CREB介导氧化应激后ERK诱导的小鼠肾小管细胞存活。

CREB mediates ERK-induced survival of mouse renal tubular cells after oxidant stress.

作者信息

Arany Istvan, Megyesi Judit K, Reusch Jane E B, Safirstein Robert L

机构信息

Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.

出版信息

Kidney Int. 2005 Oct;68(4):1573-82. doi: 10.1111/j.1523-1755.2005.00569.x.

DOI:10.1111/j.1523-1755.2005.00569.x

PMID:16164634

Abstract

BACKGROUND

We showed that extracellular signal-regulated protein kinase (ERK) is prosurvival during oxidant stress both in the kidney and in cultured mouse proximal tubule (TKPTS) cells and demonstrated concomitant activation of ERK as well as the cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB), during survival in vitro. We now show that CREB is a necessary prosurvival target of ERK.

METHODS

Ischemia/reperfusion (I/R) injury was induced in 129Sv mice. Oxidant stress was induced by hydrogen peroxide (H(2)O(2)) in TKPTS cells. Activation of CREB was determined by immunohistochemistry and Western blotting. Inhibition and activation of CREB was achieved by mutant or activated CREB-containing adenoviruses in vitro. The effects of oxidant stress on cell survival, CREB binding, and CREB-mediated transcription was determined by cell counting, gelshift analysis, and luciferase assay, respectively.

RESULTS

I/R activates CREB in the surviving distal nephron segments of the kidney. Inhibition of ERK and CREB abrogates survival after 0.5 mmol/L H(2)O(2) treatment, while overexpression of CREB ameliorates necrotic death caused by 1 mmol/L H(2)O(2). Inhibition of ERK also inhibited CREB activation. Binding of phosphorylated CREB to a CREB oligonucleotide was significantly increased after 0.5 mmol/L H(2)O(2) but decreased after 1 mmol/L H(2)O(2). Similarly, CREB-mediated transcription was significantly increased after 0.5 mmol/L H(2)O(2) treatment, while 1 mmol/L H(2)O(2) inhibited it. Interestingly, transcription from the CREB-driven bcl-2 promoter was unchanged after 0.5 mmol/L but decreased after 1 mmol/L H(2)O(2) treatment in agreement with Western blot studies.

CONCLUSION

We show that survival during oxidant stress is mediated through CREB and identification of its downstream targets will reveal important survival pathways.

摘要

背景

我们发现细胞外信号调节蛋白激酶（ERK）在肾脏和培养的小鼠近端小管（TKPTS）细胞的氧化应激过程中具有促生存作用，并且在体外存活期间证明了ERK以及环磷酸腺苷（cAMP）反应元件结合蛋白（CREB）的同时激活。我们现在表明CREB是ERK必要的促生存靶点。

方法

在129Sv小鼠中诱导缺血/再灌注（I/R）损伤。在TKPTS细胞中用过氧化氢（H₂O₂）诱导氧化应激。通过免疫组织化学和蛋白质印迹法测定CREB的激活。在体外通过含突变型或激活型CREB的腺病毒实现对CREB的抑制和激活。分别通过细胞计数、凝胶迁移分析和荧光素酶测定来确定氧化应激对细胞存活、CREB结合及CREB介导的转录的影响。

结果

I/R激活肾脏中存活的远端肾单位节段中的CREB。抑制ERK和CREB可消除0.5 mmol/L H₂O₂处理后的细胞存活，而CREB的过表达可改善1 mmol/L H₂O₂所致的坏死性死亡。抑制ERK也会抑制CREB的激活。0.5 mmol/L H₂O₂处理后，磷酸化CREB与CREB寡核苷酸的结合显著增加，但1 mmol/L H₂O₂处理后则减少。同样，0.5 mmol/L H₂O₂处理后CREB介导的转录显著增加，而1 mmol/L H₂O₂则抑制该转录。有趣的是，与蛋白质印迹研究一致，0.5 mmol/L处理后CREB驱动的bcl-2启动子的转录未改变，但1 mmol/L H₂O₂处理后则降低。