Department of Dental Pharmacology, School of Dentistry, Wonkwang University, Iksan, Chonbuk 570-749, Republic of Korea.
Int J Biochem Cell Biol. 2012 Apr;44(4):600-11. doi: 10.1016/j.biocel.2011.12.017. Epub 2012 Jan 3.
In this study, we explored the role of Bax inhibitor-1 (BI-1) on the expression of P450 2E1 and related ROS production. P450 2E1 protein, not mRNA, was expressed at relatively low levels in BI-1 plasmid-transfected cells (BI-1 cells) compared with neomycin-resistant vector-transfected cells (Neo cells). When exposed to ER stress, P450 2E1 expression and activity and ER membrane lipid peroxidation increased in both Neo cells and BI-1 cells, but to a lesser degree in BI-1 cells. This observation correlated with the lower level of ER stress in BI-1 cells than Neo cells. To examine the BI-1-associated P450 2E1 degradation mechanism, cells were treated with the lysosome inhibitor, bafilomycin and the proteasome inhibitor, MG132. Bafilomycin recovered the reduced P450 2E1 expression in BI-1 cells, but did not affect P450 2E1 expression in Neo cells. Next, proteosomal and lysosomal activities in Neo cells were compared to those in BI-1 cells. Although proteosomal activity was similar between Neo and BI-1 cells, LysoTracker and acridine orange labeling, lysosomal V-ATPase activity, and lysosomal cathepsin B expression were higher in BI-1 cells than in Neo cells. In the presence of ER stress, lysosomal activities decreased in Neo cells but did not change in BI-1 cells. P450 2E1 expression and ER membrane lipid peroxidation were greater in the hepatocytes and livers of BI-1 knock-out mice than in BI-1 wild-type cells and mice. Our results suggest that the BI-1-mediated enhancement of lysosomal activity regulates P450 2E1 expression and resultant ROS accumulation.
在这项研究中，我们探讨了 Bax 抑制剂-1（BI-1）对 P450 2E1 的表达和相关 ROS 产生的作用。与新霉素抗性载体转染细胞（Neo 细胞）相比，BI-1 质粒转染细胞（BI-1 细胞）中 P450 2E1 蛋白而非 mRNA 的表达水平相对较低。当暴露于内质网应激时，P450 2E1 的表达和活性以及内质网膜脂质过氧化作用在 Neo 细胞和 BI-1 细胞中均增加，但在 BI-1 细胞中程度较低。这一观察结果与 BI-1 细胞中的内质网应激水平低于 Neo 细胞有关。为了研究 BI-1 相关的 P450 2E1 降解机制，用溶酶体抑制剂巴弗洛霉素和蛋白酶体抑制剂 MG132 处理细胞。巴弗洛霉素恢复了 BI-1 细胞中减少的 P450 2E1 表达，但对 Neo 细胞中的 P450 2E1 表达没有影响。接下来，比较 Neo 细胞和 BI-1 细胞中的蛋白酶体和溶酶体活性。尽管 Neo 细胞和 BI-1 细胞中的蛋白酶体活性相似，但 LysoTracker 和吖啶橙标记、溶酶体 V-ATP 酶活性和溶酶体组织蛋白酶 B 的表达在 BI-1 细胞中高于 Neo 细胞。在存在内质网应激的情况下，Neo 细胞中的溶酶体活性降低，但 BI-1 细胞中的活性没有变化。BI-1 敲除小鼠的肝细胞和肝脏中的 P450 2E1 表达和内质网膜脂质过氧化作用大于 BI-1 野生型细胞和小鼠。我们的结果表明，BI-1 介导的溶酶体活性增强调节 P450 2E1 的表达和由此产生的 ROS 积累。
