Transcriptional and posttranscriptional roles for p38 mitogen-activated protein kinase in upregulation of TNF-alpha expression by deoxynivalenol (vomitoxin)

Abstract

Deoxynivalenol (DON, vomitoxin) is a trichothecene mycotoxin that potentially mediates toxicity by upregulating proinflammatory cytokine gene expression in vitro and in vivo. The purpose of this study was to test the hypothesis that DON-induced activation of mitogen-activated protein kinases (MAPKs) mediates transcriptional and posttranscriptional upregulation of TNF-alpha gene expression. RNAse protection assay revealed that DON at 100 to 500 ng/ml induced mRNA expression of TNF-alpha as well as IL-6, IFN-gamma, TGFbeta-1, and TGFbeta-3 and that these effects were potentiated by 100 ng/ml lipopolysaccharide (LPS). DON was found to induce phosphorylation of p38 kinase, extracellular signal-regulated kinases (ERKs), and c-Jun amino terminal kinases (JNKs) in a dose-dependent manner in the RAW 264.7 murine macrophage model. A luciferase reporter gene driven by the murine TNF-alpha promoter was used to assess the role of various MAPKs on DON upregulation of TNF-alpha gene transcription. The p38 inhibitor SB203580 reduced induction of luciferase activity by DON, LPS, and DON + LPS. In addition, the ERK inhibitor PD 98059 blocked DON- and DON + LPS-induced luciferase activity whereas the JNK inhibitor impaired LPS- and DON + LPS-induced luciferase activity. To study the effects of MAPKs on DON-induced TNF-alpha mRNA stability, an asynchronous model was used whereby cells were pretreated with LPS for 4 h and the medium was removed. Following incubation with medium containing a transcription inhibitor, 5,6-dichloro-beta-D-ribofuranosyl-benzimidazole, MAPK inhibitors and/or DON (250 ng/ml) cultures were monitored for TNF-alpha mRNA expression. DON-induced TNF-alpha mRNA stabilization was abrogated in the presence of SB 203580, whereas the stabilization by DON was not affected by PD 98059 or SP 600125. To verify the role of MAPKs in DON + LPS-induced TNF-alpha production, cells were incubated with LPS, DON, or LPS + DON for 18 h in the presence of inhibitors. ELISA of supernatant indicated that induction of TNF-alpha production by DON alone was significantly reduced by SB 203580 and PD 98059, whereas all three inhibitors blocked LPS- and DON + LPS-induced TNF-alpha production. Taken together, these results suggest that relative to DON-induced TNF-alpha mRNA expression, p38 and ERK activation contribute to DON-induced transcriptional upregulation whereas p38 plays a role in increasing mRNA stability.