title: Src kinase-mediates androgen receptor-dependent non-genomic activation of signaling cascade leading to endothelial nitric oxide synthase.
abstract: 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 2h 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.
authors: Department of Integrated Traditional Medicine, Graduate School of Medicine, University of Tokyo, Tokyo 113-8655, Japan.
authors: Jing
authors: J
authors: Yu
authors: Masahiro
authors: M
authors: Akishita
authors: Masato
authors: M
authors: Eto
authors: Hideki
authors: H
authors: Koizumi
authors: Ryo
authors: R
authors: Hashimoto
authors: Sumito
authors: S
authors: Ogawa
authors: Kimie
authors: K
authors: Tanaka
authors: Yasuyoshi
authors: Y
authors: Ouchi
authors: Tetsuro
authors: T
authors: Okabe
mesh_terms: D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011944:Receptors, Androgen / Q000378:metabolism*; D015398:Signal Transduction; D013739:Testosterone / Q000494:pharmacology / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D051242:Caveolin 1; D011944:Receptors, Androgen; D013739:Testosterone; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt