Abstract 388: Identification of FOXO1 as a Major Transcriptional Mediator of Angiotensin-(1-7) Signaling in Human Endothelium
Angiotensin-(1-7) has been shown to induce vasodilation, positive regulation of insulin, anti-proliferative and anti-tumorigenic activities via Mas receptor activation. However, little is known about the Ang-(1-7) signal transduction. The aim of this study was to identify proteins involved in the Ang-(1-7) signaling using mass spectrometry-based time-resolved quantitative phosphoproteome of human aortic endothelial cells (HAEC) treated with Ang-(1-7). HAEC were incubated with 10-7 M of Ang-(1-7) for 3, 5 and 20 min. After cell harvesting and protein digestion with tripsin, peptides were labeled (iTRAQ). Enriched phosphopeptides were separated by reversed-phase liquid chromatography. An LTQ Orbitrap XL instrument was used for mass-espectrometry analysis. We identified 1288 unique phosphosites on 699 different proteins with 99% certainty of correct peptide identification and phosphorylation site localization. Of these, 121 sites on 79 proteins had their phosphorylation levels significantly changed by Ang-(1-7). Among these proteins forkhead box protein O1 (FOXO1) was dephosphorylated at Ser256. Following S256 dephosphorylation, FOXO1 becomes active and then translocates to the nucleus where it can increase transcription of genes involved in apoptosis, cell arrestment, glucose metabolism, oxidative protection and tumor suppression. Indeed, additional studies in HAEC showed that following 3 minutes of Ang-(1-7) treatment, FOXO1 was accumulated in the HAEC nucleus which is in accordance with the downphosphorylation of S256. Furthermore, we observed that FOXO1 accumulated in the nucleus of A549 lung adenocarcinoma cells treated with Ang-(1-7), supporting the antitumorigenic activity of this angiotensin peptide. Moreover, Ang-(1-7) triggered changes in the phosphorylation status of several known downstream effectors of the insulin signaling, indicating an important role of Ang-(1-7) in glucose homeostasis. In summary, we performed for the first time a large-scale MS-based phosphoproteomics approach to interrogate the Ang-(1-7)/Mas signaling in human endothelial cells. Our data open new possibilities for the understanding of the mechanisms underlying Mas activation in the human endothelium.
- © 2012 by American Heart Association, Inc.