Mercuric ion attenuates nuclear factor-κB activation and DNA binding in normal rat kidney epithelial cells:: Implications for mercury-induced nephrotoxicity
Mercuric ion attenuates nuclear factor-κB activation and DNA binding in normal rat kidney epithelial cells:: Implications for mercury-induced nephrotoxicity
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DOI：
10.1006/taap.2001.9195
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发表时间：
2001-06-15
影响因子：
3.8
通讯作者：
Woods, JS
中科院分区：
文献类型：
--
作者：
Dieguez-Acuña, FJ;Ellis, ME;Woods, JS
汞离子（Hg 2+）是已知的最强硫醇结合剂之一，介导与元素、无机和有机汞化合物相关的毒性。与Hg 2+毒性相关的细胞事件的研究主要集中在细胞膜的破坏和线粒体功能的损害。相反，很少有研究试图确定特定的分子机制，通过汞2+可能会影响毒性通过改变巯基依赖的信号转导途径，调节细胞增殖和生存。在这方面特别感兴趣的是Hg 2+对核因子-κ B（NF-κ B）的影响，NF-κ B是一种多效性转录因子，已知其在活化和DNA结合的关键步骤中需要还原的半胱氨酸部分。在这里，我们评估的影响，Hg 2+对正常大鼠肾上皮细胞（NRK 52 E），Hg 2+毒性的主要目标的NF-κ B的表达。脂多糖（LPS）诱导形式的NF-kappaB在肾细胞中很容易检测到，并被表征为p50 p65异源二聚体。在体外，当Hg 2+（0-55 μ M）加入到含有非巯基还原剂三（2-羧乙基）膦盐酸盐（TCEP）的DNA结合反应中时，以剂量相关的方式阻止NF-κ B-DNA结合。类似地，在结合反应中，相同浓度的Hg 2+阻止人重组野生型p50 p50同源二聚体的DNA结合，并且使用含有cys（62）->ser（62）突变的p50蛋白的突变形式来减弱这种效应。汞离子对p50-DNA结合的抑制作用在体外可通过竞争性巯基DTT、GSH和L-半胱氨酸以剂量相关的方式逆转。与此相反，竞争性硫醇加入到核结合反应不能逆转衰减的LPS介导的NF-κ B-DNA结合的亲和力时，细胞在体内预处理与Hg 2+在浓度低至2 μ M前LPS管理。免疫印迹分析表明，Hg 2+预处理的肾细胞实质上减少，在核后LPS管理。此外，Hg 2+预处理损害了I kappaB α的磷酸化和降解，这表明在I kappaB α蛋白水解水平上对NF-κ B活化有特异性影响。最后，当在LPS处理前给予用NF-κ B驱动的荧光素酶报告基因（pLuc-4xNF-κ B）瞬时转染的肾细胞时，浓度低至5 μ M的Hg 2+显著降低NF-κ B介导的转录活性。这些发现表明，在低细胞浓度下，Hg 2+减弱了与I κ B α磷酸化和降解、p50 p65异源二聚体核转位以及p50-cys与DNA κ B结合位点相关的NF-κ B激活。Hg 2+通过这些机制减弱NF-κ B活化可能是已知与肾上皮细胞中低水平Hg 2+暴露相关的凋亡或其他细胞毒性反应的基础。(C)北京：科学出版社.
Mercuric ion (Hg2+), one of the strongest thiol-binding agents known, mediates the toxicity associated with elemental, inorganic, and organic mercurial compounds. Studies of cellular events associated with Hg2+ toxicity have focused largely on disruption of cell membranes and impairment of mitochondrial functions. In contrast, few studies have sought to define the specific molecular mechanisms through which Hg2+ might affect toxicity via alteration of thiol-dependent signal transduction pathways that regulate cell proliferation and survival. Of particular interest in this regard is the effect of Hg2+ on nuclear factor-kappaB (NF-kappaB), a pleiotropic transcriptional factor that is known to require reduced cysteine moieties at critical steps of activation and DNA binding. Here, we evaluated the effects of Hg2+ on the expression of NF-kappaB in normal rat kidney epithelial (NRK52E) cells, a principal target of Hg2+ toxicity. The lipopolysaccharide (LPS)-inducible form of NF-kappaB was readily detected in kidney cells and has been characterized as the p50p65 heterodimer. NF-kappaB-DNA binding was prevented in a dose-related manner by Hg2+ (0-55 muM) in vitro when added to DNA binding reactions containing the nonthiol reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Similarly, Hg2+ at the same concentrations prevented DNA binding of a human recombinant wild-type p50p50 homodimer in binding reactions, and this effect was attenuated using a mutant form of the p50 protein containing a cys(62)-->ser(62) mutation. The inhibition of p50-DNA binding by Hg2+ was reversible in a dose-related manner in vitro by competitive thiols DTT, GSH, and L-cysteine in binding reactions. In contrast, competitive thiols added to nuclear binding reactions were unable to reverse attenuation of LPS-mediated NF-kappaB-DNA binding affinity when cells were pretreated in vivo with Hg2+ at concentrations as low as 2 muM prior to LPS administration. Immunoblot analyses indicted that Hg2+ pretreatment of kidney cells substantially diminished, in a the nucleus following LPS administration. Additionally, Hg2+ pretreatment impaired both the phosphorylation and degradation of I kappaB alpha, suggesting a specific effect on NF-kappaB activation at the level of I kappaB alpha proteolysis. Finally, Hg2+ at concentrations as low as 5 muM significantly diminished NF-kappaB-mediated transcriptional activity when administered to kidney cells transiently transfected with an NF-kappaB-driven luciferase reporter gene (pLuc-4xNF-kappaB) prior to LPS treatment. These findings demonstrate that Hg2+, at low cellular concentrations, attenuates NF-kappaB activation at sites associated with I kappaB alpha phosphorylation and degradation, nuclear translocation of the p50p65 heterodimer, and association of p50-cys(62), with the DNA kappaB binding site. Attenuation of NF-kappaB activation by Hg2+ through these mechanisms may underlie apoptotic or other cytotoxic responses that are known to be associated with low level Hg2+ exposure in kidney epithelial cells. (C) 2001 Academic Press.