ROS-dependent activation of RhoA/Rho-kinase in pulmonary artery: Role of Src-family kinases and ARHGEF1.
ROS-dependent activation of RhoA/Rho-kinase in pulmonary artery: Role of Src-family kinases and ARHGEF1.
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DOI：
10.1016/j.freeradbiomed.2017.06.022
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发表时间：
2017-09
影响因子：
7.4
通讯作者：
Knock GA
中科院分区：
文献类型：
--
作者：
MacKay CE;Shaifta Y;Snetkov VV;Francois AA;Ward JPT;Knock GA
活性氧（ROS）在平滑肌收缩中的作用尚不清楚。我们假设 G 蛋白偶联受体 (GPCR) 激活和缺氧通过刺激 ROS 产生和随后的 Src 家族激酶 (SrcFK) 激活，诱导大鼠肺内动脉 (IPA) 中的 Rho 激酶活性和收缩。 T 型前列腺素受体激动剂 U46619 诱导肺动脉平滑肌细胞 (PASMC) 产生 ROS。 U46619 还诱导 c-Src 半胱氨酸氧化、SrcFK 自磷酸化、MYPT-1 和 MLC20 磷酸化以及 IPA 收缩，所有这些反应均被抗氧化剂（依布硒啉、Tempol）抑制。收缩和 SrcFK/MYPT-1/MLC20 磷酸化也受到组合的超氧化物歧化酶和过氧化氢酶或 SrcFK 拮抗剂 PP2 的抑制，而收缩和 MYPT-1/MLC20 磷酸化则受到 Rho 鸟嘌呤核苷酸交换因子 (RhoGEF) 抑制剂 Y16 的抑制。 H2O2 和产生超氧化物的喹啉二酮 LY83583 均诱导 c-Src 氧化、SrcFK 自磷酸化和 IPA 收缩。 LY83583和H2O2诱导的收缩被PP2抑制，而LY83583诱导的收缩也被抗氧化剂和Y16抑制。 SrcFK 自磷酸化和 MYPT-1/MLC20 磷酸化也由 IPA 中的缺氧诱导，并且被线粒体抑制剂鱼藤酮和 myxothiazol 阻断。在活 PASMC 中，U46619 和 LY83583 触发 RhoA 和 RhoGEF ARHGEF1 的亚细胞易位，并且这种易位被抗氧化剂和 PP2 阻断。 RhoA 易位也被 ARHGEF1 siRNA 抑制。 U46619 增强了 ARHGEF1 与 c-Src 的 ROS 依赖性共免疫沉淀。我们的结果证明了 GPCR 诱导的胞质 ROS 或缺氧诱导的线粒体 ROS 与 SrcFK 活性、Rho 激酶活性和收缩之间的联系。 ROS 和 SrcFK 通过 ARHGEF1 激活 RhoA。 GPCR 诱导的肺内动脉 (IPA) 收缩是 ROS 依赖性的。内源性和外源性 ROS 诱导 IPA 中的 RhoA 易位和 Rho 激酶活性。内源性和外源性 ROS 也会激活 IPA 中的 Src 家族激酶 (SrcFK)。 SrcFK 和 ARHGEF1 是 ROS 诱导的 RhoA 和 Rho 激酶活性所必需的。 SrcFK 可能通过 ARHGEF1 诱导 RhoA 的 ROS 依赖性激活。
The role of reactive oxygen species (ROS) in smooth muscle contraction is poorly understood. We hypothesised that G-protein coupled receptor (GPCR) activation and hypoxia induce Rho-kinase activity and contraction in rat intra-pulmonary artery (IPA) via stimulation of ROS production and subsequent Src-family kinase (SrcFK) activation. The T-type prostanoid receptor agonist U46619 induced ROS production in pulmonary artery smooth muscle cells (PASMC). U46619 also induced c-Src cysteine oxidation, SrcFK auto-phosphorylation, MYPT-1 and MLC20 phosphorylation and contraction in IPA, and all these responses were inhibited by antioxidants (ebselen, Tempol). Contraction and SrcFK/MYPT-1/MLC20 phosphorylations were also inhibited by combined superoxide dismutase and catalase, or by the SrcFK antagonist PP2, while contraction and MYPT-1/MLC20 phosphorylations were inhibited by the Rho guanine nucleotide exchange factor (RhoGEF) inhibitor Y16. H2O2 and the superoxide-generating quinoledione LY83583 both induced c-Src oxidation, SrcFK auto-phosphorylation and contraction in IPA. LY83583 and H2O2-induced contractions were inhibited by PP2, while LY83583-induced contraction was also inhibited by antioxidants and Y16. SrcFK auto-phosphorylation and MYPT-1/MLC20 phosphorylation was also induced by hypoxia in IPA and this was blocked by mitochondrial inhibitors rotenone and myxothiazol. In live PASMC, sub-cellular translocation of RhoA and the RhoGEF ARHGEF1 was triggered by both U46619 and LY83583 and this translocation was blocked by antioxidants and PP2. RhoA translocation was also inhibited by an ARHGEF1 siRNA. U46619 enhanced ROS-dependent co-immunoprecipitation of ARHGEF1 with c-Src. Our results demonstrate a link between GPCR-induced cytosolic ROS or hypoxia-induced mitochondrial ROS and SrcFK activity, Rho-kinase activity and contraction. ROS and SrcFK activate RhoA via ARHGEF1. GPCR-induced contraction of intra-pulmonary artery (IPA) is ROS-dependent. Endogenous and exogenous ROS induce RhoA translocation and Rho-kinase activity in IPA. Endogenous and exogenous ROS also activate Src-family kinases (SrcFK) in IPA. SrcFK and ARHGEF1 are required for ROS-induced RhoA and Rho-kinase activity. SrcFK may induce ROS-dependent activation of RhoA via ARHGEF1.
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影响因子：
10.8
作者：
Knock GA;Shaifta Y;Snetkov VA;Vowles B;Drndarski S;Ward JP;Aaronson PI
通讯作者：
Aaronson PI
DOI：
10.1161/atvbaha.112.253112
发表时间：
2012-11
期刊：
Arteriosclerosis, thrombosis, and vascular biology
影响因子：
--
作者：
Gadepalli R;Singh NK;Kundumani-Sridharan V;Heckle MR;Rao GN
通讯作者：
Rao GN
影响因子：
82.9
作者：
Guilluy, Christophe;Bregeon, Jeremy;Loirand, Gervaise
通讯作者：
Loirand, Gervaise
影响因子：
4.8
作者：
Andreev, J;Galisteo, ML;Schlessinger, J
通讯作者：
Schlessinger, J
影响因子：
5.3
作者：
Giannoni, E;Buricchi, F;Chiarugi, P
通讯作者：
Chiarugi, P