Abstract 13010: Endothelin-1 Increases ACVRL-1 Expression at the Transcriptional and Post-transcriptional Levels in Human Pulmonary Arterial Endothelial Cells via Gi, RhoA and Rho Kinase Pathway With Involvement of Sp-1

Abstract

Backgrounds: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vasoconstriction and organic stenosis due to abnormal proliferation of pulmonary vascular cells. Endothelin (ET)-1 induces pulmonary vasoconstriction hyperplasia and plays a pivotal role in the pathogenesis of PAH. The germline mutations of activin receptor-like kinase (ACVRL)-1, a serine/threonine kinase and receptor for TGF-β, have been reported in idiopathic and hereditary PAH. However, the relationship between ET-1 and ACVRL-1 in the pathogenesis of PAH is largely unknown. Thus, we investigated the molecular mechanism of ACVRL-1 expression induced by ET-1 in human pulmonary arterial endothelial cells (hPAECs). Methods: ACVRL-1 expression levels were measured using Western blotting and quantitative real-time polymerase chain reaction. The promoter activity of ACVRL-1 was evaluated using dual luciferase assay. hPAECs were pretreated with pertussis toxin (PTX), cell-permeable C3 toxin (C3T) and Y27632 to inhibit Gi, RhoA and Rho kinase, respectively. GTP-RhoA, an active form of RhoA, was assessed using pull-down assay. Results: ET-1 increased mRNA and protein expression of ACVRL-1 in hPAECs (1.8±0.2 folds, P<0.05; 1.5±0.4 folds, P<0.05, respectively). The pull-down assay showed that ET-1 induced GTP-loading of RhoA. ET-1-induced RhoA activation was suppressed by PTX pretreatment. Furthermore, PTX, C3T, and Y27632 suppressed ET-1-induced ACVRL-1 expression. The transcriptional activity of the ACVRL-1 promoter was increased by ET-1 by 1.2±0.17 folds (P<0.05 vs. control). Moreover, ACVRL-1 mRNA was stabilized by ET-1 treatment, and the effect was canceled by Y27632. Finally, the expression of Sp-1, one of known transcriptional factors for ACVRL-1, was increased with a peak at 15 min after ET-1 treatment, and PTX, C3T and Y27632 significantly inhibited the Sp-1 induction by ET-1. Conclusion: These data indicate that ET-1 increases ACVRL-1 expression both at transcriptional and post-transcriptional mechanisms via the Gi/RhoA/Rho kinase/Sp-1 axis in human PAECs.