Hypoxia-induced upregulation of eNOS gene expression is redox-sensitive: Acomparison between hypoxia and inhibitors of cell metabolism

Several papers report a hypoxia-induced upregulation of the endothelial nit ric oxide synthase (eNOS) mRNA expression. Since there is no known hypoxia- sensitive element binding site in the eNOS promoter, we reasoned that the e ffect of hypoxia could be simulated by a metabolically elicited alteration of the redox state. Therefore, cultured porcine aortic endothelial cells (P AEC) were exposed to hypoxia (1-10% O-2) Or inhibitors of cellular energy m etabolism including rotenone, 2, 4 dinitrophenol (DNP) and 2-deoxyglucose f or 6 to 24 h. Additionally, cells were treated with lactate and nicotinic a cid to alter the cellular NAD(P)H/NAD(P) ratio without changes of energy su pply. The cellular NAD(P)H/NAD(P) ratio was used as an index of the cellula r redox state and determined using the MTT-assay. Hypoxia increased eNOS mR NA transcription and MTT-reduction in a manner inversely proportional to pO (2). Exposure to rotenone, DNP, and lactate increased the NAD(P)H/NAD(P) ra tio, MTT-reduction, and eNOS mRNA also in parallel. In contrast, 2-deoxyglu cose and nicotinic acid attenuated both MTT-reduction and eNOS mRNA express ion. In order to study a potential role of the redox regulated transcriptio n factor complex AP-1 in hypoxia-induced eNOS mRNA transcription, c-jun exp ression was determined and decoy experiments were performed. c-jun expressi on paralleled changes of eNOS mRNA expression and MTT-reduction. Furthermor e, in the presence of oligodeoxynucleotides corresponding to the AP-1 bindi ng sites of the eNOS promoter, the hypoxia and chemically induced eNOS mRNA expression was completely abolished. We propose that hypoxia, by altering cellular metabolism, leads to an increase in the cellular NAD(P)H/NAD(P) ra tio which favors enhanced eNOS expression by redox-sensitive AP-1 mediated transcriptional control. (C) 2001 Wiley-Liss, Inc.