, WI). Luciferase activity was normalized against ß-galactosidase activity using the following formula: luciferase activity/ß-galactosidase activity.
Statistics.
Data were analyzed using Sigma Stat for Windows (Jandel Scientific, San Rafael, CA). For comparisons of 2 groups of data, Students t-test was performed. For comparisons of multiple groups of data, a Kruskal-Wallis 1-way analysis of variance on ranks was performed. Differences were considered significant if p < 0.05. Quantitative results were expressed as mean ± SEM.
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RESULTS |
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Role of MAPKs in VT-induced transcriptional activation of COX-2 expression.
To analyze the effect of VT on COX-2 promoter activity, the 5 COX-2 promoter region was cloned into a luciferase reporter vector (pXP2) system, and the resultant plasmid (pXP-5COX2) was transiently transfected into RAW 264.7 cells. VT, at concentrations of 10 to 250 ng/ml, significantly increased luciferase expression dose-dependently in the transfected cells at 12 h (Fig. 5). LPS (200 ng/ml) was used as the positive control, because it has also been shown to activate COX-2 promoter activity in RAW 264.7 cells (Paul et al., 1999
; Wadleigh et al., 2000
). In contrast, VT had no effect on luciferase activity in cells transfected with the empty vector, pCMV5 (data not shown).
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Role of p38 MAPK in VT-induced post-transcriptional regulation of COX-2 expression.
Stability of mRNA is also a potentially important factor in COX-2 gene regulation. The direct effect of VT on the COX-2 mRNA stability was analyzed by measuring the kinetics of mRNA decay after induction with 200 ng/ml LPS. Cells were collected at intervals following transcriptional arrest by actinomycin-D (Act-D), with or without VT, 3 h after LPS induction (Fig. 7). Marked decay of COX-2 mRNA was observed after transcriptional arrest in control cultures, whereas VT markedly delayed COX-2 mRNA degradation (Fig. 7
).
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DISCUSSION |
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The dose-response study on PGE2 production after 24 h suggests that the effects were non-linear, in that levels of this metabolite were identical at 100 and 250 ng/ml VT (Fig. 1). This contrasts with the 15 h COX-2 protein and 2 h COX-2 mRNA data, which both increased as the VT dose increased from 50 to 250 ng/ml. The PGE2 threshold effect may relate to the fact that supernatant PGE2 reflects not only the sum total of COX-2 mRNA expression and translational efficiency but also substrate availability, receptor binding, and metabolism by the cells. Another possibility is that this effect is a reflection of VT cytotoxicity. Using the 3-(4,5-di-methylthizol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay, we have found after 24 h that while 100 ng/ml of VT or less had no effect, 250 ng/ml typically inhibits the maximal response by 5 to 20 percent (data not shown). The capacity of VT to partially inhibit cell proliferation and/or reduce viability at the highest concentration is consistent with this toxins inhibitory effects on translation. Thus, it is possible that even if the amount of COX-2 per cell were higher in the 250 ng/ml VT treatment, the decreased cell number might contribute to the lack of change in PGE2 compared to the lower toxin dose. This effect might not have been observed in COX-2 protein and mRNA measurements because these employed equivalent concentrations of the respective extracted macromolecules rather than representing a culture aliquot, as does the MTT assay.
Several intestinal tumor studies have suggested that MEK and its downstream ERK-signaling pathway are essential for both increased transcription and stability of COX-2 mRNA of K-Ras-induced COX-2 (Zhang et al., 2000; Sheng et al., 2001
). ERK1/2 activation by VT was clearly involved in the transcriptional regulation of COX-2 expression but did not contribute to the mRNA stability. VT-activated ERK1/2 may be critical upstream signals regulating transcription factors rather than contributing to mRNA stability. Notably, ERK1/2 are crucial upstream kinases of CCAAAT/enhancer-binding protein-beta (C/EBPß) and cAMP response element-binding protein (CREB), which are major transcriptional factors involved in murine COX-2 expression (Davis et al., 2000
; Hu et al., 2001
). Consistent with such activity, we have recently observed that C/EBPß binding activities in RAW 264.7 cells was increased 2 and 8 h after VT exposure (Wong et al., 2002
). It is thus possible that VT-induced ERK activity contributes to increased activation of C/EPß and other transcription factors.
p38 is also known to mediate xenobiotic- or endogenous factor-induced COX-2 expression by transcriptional and post-transcriptional mechanisms (Fiebich et al., 2000; Vogel, 2000
; Yan et al., 2000
). As shown here, p38 contributed to VT-induced COX-2 expression by both mechanisms. VT-induced transactivation of the COX-2 gene may result from p38-mediated activation of NF-
B and AP-1. Activation of these transcription factors by p38 has been described in LPS-exposed macrophages (Chen et al., 1999
). This possibility is supported by our recent observation that VT exposure also increases AP-1 and NF-
B activation in RAW 264.7 cells (Wong et al., 2002
).
Relative to p38 and post-transcriptional mechanisms, the presence of multiple copies of AUUUA pentamer in the 3-untranslated region (UTR) of COX-2 mRNA suggests the involvement of mRNA stability in the VT-induced upregulation of the gene. Some proteins such as AUF1, HuR, or tristetraprolin can differentially regulate COX-2 or cytokine mRNA stability by binding to the regulatory AU-rich elements (Dixon et al., 2000, 2001
; Nabors et al., 2001
; Sirenko et al., 1997
; Zhu et al., 2001
). Notably, binding of tristetraprolin, a member of a family of zinc-finger proteins, is suppressed by p38-mediated phosphorylation (Zhu et al., 2001
). The observation that COX-2 mRNA stabilization via VT-activated p38 may be an important clue to global mechanisms of trichothecene-induced proinflammatory gene expression.
VT also activated JNK, which was transient relative to the other two MAPK families. Macrophage cells require the activation of JNK/MEKK1 in LPS-induced COX-2 transcription (Wadleigh et al., 2000), and, more specifically, blocking JNK impairs both NF-
B- and C/EBPß-mediated transcription of COX-2 (Mestre et al., 2001
). In contrast, our results showed that blocking JNK activity with dnJNK did not affect VT-induced COX-2 expression. It is possible that, in the case of VT, the activated MAPK network functions redundantly in upregulating COX-2 gene transcription. In this case, impairment of JNK might be overcome by alternate pathways involving ERK and p38.
Interestingly, trichothecenes are also thought to induce leukocyte apoptosis via the p38 and JNK signaling pathways (Shifrin et al., 1999; Yang et al., 2000). The VT concentrations employed here to induce COX-2 expression were non-cytotoxic to weakly cytotoxic. The possibility exists that trichothecene concentration may selectively dictate which MAPKs are activated and to what degree. The resultant effects may ultimately determine whether a cell generates a proinflammatory gene response or undergoes apoptosis. Further evaluation of how trichothecenes differentially regulate these two responses is warranted.
The potential exists that eicosanoid production might contribute to acute and chronic toxic effects associated with acute exposure to VT and other trichothecenes. Subchronic feeding of VT to mice results in a spectrum of immunologic effects including some manifestations that mimic human IgA nephropathy, an immune-complex disease (Dong et al., 1991). Relative to the latter, VT enhances polyclonal autoreactive immunoglobulin A, which deposit in the kidney mesangium (Rasooly et al., 1994
; Rasooly and Pestka, 1994
). Of critical importance is the capacity of VT to elevate IL-6 expression, which is a critical mediator in the VT-induced IgA production (Pestka and Zhou, 2000
; Zhou et al., 1999
). PGE2 is known to enhance IL-6 in several inflammatory models such as endotoxemia, airway inflammation, and autoimmune arthritis (Anderson et al., 1996
; Myers et al., 2000
; Tavakoli et al., 2001
). In contrast, PGE2 selectively impairs the production of IFN
and Th1 immune function in both human and murine T-cell models (Betz and Fox, 1991
; Snijdewint et al., 1993
). PGE2 suppresses interleukin-12 (IL-12) p70 heterodimer, a major Th1-driving cytokine, whereas it participates in the induction of IL-12 p40, which can function as an antagonist of biologically active IL-12p70, thus favoring a Th2 response (Kalinski et al., 2001
). Interestingly, we have previously shown that VT induces IL-12p40 but not IL-12 p35, which would be inherently required for an increase in functionally active IL-12p70 (Zhou et al., 1997
). Thus, it is feasible that VT-induced PGE2 production might alter an optimal balance between Th1 and Th2 and drive increased IgA production that is observed in the subchronic feeding models.
Although COX-2 induction can be related to adverse effects such as tissue damage, COX-2 also can play a protective role in gastrointestinal inflammation (Langenbach et al., 1999; Morteau, 1999
). It has been reported that COX-2-dependent arachidonic acid metabolites are essential modulators of the intestinal immune response to dietary antigens by promoting oral tolerance (Newberry et al., 1999
). Moreover, COX-2 products can be very important in the resolution of late-stage inflammation and may specifically involve an alternate set of prostaglandins such as those of the cyclopentenone family (Morteau, 1999
). Consistent with this observation, COX-2 knockout mice exhibit increased susceptibility to chemical-induced colitis (Morteau et al., 2000
). Therefore, the upregulation of COX-2 and its metabolites by VT as described herein might be interpretable as a protective defensive mechanism or compensatory stress response.
The mechanisms by which VT induces MAPK phosphorylation are unknown. Trichothecenes (Shifrin and Anderson, 1999; Yang et al., 2000
) and other translational inhibitors (Iordanov et al., 1997
) that bind to eukaryotic ribosomes have been previously shown to activate MAPKs. Iordanov et al.(1997) observed that anisomycin and other antibiotics that bind to 28S rRNA are potent activators of JNK. Furthermore, two ribotoxic enzymes, ricin A chain and
-sarcin, both of which catalyze sequence-specific RNA damage in the 28S rRNA, are strong agonists of JNK1 and of its activator SEK1/MKK4. Anisomycin and the ribotoxic enzymes initiate signal transduction from the damaged 28S rRNA to JNK in active, but not inactive ribosomes. These investigators described this capacity of the ribosomes to sense cellular stress as the "ribotoxic stress response." The possibility exists that VTs capacity to activate MAPKs reflects a ribotoxic stress response and that this is manifested in transcriptional and postranscriptional upregulation of COX-2 mRNA.
In conclusion, the results presented herein revealed that VT induced PGE2 production and COX-2 expression by elevating transcriptional activity and mRNA stability. Enhanced transcriptional activity was modulated by ERK and p38 signaling pathways whereas mRNA stability was promoted exclusively by VT-activated p38 phosphorylation. Future studies will be directed toward identifying MAPK-regulated transcription factors and stabilizing factors binding to 3-UTR of VT-induced COX-2 gene. Additional investigation is needed at the in vivo level relative to the role of COX-2 products in VT-induced immunopathogenic and physiological sequelae.
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ACKNOWLEDGMENTS |
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NOTES |
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