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-b inding epidermal growth factor-like growth factor (HB-EGF) in the rat kidne y, 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 investi gate whether reactive oxygen species induced transcriptional activation of HB-EGF mRNA. Hypoxia/reoxygenation increased HB-EGF expression in NRK-52E c ells, and at concentrations that induced sublethal cell injury, hydrogen pe roxide (H2O2) increased HB-EGF mRNA expression 4.7-fold. The free radical s cavengers, dimethylthiourea and N-acetylcysteine inhibited HB-EGF mRNA indu ction. In contrast, another free radical scavenger, pyrrolidine thiocarbama te (PDTC), augmented H2O2-mediated HB-EGF expression. Since PDTC has been r eported to augment AP-l-mediated transcriptional activation, we utilized an electrophoretic mobility shift assay to confirm that H2O2 administration t o NRK-52E cells did increase nuclear extract DNA-binding activity to a cons ensus AP-1 sequence. Using a CAT reporter assay coupled to the proximal 2,0 00 bp of the human HB-EGF 5'-untranslated region, we determined that H2O2 a dministration increased CAT activity 5.5-fold. Truncation or deletion mutat ions of a putative AP-1-binding site reduced the H2O2-stimulated activity b y >60%, and there was increased DNA binding of nuclear extracts from H2O2-t reated cells to a 24-bp oligonucleotide containing this putative AP-1 site. Anti-fos and jun antibodies inhibited this binding, and there was no bindi ng to an oligonucleotide in which the putative AP-1 site was mutated. The s ite of the residual activation was found to exist in the most proximal 5'-u ntranslated region (-121 to +60), which contains two putative SP1 sites. Ti ming and localization of AP-l-binding activity from nuclear extracts from t he post-ischemic tissue correlated with HB-EGF mRNA expression. Therefore, in renal epithelial cells, oxidant stress increases HB-EGF expression, whic h appears to be mediated in part by an increase in AP-1 binding. This activ ation may play an important role in the induction of HB-EGF mRNA in respons e to tissue injury and may regulate early stages of recovery following isch emic damage. Copyrighr (C) 2000 S. Karger AG, Basel.