Src kinase-mediates androgen receptor-dependent non-genomic activation of signaling cascade leading to endothelial nitric oxide synthase

Our previous study has demonstrated that testosterone rapidly activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells (ECs) via the phosphatidylinositol 3-kinase/Akt (PI3-kinase/Akt) pathway. The upstream regulators of this pathway are unknown. In this study, we further investigated the non-genomic action of testosterone in human aortic ECs. Acute (30 min) activation of eNOS caused by testosterone was unaffected by pretreatment with a transcriptional inhibitor, actinomycin D. Non-permeable testosterone-BSA rapidly induced Akt and eNOS phosphorylation. In contrast, luciferase reporter assay showed that the transcriptional activity of the androgen-responsive element (ARE) was increased by testosterone, but not by testosterone-BSA at 2 h after stimulation. Immunostaining displayed co-localization of androgen receptor (AR) with caveolin-1. Fractional analysis showed that AR was expressed in caveolae-enriched membrane fractions. Immunoprecipitation assays revealed the association of AR with caveolin-1 and c-Src, suggesting complex formation among them. Testosterone rapidly increased the phosphorylation of c-Src on Tyr416, which was inhibited by an AR antagonist and by siRNA for AR. PP2, a specific-inhibitor of Src kinase, abolished the testosterone-induced phosphorylation of Akt and eNOS. Our data indicate that testosterone induces rapid assembly of a membrane signaling complex among AR, caveolin-1 and c-Src, which then facilitates activation of the c-Src/ PI3-kinase/Akt cascade, resulting in activation of eNOS.

► The effects of testosterone on acute eNOS activation are a non-genomic action.
► Testosterone induces rapid assembly of a membrane signaling complex among AR, caveolin-1 and c-Src.
► c-Src kinase is a critical upstream regulator of the Akt phosphorylation cascade for eNOS activation in endothelial ECs.