Androgen Receptor-Dependent Activation of Endothelial Nitric Oxide Synthase in Vascular Endothelial Cells: Role of Phosphatidylinositol 3-Kinase/Akt Pathway

Abstract

The mechanisms of testosterone-induced vasodilatation are not fully understood. This study investigated the effect of testosterone on nitric oxide (NO) synthesis and its molecular mechanism using human aortic endothelial cells (HAEC). Testosterone at physiological concentrations (1-100 nm) induced a rapid (15-30 min) increase in NO production, which was associated with phosphorylation and activation of endothelial NO synthase (eNOS). Then, the involvement of the androgen receptor (AR), which is abundantly expressed in HAEC, was examined. The effect of testosterone on eNOS activation and NO production were abolished by pretreatment with an AR antagonist nilutamide and by transfection with AR small interference RNA. In contrast, testosterone-induced eNOS phosphorylation was unchanged by pretreatment with an aromatase inhibitor or by transfection with ERalpha small interference RNA. 5alpha-Dihydrotestosterone, a nonaromatizable androgen, also stimulated eNOS phosphorylation. Next, the signaling cascade that leads to eNOS phosphorylation was explored. Testosterone stimulated rapid phosphorylation of Akt in a time- and dose-dependent manner, with maximal response at 15-60 min. The rapid phosphorylation of eNOS or NO production induced by testosterone was inhibited by Akt inhibitor SH-5 or by phosphatidylinositol (PI) 3-kinase inhibitor wortmannin. Co-immunoprecipitation assays revealed a testosterone-dependent interaction between AR and the p85alpha subunit of PI3-kinase. In conclusion, testosterone rapidly induces NO production via AR-dependent activation of eNOS in HAEC. Activation of PI3-kinase/Akt signaling and the direct interaction of AR with p85alpha are involved, at least in part, in eNOS phosphorylation.

Hormonal effects on blood vessels

“…101 We recently demonstrated that testosterone rapidly induces NO production via AR-mediated activation of eNOS in human aortic ECs. 89 Furthermore, we showed that AR directly interacts with the p85 subunit of phosphatidylinositol 3-kinase, resulting in phosphorylation/activation of Akt/eNOS signaling. Taking together with our preliminary observation about the involvement of extracellular signal-regulated kinase 1/2 signaling and [Ca2+]i in AR-dependent eNOS activation, quite similar signaling pathways to those for estrogen can be proposed for testosterone (Figure 1), although some of these pathways should be verified in further studies.…”

“…Indeed, androgen receptor (AR) has been shown to be expressed in these cells. [89][90][91] Effects of testosterone on animal models of atherosclerosis and neointima formation It has been demonstrated that the administration of testosterone in castrated male rabbits that were fed a high-cholesterol diet reduced aortic atherosclerosis, largely independent of plasma lipids. 92,93 In addition, neointima formation after coronary balloon injury in swine was increased by castration and was reversed by testosterone replacement.…”

Testosterone: a vascular hormone in health and disease

“…NO is a potent vasodilator that can be synthesised by NO synthase (NOS) and is released by the vascular endothelium, among other tissues, and NO-induced vasodilation is often therapeutically targeted in hypertension and angina (Miller & Megson 2007). In vitro, cultured human endothelial cells increase NO synthesis in response to physiological concentrations of testosterone via nongenomic activation of intracellular signalling pathways and Ca 2C influx (Goglia et al 2010, Yu et al 2010, Campelo et al 2012. Moreover, increased endothelial NOS (eNOS) expression and phosphorylation were observed in testosterone-and DHT-treated human umbilical vein endothelial cells (HUVECs) stimulated with hydrogen peroxide to induce senescence (Ota et al 2012).…”

“…In addition to slower acting gene regulation, rapid AR activation of intracellular signalling pathways has also been suggested. Yu et al (2010) described how the rapid phosphorylation of eNOS or NO production was abolished by pre-treatment with an AR antagonist, nilutamide, or by transfection with AR siRNA and that DHT also stimulated eNOS phosphorylation in human aortic endothelial cells (HAECs). The authors suggest that the activation of PI3-kinase/Akt intracellular signalling pathway and the direct interaction of the AR with p85a (the regulatory subunit of PI3-kinase) were responsible for the observed effects (Yu et al 2010).…”

Roles of the Androgen – Androgen Receptor System in Vascular Angiogenesis

“…These cells express ARs and are targets for androgen action 1) . Regarding ECs, it has been reported that androgens rapidly induce nitric oxide (NO) production in human endothelial cells via the phosphorylation/activation of endothelial nitric oxide synthase (eNOS) and that the activation of eNOS is inhibited by an antiandrogen drug or AR knockdown [58][59][60][61][62] . Yu et al demonstrated that testosterone induces the rapid assembly of a membrane-signaling complex among AR, c-Src, and caveolin-1, which facilitates activation of the c-Src/phosphoinositide-3-kinase (PI3K)/serine/threonine protein kinase (Akt) cascade, with the consequent activation of eNOS in vascular ECs 58) .…”