20-HETE Activates the Transcription of Angiotensin-Converting Enzyme via Nuclear Factor-κB Translocation and Promoter Binding

Increased vascular 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, promotes vascular dysfunction, injury, and hypertension that is dependent, in part, on the renin angiotensin system (RAS). We have shown that, in human microvascular endothelial cells, 20-HETE increases angiotensin-converting enzyme (ACE) mRNA, protein, and ACE activity via an epidermal growth factor receptor (EGFR)/ tyrosine kinase/mitogen-activated protein kinase (MAPK)/ inhibitor of κB kinase (IKK)b'mediated signaling pathway. In this work, we show that, similar to epidermal growth factor (EGF), 20-HETE (10 nM) activates EGFR by stimulating tyrosine phosphorylation; however, unlike 20-HETE, EGF does not induce ACE expression, and pretreatment with a neutralizing antibody against EGF does not prevent the 20-HETE-mediated ACE induction. Inhibition of nuclear factor κB (NF-κB) activation prevented the 4.58-fold (±0.78; P < 0.05) 20-HETE-mediated induction of ACE. The 20-HETE increased NF-κB'binding activity in nuclear extracts and the activity of both the somatic and germinal ACE promoters by 4.37-fold (60.18; P < 0.05) and 2.53-fold (60.24; P,0.05), respectively. The 20-HETE'stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-hydroxy-6,15-eicosadienoic acid and by inhibitors of EGFR, MAPK, IKKb, and NF-κB activation. Sequence analysis demonstrated the presence of two and one putative NF-κB binding sites on the human somatic and germinal ACE promoters, respectively. Chromatin immunoprecipitation assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-κB to each of the putative binding sites (S1, 3.43 ± 0.3-fold enrichment versus vehicle; S2, 3.72 ± 0.68-fold enrichment versus vehicle; S3, 3.20 ± 0.18-fold enrichment versus vehicle; P < 0.05). This is the first study to identify NF-κB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-κB signaling cascade underlying 20-HETE'mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.