Testosterone (T) deficiency is positively correlated with the increased incidence of cardiovascular disease. However, the effects of T on vascular endothelial cells remain obscure. Tube formation capacity is critical for vascular regeneration/repair and Smad1 plays an important role in these events. In this study, we investigated the effects of T on Smad1 activation and tube formation of cultured human umbilical endothelial cells (HUVECs). Our results showed that T rapidly increased endothelial Smad1 phosphorylation. This effect was mimicked by cell-impermeable T-BSA conjugates and was not altered by transcriptional inhibitor actinomycin D or translational inhibitor cycloheximide. T-induced Smad1 phosphorylation was blocked by ERK1/2 and c-Src inhibitors or their specific siRNAs, while it was reinforced by ERK1/2 or c-Src overexpression. Indeed, T rapidly activated ERK1/2 and c-Src signalings and c-Src was confirmed as the upstream of ERK1/2. Moreover, caveolae disruptor methyl-β-cyclodextrin (β-MCD) blocked Smad1 activation induced by T. The association of caveolin-1 with androgen receptor (AR) or c-Src was detected by immunoprecipitation and it was significantly increased by rapid T stimulation. Furthermore, fractional analysis showed that AR and c-Src were expressed in caveolae-enriched membrane fractions. T promoted tube formation of HUVECs, which was blocked by c-Src and ERK1/2 inhibitors or by the knockdown of Smad1. In conclusion, T increased tube formation of endothelial cells isolated from veins by stimulating Smad1 phosphorylation in a nongenomic manner, which was mediated by signals from AR/c-Src located in caveolae to ERK1/2 cascade. These findings may shed new light on the relevance of T to its vascular functions.
Molecular and cellular endocrinology. 2017 Feb 03 [Epub ahead of print]
Pei Liu, Xiaosa Li, Fuhu Song, Ping Li, Jinzhi Wei, Qing Yan, Xingyan Xu, Jun Yang, Chuanxiang Li, Xiaodong Fu
Department of Anesthesiology, The Third Affiliated Hospital of Southern Medical University, Guangzhou City, Guangdong Province, 510630, China., Key Laboratory of Cardiovascular Diseases, School of Basic Medical Sciences; Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou 511436, China., Department of Anesthesiology, The Third Affiliated Hospital of Southern Medical University, Guangzhou City, Guangdong Province, 510630, China. Electronic address: ., Key Laboratory of Cardiovascular Diseases, School of Basic Medical Sciences; Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou 511436, China. Electronic address: .