Endothelin-1 upregulates activin receptor-like kinase-1 expression via Gi/RhoA/Sp-1/Rho kinase pathways at transcriptional and post-transcriptional levels in human pulmonary arterial endothelial cells

BackgroundsPulmonary arterial hypertension (PAH) is a disease with poor prognosis that is characterized by pulmonary vasoconstriction and organic stenosis due to abnormal proliferation of pulmonary vascular cells. It has been demonstrated that endothelin (ET)-1 induces pulmonary vasoconstriction through activation of RhoA. Moreover, we previously demonstrated that Gi, a heterotrimeric G protein, functions upstream of RhoA activation. A gene mutation of activin receptor-like kinase (ACVRL)-1 is recognized in idiopathic or heritable PAH patients. However, little is known about the association between ET-1 and ACVRL-1. In the present study, we investigated the effect of ET-1 on ACVRL-1 expression and aimed to delineate the involvement of the Gi/RhoA/Rho kinase pathway.MethodsET-1 was added to culture medium of human pulmonary arterial endothelial cells (PAECs), and ACVRL-1 expression levels were analyzed using western blotting and quantitative polymerase chain reaction. The promoter activity of ACVRL-1 was evaluated by dual luciferase assay. Before adding ET-1 to the PAECs, pretreatment with pertussis toxin (PTX) or exoenzyme C3 transferase (C3T) was performed for the inhibition of Gi or RhoA, respectively. Rho kinase was inhibited by Y27632. Active form of RhoA (GTP-RhoA) was assessed by pull-down assay.ResultsACVRL-1 expression was increased by ET-1 in the PAECs. Pull-down assay revealed that ET-1 rapidly induced a GTP-loading of RhoA. The ET-1-induced RhoA activation was suppressed by pretreatment with PTX or C3T. Further, PTX, C3T, and Y27632 suppressed the ET-1-induced ACVRL-1 expression. The activity of ACVRL-1 promotor and the lifespan of ACVRL-1 mRNA was increased by ET-1. Sp-1, which is one of the transcriptional factors of ACVRL-1, peaked 15 min after adding ET-1 to the PAECs. PTX and C3T prevented the increase of Sp-1 induced by ET-1.ConclusionThe present study demonstrated that ET-1 increases ACVRL-1 expression at the transcriptional and post-transcriptional levels in human PAECs via the Gi/RhoA/Rho kinase pathway with involvement of Sp-1.