Regulation Of Nuclear Factor-kappa B Activity

IkappaB激酶（IKK）催化亚基在细胞因子介导的核因子(NF)-kappaB信号传导中起关键作用，NF- kb功能的丧失似乎可以抑制炎症和肿瘤的发生。马霉素A是一种天然的环氧醌类化合物，是一种有效的选择性法尼基转移酶抑制剂，具有抗肿瘤活性。我们最近的研究结果表明，在许多细胞系和原代培养的大鼠肝细胞中，马霉素A治疗导致了TNF α刺激的IKK活性的快速、选择性和有效的抑制。出乎意料的是，其他种类的法尼基转移酶抑制剂没有抑制作用。为了确定马霉素A作用的分子机制，我们用IKKalpha和ikkβ构建体转染人HepG2肝癌细胞系，发现在马霉素A存在的情况下，ikkβ活性直接受到抑制，同时形成稳定的同型ikkβ二聚体。为了研究ikkβ的一些Cys-> α点突变体是由对马霉素A的环氧类核心的亲核攻击导致ikkβ共价二聚体的可能性，表达ikkβ的细胞在Cys-179激活环中发生突变Cys-662和？在马霉素a处理的细胞中，IKK与衔接蛋白IKKgamma/NEMO的相互作用也被破坏。因此，马霉素A通过不同于法尼化抑制剂的途径在IKK信号传导中发挥重要的调节作用。

IkappaB kinase (IKK) catalytic subunits play a key role in cytokine-mediated nuclear factor (NF)-kappaB signaling, and a loss of NF-kB function appears to inhibit inflammation and oncogenesis. Manumycin A, a natural epoxyquinoid compound, is a potent and selective farnesyltransferase inhibitor with antitumor activity. Our recent findings indicate that treatment with manumycin A resulted in a rapid, selective and potent inhibition of TNF alpha-stimulated IKK activity in a number of cell lines and a primary culture of rat hepatocytes. Unexpectedly, other classes of farnesyltransferase inhibitors had no inhibitory effect. To identify the molecular mechanisms of manumycin A action, we transfected human HepG2 hepatoma cell line 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. A number of Cys-> Ala point mutants of IKKbeta were generated to investigate the possibility that IKKbeta covalent dimerization results from nucleophilic attack on the epoquinoid core of manumycin A. Cells expressing IKKbeta mutated in the activation loop at Cys-179 exhibited similar dimer formation, whereas double substitution of Cys-662 and ?716 conferred protection against homotypic dimerization by manumycin A. IKK interaction with the adaptor protein IKKgamma/NEMO was also disrupted in manumycin A-treated cells. Thus, manumycin A plays important regulatory function in IKK signaling through pathways distinct from its role as farnesylation inhibitor. We are currently using an in vivo approach to investigate the hypothesis that manumycin A affects the activation of IKKbeta, a major contributor to inflammation-induced insulin resistance. Injection of various concentrations of manumycin A has been carried out in murine B16F10 melanoma implanted s.c. in the back of several mice. The tumor masses were excised, weighted and are currently being processed for Western blot, in vitro IKK activity and EMSA (NF-kappaB) studies. It is anticipated that our results will demonstrate that IKKbeta is a direct target of manumycin A in vivo. Future plans include the study of the effect of manumycin A in the regulation of insulin responsiveness in physiological and pathophysiological pro-inflammatory states such as diet-induced obesity and aging.