Quantitative Phosphoproteomics of the Angiotensin AT2-Receptor Signaling Network Identifies HDAC1 (Histone-Deacetylase-1) and p53 as Mediators of Antiproliferation and Apoptosis.

BACKGROUND: Angiotensin AT2-receptor signaling is atypical for a G-protein coupled receptor and incompletely understood. To obtain novel insights into AT2-receptor signaling, we mapped changes in the phosphorylation status of the entire proteome of human aortic endothelial cells in response to AT2-receptor stimulation. METHODS: Phosphorylation status of human aortic endothelial cells after stimulation with C21 (1 µM; 0, 1, 3, 5, 20 minutes) was determined utilizing time-resolved quantitative phosphoproteomics. Specific changes in protein phosphorylation and acetylation were confirmed by Western Blotting. Functional tests included resazurin assay for cell proliferation, and caspase 3/7 luminescence assay or FACS analysis of annexin V expression for apoptosis. RESULTS: AT2-receptor stimulation significantly altered the phosphorylation status of 172 proteins (46% phosphorylations, 54% dephosphorylations). Bioinformatic analysis revealed a cluster of phospho-modified proteins involved in antiproliferation and apoptosis. Among these proteins, HDAC1 (histone-deacetylase-1) was dephosphorylated at serine421/423 involving serine/threonine phosphatases. Resulting HDAC1 inhibition led to p53 acetylation and activation. AT2-receptor stimulation induced antiproliferation and apoptosis, which were absent when cells were co-incubated with the p53 inhibitor pifithrin-α, thus indicating p53-dependence of these AT2-receptor mediated functions. CONCLUSIONS: Contrary to the prevailing view that AT2-receptor signaling largely involves phosphatases, our study revealed significant involvement of kinases. HDAC1 inhibition and resulting p53 activation were identified as novel, AT2-receptor coupled signaling mechanisms. Furthermore, the study created an openly available dataset of AT2-receptor induced phospho-modified proteins, which has the potential to be the basis for further discoveries of currently unknown, AT2-receptor coupled signaling mechanisms.

Aldosterone Induces Vasoconstriction in Individuals with Type 2 Diabetes: Effect of Acute Antioxidant Administration.

CONTEXT: Individuals with type 2 diabetes have an increased risk of endothelial dysfunction and cardiovascular disease. Plasma aldosterone could contribute by reactive oxygen species-dependent mechanisms by inducing a shift in the balance between a vasoconstrictor and vasodilator response to aldosterone. OBJECTIVE: We aimed to investigate the acute vascular effects of aldosterone in individuals with type 2 diabetes compared with healthy controls and if infusion of an antioxidant (n-acetylcysteine [NAC]) would alter the vascular response. METHODS: In a case-control design, 12 participants with type 2 diabetes and 14 healthy controls, recruited from the general community, were studied. Leg hemodynamics were measured before and during aldosterone infusion (0.2 and 5 ng min-1 [L leg volume]-1) for 10 minutes into the femoral artery with and without coinfusion of NAC (125 mg kg-1 hour-1 followed by 25 mg kg-1 hour-1). Leg blood flow and arterial blood pressure was measured, and femoral arterial and venous blood samples were collected. RESULTS: Compared with the control group, leg blood flow and vascular conductance decreased during infusion of aldosterone at the high dose in individuals with type 2 diabetes, whereas coinfusion of NAC attenuated this response. Plasma aldosterone increased in both groups during aldosterone infusion and there was no difference between groups at baseline or during the infusions. CONCLUSION: These results suggests that type 2 diabetes is associated with a vasoconstrictor response to physiological levels of infused aldosterone and that the antioxidant NAC diminishes this response.