ToxSci Advance Access originally published online on January 31, 2003
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Toxicological Sciences 72, 130-142 (2003)
Copyright © 2003 by the Society of Toxicology
MOLECULAR AND GENETIC TOXICOLOGY |
Rapid, Sequential Activation of Mitogen-Activated Protein Kinases and Transcription Factors Precedes Proinflammatory Cytokine mRNA Expression in Spleens of Mice Exposed to the Trichothecene Vomitoxin
,
,1
* Department of Food Science and Human Nutrition,
Department of Microbiology and Molecular Genetics, and
Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824-1224
Since proinflammatory cytokine mRNA expression is induced within lymphoid tissue in vivo by the trichothecene vomitoxin (VT) in a rapid (1–2 h) and transient (4–8 h) fashion, it was hypothesized that mitogen-activated protein kinases (MAPKs) and transcription factors associated upstream with gene transcription of these cytokines are activated prior to or within these time windows. To test this hypothesis, mice were first treated with a single oral dose of VT and then analyzed for MAPK phosphorylation in the spleen. As little as 1 mg/kg of VT induced JNK 1/2, ERK 1/2, and p38 phosphorylation with maximal effects being observed at 5 to 100 mg/kg of VT. VT transiently induced JNK and p38 phosphorylation over a 60-min time period with peak effects being observed at 15 and 30 min, respectively. In contrast, ERK remained phosphorylated from 15 to 120 min. Next, the binding of activating protein 1 (AP-1), CCAAT enhancer-binding protein (C/EBP), CRE-binding protein (CREB), and nuclear factor-
B (NF-B) was measured by electrophoretic mobility shift assay (EMSA) using four different consensus transcriptional control motifs at 0, 0.5, 1.5, 4, and 8 h after oral exposure to 25 mg/kg of VT. AP-1 binding activity was differentially elevated from 0.5 h to 8 h, whereas C/EBP binding was elevated only at 0.5 h. CREB binding decreased slightly at 0.5 h but gradually increased, reaching a maximum at 4 h. NF-B binding was increased only slightly at 4 and 8 h. The specificities of AP-1, C/EBP, CREB, and NF-B for relevant DNA motifs were verified by competition assays, using an excess of unlabeled consensus and mutant oligonucleotides. Supershift EMSAs and Western blot analysis identified specific VT-inducible DNA binding proteins for AP-1 (cJun, phospho c-jun, JunB, and JunD), C/EBP (C/EBPß), CREB (CREB-1 and ATF-2), and NF-B (p50 and cRel). Finally, when the effects of oral VT exposure on proinflammatory gene expression were assessed at 3, 6, and 9 h, splenic TNF-
, IL-1ß, and IL-6 mRNA were found to peak at 3 h and were still significantly elevated at 6 h but not at 9 h. Taken together, VT first activated MAPKs in vivo and either concurrently (AP-1, C/EBP) or subsequently (AP-1, CREB, NF-B) modulated binding activities of transcription factors specific for potential regulatory motifs in cytokine promoters. The timing of these events was highly consistent with the kinetics of proinflammatory gene expression in the spleens of mice exposed to VT. This study provides a novel model for studying the interrelationship of MAPK phosphorylation, transcription factor activation, and cytokine gene expression in an intact animal exposed to a toxic compound.
Key Words: transcription factor; mitogen-activated protein kinases; trichothecene; mycotoxin; vomitoxin; immunotoxicity; TNF-; IL-1ß; IL-6.
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