Xie Jianxun, Shaikh Zahir A
Department of Biomedical and Pharmaceutical Sciences and Center for Molecular Toxicology, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, USA.
Toxicol Sci. 2006 May;91(1):299-308. doi: 10.1093/toxsci/kfj131. Epub 2006 Feb 14.
Renal epithelial cells undergo apoptosis upon exposure to cadmium (Cd). Transcription factors, such as nuclear factor-kappa B (NF-kappaB), mediate the expression of a number of genes involved in apoptosis. The present study was designed to examine the involvement of this transcription factor in Cd-induced apoptosis. Rat kidney proximal tubular epithelial cells, NRK-52E, were incubated with up to 20 microM CdCl2 in serum-free medium for 5 h followed by incubation in serum-containing medium (without Cd) for an additional 12 h. The cells accumulated 582 +/- 19 ng Cd/mg protein after 5-h exposure to 20 microM Cd. As a result of Cd exposure, the DNA-binding activity of the p65 subunit of NF-kappaB was decreased in a concentration- and time-dependent manner. The activity of tumor necrosis factor-alpha-induced inhibitor of kappa B (IkappaB) kinase alpha was also inhibited by Cd. In addition, the phosphorylation of IkappaB-alpha and NF-kappaB p65, as well as the levels of NF-kappaB target gene products, cIAP-1 and cIAP-2, were reduced. Pretreatment of the cells with the antioxidant U83836E or butylated hydroxytoluene preserved the DNA-binding activity and blocked the Cd-induced decease in IkappaB-alpha phosphorylation. Cd exposure caused the activation of caspase-3, -7, and -9 and DNA fragmentation. By flow cytometry, 14.6 and 30.5% apoptosis was detected at 6 and 12 h after stopping the Cd exposure. Overexpression of NF-kappaB p65 by transient transfection protected the cells from the Cd-induced apoptosis. Conversely, attenuation of NF-kappaB activity by pretreatment with SN50, an NF-kappaB nuclear translocation inhibitor, potentiated apoptosis. These results suggest that Cd-induced apoptosis involves suppression of NF-kappaB activity which may be mediated by oxidative stress.
肾上皮细胞在接触镉（Cd）后会发生凋亡。转录因子，如核因子-κB（NF-κB），介导许多参与凋亡的基因的表达。本研究旨在检测该转录因子在镉诱导的凋亡中的作用。将大鼠肾近端小管上皮细胞NRK-52E在无血清培养基中与高达20μM的CdCl2孵育5小时，然后在含血清培养基（不含镉）中再孵育12小时。在接触20μM镉5小时后，细胞积累了582±19 ng镉/毫克蛋白质。由于镉暴露，NF-κB的p65亚基的DNA结合活性以浓度和时间依赖性方式降低。肿瘤坏死因子-α诱导的κB激酶α（IkappaB）的活性也被镉抑制。此外，IkappaB-α和NF-κB p65的磷酸化以及NF-κB靶基因产物cIAP-1和cIAP-2的水平均降低。用抗氧化剂U83836E或丁基羟基甲苯预处理细胞可保留DNA结合活性并阻断镉诱导的IkappaB-α磷酸化降低。镉暴露导致半胱天冬酶-3、-7和-9的激活以及DNA片段化。通过流式细胞术，在停止镉暴露后6小时和12小时检测到14.6%和30.5%的凋亡。通过瞬时转染过表达NF-κB p65可保护细胞免受镉诱导的凋亡。相反，用NF-κB核转位抑制剂SN50预处理减弱NF-κB活性可增强凋亡。这些结果表明，镉诱导的凋亡涉及NF-κB活性的抑制，这可能由氧化应激介导。
