SphK1/S1P信号通路介导肺动脉平滑肌细胞增殖的分子机制研究

Recent studies have indicated that SphK1/S1P signaling pathway is involved in the development of pulmonary vascular remodeling, while the molecular mechanisms responsible for up-regulation of SphK1 expression in pulmonary arterial smooth muscle cell and S1P-inducing pulmonary arterial smooth muscle cell proliferation are still unclear. The aims of the present proposal will explore: (1) whether PDGF up-regulates SphK1 expression and increases S1P production in pulmonary arterial smooth muscle cell, and whether activation of Akt or ERK1/2 signaling mediates PDGF-induced SphK1 expression; (2) whether S1P binds to specific S1P receptor and further activates STAT3 cascades，which in turn modulates Skp2/p27 axis leading to pulmonary arterial smooth muscle cell proliferation;(3) whether intervention of SphK1/S1P pathway in animal model of pulmonary arterial hypertension inhibits pulmonary arterial smooth muscle cell proliferation and pulmonary arterial remodeling, and therefore prevent and treat the development of pulmonary arterial hypertension, and whether this mechanism is associated with the suppression of STAT3 regulation of Skp2/p27 axis. The present study will address the role and mechanisms of SphK1/S1P signaling in the proliferation of pulmonary arterial smooth muscle cell in cultured pulmonary arterial smooth muscle cell and animal model of pulmonary arterial hypertension, our study will provide novel theoretical evidences and new targets for the treatment of pulmonary arterial hypertension.

新近的研究提示SphK1/S1P信号通路介导肺血管重塑的发生，但目前仍不清楚上调肺动脉平滑肌细胞中SphK1表达及S1P诱导肺动脉平滑肌细胞增殖的分子信号机制。本研究将探讨：（1）PDGF是否可上调肺动脉平滑肌细胞中SphK1表达，增加S1P产生；激活何种信号通路（Akt 或ERK）介导PDGF诱导的SphK1表达；（2）S1P是否通过S1P受体激活与之偶联的STAT3信号通路，进而调控Skp2/p27轴，诱导肺动脉平滑肌细胞增殖；（3）干预SphK1/S1P信号通路可否抑制动物模型中肺动脉平滑肌细胞增殖及肺血管重塑，预防及治疗肺动脉高压，其机制是否与抑制STAT3调控的Skp2/p27变化相关。本研究将从培养的肺动脉平滑肌细胞及肺动脉高压动物模型两个方面探索SphK1/S1P信号通路在肺动脉平滑肌细胞增殖中的作用及机制，本研究将为肺动脉高压的治疗提供新的理论基础、实验依据及治疗靶点。

S1P作为重要的磷脂类生物活性物质参与肺动脉高压的发病，但其分子生物学机制尚不完全清楚。本研以培养的肺动脉平滑肌细胞及肺动脉高压动物模型为研究对象，探索S1P介导肺动脉平滑肌细胞增殖、肺血管重塑的机制，为肺动脉高压的治疗提供理论基础及实验依据。本项目获得了以下研究结果：（1）野百合碱诱发的大鼠肺动脉高压中，SphK1表达上调，S1P产生增加，进而通过激活NF-KB信号通路，上调细胞周期蛋白cyclinD1 表达，促进肺动脉平滑肌细胞增殖，参与肺血管重塑引发肺动脉高压；白芦藜醇可通过抑制SphK1表达，抑制S1P产生、NF-KB激活、cyclinD1上调，抑制肺动脉高压的发生发展；（2）TGF-b可上调肺动脉平滑肌细胞中SphK1的表达，增加S1P的产生，进而上调Notch3表达及激活，导致细胞内NICD3增加，促进了肺动脉平滑肌细胞的增殖；抑制SMAD2/3信号通路或抑制SphK1活性，可抑制TGF-b 诱导的Notch3的激活及肺动脉平滑肌细胞增殖；（3）原代培养的肺动脉平滑肌细胞中S1P可通过激活PLC，导致细胞内IP3产生增加，进而导致细胞内钙离子浓度增加，激活calcineurin/NFAT3信号通路，从而上调OPN的表达，导致了肺动脉平滑肌细胞的增殖；进一步我们发现以罗格列酮激活PPARgamma 可抑制S1P刺激的肺动脉平滑肌细胞的增殖，其机制与PPARgamma抑制S1P激活的calcineurin/NFAT3 信号通路，进而抑制OPN的表达相关；（4）原代培养的肺动脉平滑肌细胞中，S1P可剂量及时间依赖性的下调CDH1的表达，同时S1P可上调肺动脉平滑肌细胞中TRAF2的表达，上调自噬相关蛋白BECN1表达；以小干扰RNA沉默TRAF2的表达，可抑制S1P刺激的BECN1表达增加，逆转S1P导致的CDH1下调；进一步的研究发现，沉默BECN1的表达或以氯喹抑制自噬的活性，亦可抑制S1P诱发的CDH1表达下调。我们的研究提示S1P可通过上调TRAF2，进而上调BECN1激活自噬，降解肺动脉平滑肌细胞中CDH1；（5）PDGF通过激活ERK信号通路上调SphK1蛋白表达、增加S1P产生，S1P进而上调miRNA21表达，miRNA21可降低BMPRII蛋白表达，进而导致Id1蛋白表达下降，从而促进肺动脉平滑肌细胞增殖。