Liu Fang, Inageda Kiyoshi, Nishitai Gen, Matsuoka Masato
Department of Hygiene and Public Health I, School of Medicine, Tokyo Women's Medical University, Tokyo, Japan.
Environ Health Perspect. 2006 Jun;114(6):859-64. doi: 10.1289/ehp.8920.
To reveal the effects of cadmium exposure on the endoplasmic reticulum (ER) stress response, we examined the expression and function of 78-kDa glucose-regulated protein (Grp78) , an ER-resident molecular chaperone, in LLC-PK1 cells. In cells treated with 10 microM cadmium chloride, Grp78 protein levels increased after 6 hr and remained elevated at 24 hr. When cells were incubated with 1-20 microM CdCl2 for 6 hr, Grp78 increased in a dose-dependent manner. In addition, Grp78 mRNA levels were elevated in response to CdCl2 exposure. After exposure to 10 microM CdCl2, the levels of activating transcription factor 4 (ATF4) were increased at 2 hr, with a further enhancement after that ; this accumulation followed the transient but marked phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2(alpha)) on serine 51. Although ATF4 mRNA levels increased mildly by CdCl2 exposure, treatment with actinomycin D did not suppress CdCl2-induced accumulation of ATF4 protein, suggesting the involvement of posttranscriptional and, in part, transcriptional mechanisms. Compared with other heavy-metal compounds such as manganese chloride, zinc chloride, mercuric chloride, and lead chloride, CdCl2 could increase the levels of Grp78, ATF4, and the phosphorylated form of eIF2(alpha) more markedly without definite cellular damage. The silencing of Grp78 expression using short-interference RNA enhanced CdCl2-induced cellular damage. These results show that cadmium induces the expression of Grp78 probably via phosphorylation of eIF2(alpha) and resultant translation of ATF4, and this ER stress response plays a role in protection against cadmium cytotoxicity in this renal epithelial cell.
为揭示镉暴露对内质网（ER）应激反应的影响，我们检测了内质网驻留分子伴侣78 kDa葡萄糖调节蛋白（Grp78）在LLC-PK1细胞中的表达及功能。在用10微摩尔氯化镉处理的细胞中，Grp78蛋白水平在6小时后升高，并在24小时时保持升高状态。当细胞与1 - 20微摩尔氯化镉孵育6小时时，Grp78呈剂量依赖性增加。此外，Grp78 mRNA水平也因镉暴露而升高。暴露于10微摩尔氯化镉后，激活转录因子4（ATF4）的水平在2小时时升高，之后进一步增强；这种积累伴随着真核翻译起始因子2（eIF2(α)）的α亚基在丝氨酸51位点的短暂但显著的磷酸化。尽管镉暴露使ATF4 mRNA水平略有升高，但用放线菌素D处理并不能抑制镉诱导的ATF4蛋白积累，这表明转录后机制以及部分转录机制均参与其中。与其他重金属化合物如氯化锰、氯化锌、氯化汞和氯化铅相比，氯化镉能更显著地增加Grp78、ATF4和eIF2(α)磷酸化形式的水平，且无明显的细胞损伤。使用短干扰RNA沉默Grp78表达会增强镉诱导的细胞损伤。这些结果表明，镉可能通过eIF2(α)的磷酸化以及由此导致的ATF4翻译来诱导Grp78的表达，并且这种内质网应激反应在保护该肾上皮细胞免受镉细胞毒性方面发挥作用。
