Regulation Of Nuclear Factor-kappa B Activity

IkappaB激酶(IKK)催化亚基IKKβ在细胞因子介导的核因子-kappaB信号的多效性增强中起关键作用。IKKβ活性缺陷抑制了核因子-kappaB介导的炎症和肿瘤发生，同时促进了细胞凋亡。甘露霉素是一种天然的环氧奎宁类化合物，对法尼基转移酶具有很强的选择性抑制活性，已在多种实验模型中用于抗肿瘤作用。我们最近的发现表明，在一些细胞系和原代培养的大鼠肝细胞中，甘露霉素能迅速而有效地抑制肿瘤坏死因子α刺激的IKK活性。此外，对移植了小鼠B16F10肿瘤的小鼠给予甘露霉素可产生有效的IKK抑制效应(Bernier等人，2006年)。出乎意料的是，在这些实验条件下，其他类别的法尼基转移酶抑制剂没有抑制作用。为了确定甘露霉素作用的分子机制，我们将IKKalpha和IKKβ载体导入培养的人肝癌细胞中，发现在甘露霉素存在的情况下，IKK活性被直接抑制，同时伴随着稳定的同型IKKβ二聚体的形成。为了研究IKKβ的共价二聚化是由于对manumycin的环氧奎宁核心的亲核攻击而导致的，我们产生了一系列IKKβ的Cys-&Gt；Ala点突变。表达IKKβ的细胞在Cys-179的激活环中发生突变，表现出类似的二聚体形成，而在IKKβ的羧基末端区域的Cys-662/716的双取代对甘露霉素引起的同型二聚体具有保护作用。二硫苏糖醇的前处理实质上阻断了免疫沉淀物中甘露霉素介导的共价IKKβ二聚体的形成。利用质谱学技术，我们鉴定了谷胱甘肽(GSH)是一种容易通过甘露霉素的环氧奎宁功能进行修饰的分子，这表明细胞内硫醇的枯竭至少在一定程度上对甘露霉素的抗肿瘤特性起到了作用。这些结果表明，甘露霉素在IKK/NFkappaB信号转导中起着重要的调节作用，其途径有别于法尼化抑制因子。 尽管取得了这些进展，但仍不清楚甘露霉素如何影响IKK/NF-kappaB途径的其他特定元件的功能。目前正在进行生化和分子生物学研究，以确定甘露霉素对IKK多聚体复合体的失活作用，以及它在培养的细胞系和移植到免疫低下小鼠的癌细胞中诱导凋亡的途径。我们的数据应该为核因子-kappaB信号的关键方面提供新的见解，并可能具有特别的临床相关性。

The IkappaB kinase (IKK) catalytic subunit, IKKbeta, plays a key role in cytokine-mediated increase in the pleiotropic responses of nuclear factor (NF)-kappaB signaling. Defects in IKKbeta activity inhibit NF-kappaB-mediated inflammation and oncogenesis, while promoting apoptosis. Manumycin is a natural epoxyquinoid compound that exhibits potent and selective inhibitory activity against farnesyltransferase, and it has been used for its antineoplastic action in a variety of experimental models. Our recent findings indicate that manumycin elicited a rapid and potent inhibition of TNF alpha-stimulated IKK activity in a number of cell lines and a primary culture of rat hepatocytes. Moreover, administration of manumycin to mice xenografted with murine B16F10 tumors caused potent IKK-suppressive effects (Bernier et al., 2006). Unexpectedly, other classes of farnesyltransferase inhibitors had no inhibitory effect under these experimental conditions. To identify the molecular mechanisms of manumycin action, we transfected cultured human HepG2 hepatoma cells with IKKalpha and IKKbeta constructs and demonstrated direct inhibition of IKK activity with concomittant formation of stable homotypic IKKbeta dimers in the presence of manumycin. A number of Cys-> Ala point mutants of IKKbeta were generated to investigate the possibility that IKKbeta covalent dimerization resulted from nucleophilic attack on the epoxyquinoid core of manumycin. Cells expressing IKKbeta mutated in the activation loop at Cys-179 exhibited similar dimer formation, whereas double substitution of Cys-662/716 in the carboxy-terminal domain of IKKbeta conferred protection against homotypic dimerization by manumycin. Dithiothreitol pre-treatment substantially blocked manumycin-mediated formation of covalent IKKbeta dimers in immunoprecipitates. Using mass spectrometry technique, we identified glutathione (GSH) as a molecule susceptible to modification via the epoxyquinoid function of manumycin, which suggests that the depletion of intracellular thiols could contribute, at least in part, to manumycin's antineoplastic properties. These results indicated that manumycin plays important regulatory function in IKK/NFkappaB signaling through pathways distinct from its role as farnesylation inhibitor. Despite these advances, it remains to be understood how manumycin affects the function of other specific elements of the IKK/NF-kappaB pathway. Biochemical and molecular biological studies are being carried out to characterize the inactivation of the IKK multimeric complex by manumycin and the pathways that it uses to induce apoptosis both in cultured cell lines and in cancer cells xenotransplanted into immunocompromised mice. Our data should provide novel insights into key aspects of NF-kappaB signaling, and may be of particular clinical relevance.