Abstract 024: 20-HETE Activates the Transcription of Endothelial Angiotensin Converting Enzyme (ACE) via NF-kB 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 showed that in cultured human microvascular endothelial cells (HMVEC), 20-HETE (5 nM) increases angiotensin converting enzyme (ACE) mRNA, protein, and cellular and extracellular ACE activity via EGFR-tyrosine kinase-MAPK-IKKβ- signaling pathway. Here we show that treatment of HMVEC with EGF (100 ng/ml) alone or with 20-HETE (10 nM) did not induce or further increase ACE mRNA levels. Pretreatment of cells with a neutralizing antibody against EGF did not prevent the 20-HETE- or 20-HETE+EGF-mediated ACE induction, indicating that the induction of ACE by 20-HETE is EGF-independent. The NF-kB inhibitor JSH-23 prevented the 4.58-fold (±0.78; p<0.05) 20-HETE (10nM)-mediated induction of ACE. EMSA of 20-HETE-treated cells showed increased NF-kB binding activity in nuclear extracts. In cells transfected with luciferase promoter-reporter constructs for the somatic or germinal ACE promoter regions, 20-HETE (10 nM) increased promoter activity by 4.37-fold (±0.18; p<0.05) and 2.53-fold (± 0.24; p<0.05), respectively. 20-HETE-stimulated ACE promoter activity was abrogated by the 20-HETE antagonist 20-HEDE and by inhibitors of EGFR, MAPK, IKKβ and NF-kB activation. Sequence analysis demonstrated the presence of two (Chr 17: S1: 61554061-61554075 , S2: 61554157-61554171) and one (Chr 17: S3: 61562250-61562264) putative NF-kB binding sites on the human somatic and germinal ACE promoters, respectively. CHIP assay indicated that 20-HETE stimulates the translocation and subsequent binding of NF-kB to each of the putative binding sites (S1: 3.43±0.3, S2: 3.72±0.68, S3: 3.20±0.18-fold enrichment vs vehicle; p<0.05). In vivo, increased vascular 20-HETE levels are associated with increased phospho-IKK and ACE levels that are prevented by treatment with 20-HETE antagonists. This is the first study to identify NF-kB as a transcriptional factor for ACE and to implicate a distinct EGFR/MAPK/IKK/NF-kB signaling cascade underlying 20-HETE-mediated transcriptional activation of ACE mRNA and stimulation of ACE activity.
Author Disclosures: V. Garcia: None. L. Milhau: None. J.R. Falck: None. M.L. Schwartzman: None.
- © 2014 by American Heart Association, Inc.