氧化应激激活AP-1和肝素结合表皮生长因子样生长因子在肾上皮细胞中的转录。

M Sakai, T Tsukada, R C Harris
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引用次数: 36

摘要

缺血/再灌注损伤可增加大鼠肾脏中生物活性肝素结合表皮生长因子样生长因子(HB-EGF)的表达,提示氧化应激或氧化应激相关的细胞损伤可能影响损伤肾实质中HB-EGF的表达。我们利用未转化的大鼠肾上皮细胞系(NRK-52E细胞)来研究活性氧是否诱导HB-EGF mRNA的转录激活。缺氧/再氧化增加了NRK-52E细胞中HB-EGF的表达,在诱导亚致死细胞损伤的浓度下,过氧化氢(H(2)O(2))使HB-EGF mRNA表达增加4.7倍。自由基清除剂、二甲基硫脲和n -乙酰半胱氨酸抑制HB-EGF mRNA的诱导。相反,另一种自由基清除剂吡咯烷硫代氨基甲酸酯(PDTC)增强了H(2)O(2)介导的HB-EGF表达。由于PDTC已被报道增强AP-1介导的转录激活,我们利用电泳迁移率转移试验证实,H(2)O(2)给药NRK-52E细胞确实增加了核提取物dna结合活性,达到一致的AP-1序列。通过与人HB-EGF 5'-未翻译区近2000 bp相结合的CAT报告基因试验,我们确定H(2)O(2)使CAT活性增加了5.5倍。假设AP-1结合位点的截断或缺失突变使H(2)O(2)刺激的活性降低了60%以上,并且H(2)O(2)处理细胞的核提取物与含有该假设AP-1位点的24bp寡核苷酸的DNA结合增加。抗fos和jun抗体抑制了这种结合,并且没有与推定的AP-1位点突变的寡核苷酸结合。残馀激活位点存在于最近的5′-未翻译区(-121 ~ +60),其中包含两个假定的SP1位点。缺血后组织核提取物ap -1结合活性的时间和定位与HB-EGF mRNA表达相关。因此,在肾上皮细胞中,氧化应激增加HB-EGF的表达,这似乎部分是由AP-1结合的增加介导的。这种激活可能在组织损伤诱导HB-EGF mRNA中发挥重要作用,并可能调节缺血损伤后恢复的早期阶段。
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Oxidant stress activates AP-1 and heparin-binding epidermal growth factor-like growth factor transcription in renal epithelial cells.

Ischemia/reperfusion injury increases the expression of bioactive heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the rat kidney, suggesting that oxidant stress or cell injury related to oxidant stress might affect HB-EGF expression in the injured renal parenchyma. We utilized a nontransformed rat renal epithelial cell line (NRK-52E cells) to investigate whether reactive oxygen species induced transcriptional activation of HB-EGF mRNA. Hypoxia/reoxygenation increased HB-EGF expression in NRK-52E cells, and at concentrations that induced sublethal cell injury, hydrogen peroxide (H(2)O(2)) increased HB-EGF mRNA expression 4.7-fold. The free radical scavengers, dimethylthiourea and N-acetylcysteine inhibited HB-EGF mRNA induction. In contrast, another free radical scavenger, pyrrolidine thiocarbamate (PDTC), augmented H(2)O(2)-mediated HB-EGF expression. Since PDTC has been reported to augment AP-1-mediated transcriptional activation, we utilized an electrophoretic mobility shift assay to confirm that H(2)O(2) administration to NRK-52E cells did increase nuclear extract DNA-binding activity to a consensus AP-1 sequence. Using a CAT reporter assay coupled to the proximal 2,000 bp of the human HB-EGF 5'-untranslated region, we determined that H(2)O(2) administration increased CAT activity 5.5-fold. Truncation or deletion mutations of a putative AP-1-binding site reduced the H(2)O(2)-stimulated activity by >60%, and there was increased DNA binding of nuclear extracts from H(2)O(2)-treated cells to a 24-bp oligonucleotide containing this putative AP-1 site. Anti-fos and jun antibodies inhibited this binding, and there was no binding to an oligonucleotide in which the putative AP-1 site was mutated. The site of the residual activation was found to exist in the most proximal 5'-untranslated region (-121 to +60), which contains two putative SP1 sites. Timing and localization of AP-1-binding activity from nuclear extracts from the post-ischemic tissue correlated with HB-EGF mRNA expression. Therefore, in renal epithelial cells, oxidant stress increases HB-EGF expression, which appears to be mediated in part by an increase in AP-1 binding. This activation may play an important role in the induction of HB-EGF mRNA in response to tissue injury and may regulate early stages of recovery following ischemic damage.

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