Department of Nephrology, State Key Laboratory of Kidney Diseases, Chinese PLA General Hospital, Beijing, PR China.
Am J Nephrol. 2012;35(5):456-65. doi: 10.1159/000338302. Epub 2012 Apr 30.
Nephrotoxicity of cisplatin limits its clinical application. Cisplatin-induced acute renal tubular epithelial cell apoptosis is one of the major mechanisms of cisplatin nephrotoxicity. Here, the role and regulation of death-associated protein 5 (DAP5) in cisplatin-induced tubular cell apoptosis were investigated.
After upregulation of DAP5 expression by plasmid transfection and downregulation of DAP5 expression by small interfering RNA in human kidney tubular epithelial cell line (HKC) cells, the degree of cell apoptosis was assessed by flow cytometric analysis. The expression of Bax and Bcl-2 proteins was detected by Western blot analysis. The relationship between the PI3K/Akt/mTOR signaling pathway and DAP5 was also evaluated.
During cisplatin-induced apoptosis in HKC cells, DAP5 underwent proteolytic fragmentation, yielding an 86-kDa species, DAP5/p86. Overexpression of DAP5/p97 and DAP5/p86 increased the translation of Bcl-2 and reduced the extent of cisplatin-induced apoptosis. Knockdown of DAP5 expression using small interfering RNA decreased the translation of Bcl-2 and increased the degree of apoptosis. Neither manipulation affected the expression of Bax. DAP5 expression was positively regulated by the PI3K/Akt/mTOR signaling pathway.
Collectively, the results from the present study revealed a new role for DAP5 in cisplatin-induced apoptosis: DAP5/p97 and DAP5/p86 enhanced the translation of the anti-apoptotic protein Bcl-2 and inhibited cisplatin-induced apoptosis. The PI3K/Akt/mTOR signaling pathway may positively regulate the expression of DAP5.
顺铂的肾毒性限制了其临床应用。顺铂诱导的急性肾小管上皮细胞凋亡是顺铂肾毒性的主要机制之一。本研究旨在探讨凋亡相关蛋白 5（DAP5）在顺铂诱导的肾小管细胞凋亡中的作用及其调控机制。
通过质粒转染上调人肾小管上皮细胞系（HKC）细胞中 DAP5 的表达，用小干扰 RNA 下调 DAP5 的表达，流式细胞术检测细胞凋亡程度，Western blot 检测 Bax 和 Bcl-2 蛋白的表达。评估 PI3K/Akt/mTOR 信号通路与 DAP5 的关系。
在 HKC 细胞顺铂诱导的凋亡过程中，DAP5 发生蛋白水解片段化，产生 86 kDa 的 DAP5/p86 片段。DAP5/p97 和 DAP5/p86 的过表达增加了 Bcl-2 的翻译，减少了顺铂诱导的凋亡程度。用小干扰 RNA 下调 DAP5 表达降低了 Bcl-2 的翻译，增加了凋亡程度。两种操作均不影响 Bax 的表达。DAP5 的表达受 PI3K/Akt/mTOR 信号通路的正调控。
综上所述，本研究揭示了 DAP5 在顺铂诱导的凋亡中的新作用：DAP5/p97 和 DAP5/p86 增强了抗凋亡蛋白 Bcl-2 的翻译，抑制了顺铂诱导的凋亡。PI3K/Akt/mTOR 信号通路可能正向调节 DAP5 的表达。
