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.
Sakai
M
M
Department of Medicine and Department of Veterans Affairs Medical Center, Nashville, Tenn., USA.
Tsukada
T
T
Harris
R C
RC
D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000235:genetics*; D004847:Epithelial Cells / Q000502:physiology; D016166:Free Radical Scavengers / Q000494:pharmacology; D066257:Heparin-binding EGF-like Growth Factor; D006861:Hydrogen Peroxide / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D007668:Kidney / Q000166:cytology / Q000502:physiology*; D008297:Male; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000502:physiology*; D010100:Oxygen / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018808:Transcription Factor AP-1 / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects*
D016166:Free Radical Scavengers; C587883:HBEGF protein, human; C587885:Hbegf protein, rat; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D016877:Oxidants; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D004815:Epidermal Growth Factor; D006861:Hydrogen Peroxide; D010100:Oxygen