背景:肝纤维化,一种普遍的慢性肝病,涉及与异常伤口愈合相关的过度细胞外基质产生。肝星状细胞(HSC)在肝纤维化中起关键作用,被1-磷酸鞘氨醇(S1P)等炎症因子激活。尽管S1P参与纤维化,其在HSCs中的具体作用和下游通路仍存在争议.
方法:在本研究中,我们研究了S1P/S1P受体(S1PR)在LX-2细胞系和原代HSCHippo-YAP激活中的调节作用.实时PCR,westernblot,药理学抑制剂,siRNA,并通过Rho活性测定来探讨S1P介导YAP活化的分子机制。
结果:血清和外源性S1P显著增加了HSCs中YAP靶基因的表达。S1P受体的药理学抑制剂和siRNA介导的敲除显示S1P受体2(S1PR2)是S1P诱导的HSC中CTGF表达的主要介质。使用siRNA介导的敲减的结果,Verteporfin,和磷酸化标记免疫印迹显示S1P-S1PR2信号有效抑制Hippo激酶级联,从而激活YAP。此外,S1P增加细胞中的RhoA活性,ROCK抑制剂有效阻断CTGF诱导。细胞骨架扰动试剂被证明可以极大地调节CTGF的诱导,提示肌动蛋白细胞骨架在S1P诱导的YAP激活中的重要作用。外源S1P处理足以增加COL1A1和α-SMA的表达,被YAP特异性抑制剂阻断。
结论:我们的数据表明S1P/S1PR2-Src-RhoA-ROCK轴导致Hippo-YAP激活,导致CTGF的上调,COL1A1和α-SMA在HSC中的表达。因此,S1PR2可能代表肝纤维化的潜在治疗靶标。
BACKGROUND: Hepatic fibrosis, a prevalent chronic liver condition, involves excessive extracellular matrix production associated with aberrant wound healing. Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis, activated by inflammatory factors such as sphingosine 1-phosphate (S1P). Despite S1P\'s involvement in fibrosis, its specific role and downstream pathway in HSCs remain controversial.
METHODS: In this study, we investigated the regulatory role of S1P/S1P receptor (S1PR) in Hippo-YAP activation in both LX-2 cell lines and primary HSCs. Real-time PCR, western blot, pharmacological inhibitors, siRNAs, and Rho activity assays were adopted to address the molecular mechanisms of S1P mediated YAP activation.
RESULTS: Serum and exogenous S1P significantly increased the expression of YAP target genes in HSCs. Pharmacologic inhibitors and siRNA-mediated knockdowns of S1P receptors showed S1P receptor 2 (S1PR2) as the primary mediator for S1P-induced CTGF expression in HSCs. Results using siRNA-mediated knockdown, Verteporfin, and Phospho-Tag immunoblots showed that S1P-S1PR2 signaling effectively suppressed the Hippo kinases cascade, thereby activating YAP. Furthermore, S1P increased RhoA activities in cells and ROCK inhibitors effectively blocked CTGF induction. Cytoskeletal-perturbing reagents were shown to greatly modulate CTGF induction, suggesting the important role of actin cytoskeleton in S1P-induced YAP activation. Exogeneous S1P treatment was enough to increase the expression of COL1A1 and α-SMA, that were blocked by YAP specific inhibitor.
CONCLUSIONS: Our data demonstrate that S1P/S1PR2-Src-RhoA-ROCK axis leads to Hippo-YAP activation, resulting in the up-regulation of CTGF, COL1A1 and α-SMA expression in HSCs. Therefore, S1PR2 may represent a potential therapeutic target for hepatic fibrosis.