Transcriptional and posttranscriptional roles for p38 mitogen-activated protein kinase in upregulation of TNF-α expression by deoxynivalenol (vomitoxin)
Transcriptional and posttranscriptional roles for p38 mitogen-activated protein kinase in upregulation of TNF-α expression by deoxynivalenol (vomitoxin)
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DOI：
10.1016/s0041-008x(03)00299-0
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发表时间：
2003-12-01
影响因子：
3.8
通讯作者：
Pestka, JJ
中科院分区：
文献类型：
--
作者：
Chung, YJ;Zhou, HR;Pestka, JJ
脱氧雪腐镰刀菌烯醇（DON，vomitoxin）是一种具有潜在毒性的二孢霉烯类真菌毒素，在体内和体外通过上调促炎细胞因子基因表达而介导毒性。本研究的目的是检验这一假设，即DON诱导的丝裂原活化蛋白激酶（MAPKs）激活介导TNF-α基因表达的转录和转录后上调。RNA酶保护试验显示，100至500 ng/ml的DON诱导TNF-α以及IL-6、IFN-γ、TGF β-1和TGF β-3的mRNA表达，并且这些作用被100 ng/ml脂多糖（LPS）增强。发现DON在RAW 264.7鼠巨噬细胞模型中以剂量依赖性方式诱导p38激酶、细胞外信号调节激酶（ERK）和c-Jun氨基末端激酶（JNKs）的磷酸化。由鼠TNF-α启动子驱动的荧光素酶报告基因用于评估各种MAPK对TNF-α基因转录的DON上调的作用。p38抑制剂SB 203580减少DON、LPS和DON + LPS对荧光素酶活性的诱导。此外，ERK抑制剂PD 98059阻断DON和DON + LPS诱导的荧光素酶活性，而JNK抑制剂损害LPS和DON + LPS诱导的荧光素酶活性。为了研究MAPK对DON诱导的TNF-α mRNA稳定性的影响，使用了异步模型，其中细胞用LPS预处理4 μ l并除去培养基。在用含有转录抑制剂、5，6-二氯-β-D-呋喃核糖基-苯并咪唑、MAPK抑制剂和/或DON（250 ng/ml）的培养基孵育后，监测培养物的TNF-α mRNA表达。在SB 203580的存在下，DON诱导的TNF-α mRNA稳定被废除，而PD 98059或SP 600125不影响DON的稳定。为了验证MAPK在DON + LPS诱导的TNF-α产生中的作用，在抑制剂存在下，将细胞与LPS、DON或LPS + DON孵育18小时。上清液的ELISA表明，SB 203580和PD 98059显著降低了单独由DON诱导的TNF-α产生，而所有三种抑制剂都阻断了LPS和DON + LPS诱导的TNF-α产生。总之，这些结果表明，相对于DON诱导的TNF-α mRNA表达，p38和ERK激活有助于DON诱导的转录上调，而p38在增加mRNA稳定性中起作用。(C)2003年爱思唯尔公司All rights reserved.
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 It 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 It in the presence of inhibitors. ELISA of supernatant indicated that induction of TNF-a 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. (C) 2003 Elsevier Inc. All rights reserved.