Sugimoto Koichi, Yokokawa Tetsuro, Misaka Tomofumi, Kaneshiro Takashi, Yamada Shinya, Yoshihisa Akiomi, Nakazato Kazuhiko, Takeishi Yasuchika
Department of Cardiovascular Medicine, Fukushima Medical University, Fukushima, Japan.
Department of Pulmonary Hypertension, Fukushima Medical University, Fukushima, Japan.
Front Cardiovasc Med. 2021 Feb 23;8:648981. doi: 10.3389/fcvm.2021.648981. eCollection 2021.
Pulmonary arterial hypertension (PAH) is characterized by pulmonary vasoconstriction and organic stenosis. It has been demonstrated that endothelin-1 (ET-1) induces pulmonary vasoconstriction through the activation of RhoA. In addition, a gene mutation of activin receptor-like kinase (ACVRL)-1 is recognized in PAH patients. However, little is known about the association between ET-1 and ACVRL-1. In the present study, we aimed to investigate the effect of ET-1 on ACVRL-1 expression and delineate the involvement of the G/RhoA/Rho kinase pathway. ET-1 was added to culture medium of human pulmonary arterial endothelial cells (PAECs). Pre-treatment with pertussis toxin (PTX) or exoenzyme C3 transferase (C3T) was performed for inhibition of G or RhoA, respectively. Rho kinase was inhibited by Y27632. Mithramycin A was used for inhibition of Sp-1, which is a transcriptional factor of ACVRL-1. The active form of RhoA (GTP-RhoA) was assessed by pull-down assay. ACVRL-1 expression was increased by ET-1 in the PAECs. Pull-down assay revealed that ET-1 induced GTP-loading of RhoA, which was suppressed by pre-treatment with PTX or C3T. Further, PTX, C3T, and Y27632 suppressed the ET-1-induced ACVRL-1 expression. ET-1 increased the activity of the ACVRL-1 promoter and stabilized the ACVRL-1 mRNA. Sp-1 peaked 15 min after adding ET-1 to the PAECs. PTX and C3T prevented the increase of Sp-1 induced by ET-1. Inhibition of Sp-1 by mithramycin A suppressed ET-1-induced ACVRL-1 upregulation. The present study demonstrated that ET-1 increases ACVRL-1 expression in human PAECs the G/RhoA/Rho kinase pathway with the involvement of Sp-1.
肺动脉高压（PAH）的特征是肺血管收缩和器质性狭窄。已有研究表明，内皮素-1（ET-1）通过激活RhoA诱导肺血管收缩。此外，在PAH患者中发现了激活素受体样激酶（ACVRL）-1的基因突变。然而，关于ET-1与ACVRL-1之间的关联知之甚少。在本研究中，我们旨在研究ET-1对ACVRL-1表达的影响，并阐明G/RhoA/Rho激酶途径的参与情况。将ET-1添加到人肺动脉内皮细胞（PAECs）的培养基中。分别用百日咳毒素（PTX）或外切酶C3转移酶（C3T）进行预处理以抑制G或RhoA。用Y27632抑制Rho激酶。用丝裂霉素A抑制ACVRL-1的转录因子Sp-1。通过下拉试验评估RhoA的活性形式（GTP-RhoA）。ET-1使PAECs中的ACVRL-1表达增加。下拉试验显示，ET-1诱导RhoA的GTP负载，这被PTX或C3T预处理所抑制。此外，PTX、C3T和Y27632抑制了ET-1诱导的ACVRL-1表达。ET-1增加了ACVRL-1启动子的活性并稳定了ACVRL-1 mRNA。在向PAECs中添加ET-1后15分钟，Sp-1达到峰值。PTX和C3T阻止了ET-1诱导的Sp-1增加。丝裂霉素A对Sp-1的抑制抑制了ET-1诱导的ACVRL-1上调。本研究表明，ET-1通过Sp-1参与G/RhoA/Rho激酶途径增加人PAECs中ACVRL-1的表达。
