Department of Health Science and Technology, SAIHST, Sungkyunkwan University, 81 Irwon-Ro, Gangnam-Gu, Seoul, 06351, South Korea.
Samsung Medical Center, 81 Irwon- Ro, Gangnam-Gu, Seoul, 06351, South Korea.
Mol Biol Rep. 2024 Sep 2;51(1):950. doi: 10.1007/s11033-024-09868-w.
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.
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.
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.
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.
肝纤维化是一种常见的慢性肝病，涉及与异常伤口愈合相关的细胞外基质过度产生。肝星状细胞（HSCs）在肝纤维化中起着关键作用，被诸如鞘氨醇 1-磷酸（S1P）等炎症因子激活。尽管 S1P 参与纤维化，但它在 HSCs 中的具体作用和下游途径仍存在争议。
在这项研究中，我们研究了 S1P/S1P 受体（S1PR）在 LX-2 细胞系和原代 HSCs 中 Hippo-YAP 激活中的调节作用。采用实时 PCR、western blot、药理学抑制剂、siRNA 和 Rho 活性测定来解决 S1P 介导的 YAP 激活的分子机制。
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 介导的敲低显示，S1P 受体 2（S1PR2）是 S1P 诱导 HSCs 中 CTGF 表达的主要介质。使用 siRNA 介导的敲低、verteporfin 和磷酸化标签免疫印迹显示，S1P-S1PR2 信号有效地抑制了 Hippo 激酶级联反应，从而激活了 YAP。此外，S1P 增加了细胞中的 RhoA 活性，而 ROCK 抑制剂有效地阻断了 CTGF 的诱导。细胞骨架扰乱试剂显示极大地调节了 CTGF 的诱导，表明肌动蛋白细胞骨架在 S1P 诱导的 YAP 激活中起着重要作用。外源性 S1P 处理足以增加 COL1A1 和 α-SMA 的表达，而 YAP 特异性抑制剂则阻断了它们的表达。
我们的数据表明，S1P/S1PR2-Src-RhoA-ROCK 轴导致 Hippo-YAP 激活，导致 HSCs 中 CTGF、COL1A1 和 α-SMA 的表达上调。因此，S1PR2 可能是肝纤维化的一个潜在治疗靶点。
