Aeromonas hydrophila cytotoxic enterotoxin activates mitogen-activated protein kinases and induces apoptosis in murine macrophages and human intestinal epithelial cells
Aeromonas hydrophila cytotoxic enterotoxin activates mitogen-activated protein kinases and induces apoptosis in murine macrophages and human intestinal epithelial cells
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DOI：
10.1074/jbc.m404641200
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发表时间：
2004-09-03
影响因子：
4.8
通讯作者：
Chopra, AK
中科院分区：
文献类型：
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作者：
Galindo, CL;Fadl, AA;Chopra, AK
嗜水气单胞菌的一种细胞毒性肠毒素(Act)具有多种生物活性，可在宿主体内诱导炎症反应，并引起小鼠巨噬细胞的凋亡。在这项研究中，我们利用五种靶细胞类型(一种小鼠巨噬细胞系(RAW 264.7)、骨髓源性转化的巨噬细胞、小鼠腹膜巨噬细胞和两种人肠上皮细胞系(T84和HT-29))来研究ACT对丝裂原活化蛋白激酶(MAPK)通路和导致细胞凋亡的机制的影响。免疫沉淀和蛋白印迹分析表明，Act激活了这些细胞中的应激相关p38、c-jun氨基末端激酶(JNK)和细胞外信号调节蛋白1/2(ERK1/2)。ACT还可诱导上游MAPK因子(MAPK激酶3/6(MKK3/6)、MKK4和MAP/ERK激酶1(MEK1))和下游效应因子(MAPK激活蛋白激酶-2、激活转录因子-2和c-jun)的磷酸化。ACT可引起细胞膜起泡，caspase3裂解，并激活caspase8和caspase9。在不表达功能性肿瘤坏死因子受体的巨噬细胞中，细胞凋亡和caspase活性显著降低。对宿主全细胞裂解产物的免疫印迹显示，Act诱导的凋亡相关蛋白表达上调，包括线粒体蛋白细胞色素c和凋亡诱导因子。然而，ACT对线粒体膜的去极化作用不明显。综上所述，这些数据首次证明了Act诱导巨噬细胞和肠上皮细胞MAPK信号的激活和经典的caspase相关的凋亡。鉴于MAPK通路和细胞凋亡在炎症相关疾病中的重要性，本研究为Act对宿主细胞的作用机制提供了新的见解。
A cytotoxic enterotoxin ( Act) of Aeromonas hydrophila possesses several biological activities, induces an inflammatory response in the host, and causes apoptosis of murine macrophages. In this study, we utilized five target cell types ( a murine macrophage cell line ( RAW 264.7), bone marrow-derived transformed macrophages, murine peritoneal macrophages, and two human intestinal epithelial cell lines (T84 and HT-29)) to investigate the effect of Act on mitogen-activated protein kinase ( MAPK) pathways and mechanisms leading to apoptosis. As demonstrated by immunoprecipitation/kinase assays or Western blot analysis, Act activated stress-associated p38, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) in these cells. Act also induced phosphorylation of upstream MAPK factors ( MAPK kinase 3/6 (MKK3/6), MKK4, and MAP/ERK kinase 1 (MEK1)) and downstream effectors (MAPK-activated protein kinase-2, activating transcription factor-2, and c-Jun). Act evoked cell membrane blebbing, caspase 3-cleavage, and activation of caspases 8 and 9 in these cells. In macrophages that do not express functional tumor necrosis factor receptors, apoptosis and caspase activities were significantly decreased. Immunoblotting of host whole cell lysates revealed Act-induced up-regulation of apoptosis-related proteins, including the mitochondrial proteins cytochrome c and apoptosis-inducing factor. However, mitochondrial membrane depolarization was not detected in response to Act. Taken together, the data demonstrated for the first time Act-induced activation of MAPK signaling and classical caspase-associated apoptosis in macrophages and intestinal epithelial cells. Given the importance of MAPK pathways and apoptosis in inflammation-associated diseases, this study provided new insights into the mechanism of action of Act on host cells.