Qanungo Suparna, Starke David W, Pai Harish V, Mieyal John J, Nieminen Anna-Liisa
Department of Anatomy, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Department of Pharmacology, Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
J Biol Chem. 2007 Jun 22;282(25):18427-18436. doi: 10.1074/jbc.M610934200. Epub 2007 Apr 27.
In murine embryonic fibroblasts, N-acetyl-L-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFkappaB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFkappaB that are responsible for NFkappaB inactivation. In MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFkappaB DNA binding and NFkappaB transactivation by 2.6- and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFkappaB protein levels and p65-NFkappaB nuclear translocation during hypoxia. Pharmacological inhibition of the NFkappaB pathway also induced hypoxic apoptosis, indicating that the NFkappaB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after hypoxia and treatment with N-ethylmaleimide (thiol alkylating agent), dithiothreitol (disulfide reducing agent) was not able to increase binding of p65-NFkappaB to DNA, suggesting that most sulfhydryls in p65-NFkappaB protein were in reduced and activated forms after hypoxia, thereby being blocked by N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC, dithiothreitol increased p65-NFkappaB DNA binding. Glutaredoxin (GRx), which specifically catalyzes reduction of protein-SSG mixed disulfides, reversed inhibition of p65-NFkappaB DNA binding in extracts from cells treated with hypoxia plus NAC and restored NFkappaB activity. This finding indicated that p65-NFkappaB-SSG was formed in situ under hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFkappaB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFkappaB is most likely responsible for NAC-mediated NFkappaB inactivation and enhanced hypoxic apoptosis.
在小鼠胚胎成纤维细胞中，谷胱甘肽生成剂N - 乙酰 - L - 半胱氨酸（NAC）通过阻断NFκB生存途径增强缺氧诱导的细胞凋亡（Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455 - 50464）。在此，我们研究了负责NFκB失活的NFκB p65亚基的巯基修饰。在MIA PaCa - 2胰腺癌细胞中，缺氧分别使p65 - NFκB与DNA的结合及NFκB的反式激活增加了2.6倍和2.8倍。NAC可阻断这些事件，且对缺氧期间p65 - NFκB蛋白水平和p65 - NFκB核转位无影响。对NFκB途径的药理学抑制也可诱导缺氧诱导的细胞凋亡，这表明NFκB信号通路是对抗缺氧诱导细胞凋亡的主要保护机制。在用N - 乙基马来酰亚胺（硫醇烷基化剂）处理后的缺氧细胞裂解物中，二硫苏糖醇（二硫化物还原剂）无法增加p65 - NFκB与DNA的结合，这表明缺氧后p65 - NFκB蛋白中的大多数巯基处于还原和激活状态，因此被N - 乙基马来酰亚胺阻断。相反，在用NAC处理的缺氧细胞中，二硫苏糖醇增加了p65 - NFκB与DNA的结合。谷氧还蛋白（GRx）可特异性催化蛋白质 - SSG混合二硫化物的还原，它逆转了缺氧加NAC处理的细胞提取物中p65 - NFκB与DNA结合的抑制，并恢复了NFκB活性。这一发现表明，在缺氧加NAC条件下原位形成了p65 - NFκB - SSG。在细胞中，敲低内源性GRx1（在缺氧自由基生成条件下也促进蛋白质谷胱甘肽化）可阻止NAC诱导的NFκB失活和缺氧诱导的细胞凋亡。结果表明，p65 - NFκB的GRx依赖性S - 谷胱甘肽化很可能是NAC介导的NFκB失活和增强缺氧诱导细胞凋亡的原因。
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