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Oxidant stress activates AP-1 and heparin-binding epidermal growth factor-like growth factor transcription in renal epithelial cells.

Sakai M, Tsukada T, Harris RC
Exp Nephrol. 2001 9 (1): 28-39

PMID: 11053978 · DOI:10.1159/000020705

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.

Copyright 2000 S. Karger AG, Basel

MeSH Terms (20)

Animals Cell Hypoxia Cell Line Epidermal Growth Factor Epithelial Cells Free Radical Scavengers Heparin-binding EGF-like Growth Factor Hydrogen Peroxide Intercellular Signaling Peptides and Proteins Kidney Male Oxidants Oxidative Stress Oxygen Promoter Regions, Genetic Rats Rats, Sprague-Dawley RNA, Messenger Transcription, Genetic Transcription Factor AP-1

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