Inflammation Research Unit, Pfizer Global Research and Development, Pfizer Inc., 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA.
J Hepatol. 2011 Mar;54(3):521-8. doi: 10.1016/j.jhep.2010.07.026. Epub 2010 Sep 19.
BACKGROUND & AIMS: TGF-β1 a key pro-fibrotic factor activates signaling via the canonical ALK/SMAD as well as the Rho GTPase pathways. Rho kinase is a major downstream effector of Rho GTPase signaling. To understand the contribution of Rho kinase activation towards the synthesis of fibrotic mediators by hepatic stellate cells (HSC), we first profiled activated HSC and fibrotic liver tissues to identify common transcripts that were most significantly up-regulated across all samples. We then applied a pharmacologic as well as a genomics approach in a TGF-β1 activated human HSC line (LX-2) to study the involvement of Rho kinase signaling in the expression of a subset of these up-regulated fibrotic genes.
Total RNA was profiled using microarray chips. Data analysis was performed using Ingenuity Pathway Analysis software. LX-2 cells were activated with 10 ng/ml of TGF-β1 for 24 h. Activation of downstream pathways was assessed by Western blotting with phospho-specific target biomarker antibodies. Targeted knockdown of Rho kinase isoforms 1 and 2 was achieved with RNAi. Secreted levels of endothelin-1, TGF-β2, and thrombospondin-1 were measured by ELISA.
TGF-β1 activated Rho kinase and Smad pathways in LX-2 cells. The syntheses of endothelin-1 and TGF-β2 were significantly inhibited in TGF-β1 treated LX-2 cells, by isoform non-selective Rho kinase inhibitors. siRNA knockdown of each isoform suggested that endothelin-1 synthesis was largely mediated by the Rho kinase-1 isoform, while both isoforms contributed to the synthesis of TGF-β2.
The TGF-β1 mediated secretion of endothelin-1 and TGF-β2 is mediated by Rho kinase activation in human HSC.
TGF-β1 是一种关键的促纤维化因子，可通过经典的 ALK/SMAD 以及 Rho GTPase 信号通路激活信号。Rho 激酶是 Rho GTPase 信号的主要下游效应物。为了了解 Rho 激酶激活对肝星状细胞（HSC）合成纤维化介质的贡献，我们首先对激活的 HSC 和纤维化肝脏组织进行了分析，以确定在所有样本中上调最显著的常见转录本。然后，我们在 TGF-β1 激活的人 HSC 系（LX-2）中应用药理学和基因组学方法，研究 Rho 激酶信号在这些上调的纤维化基因子集表达中的作用。
使用微阵列芯片对总 RNA 进行分析。使用 Ingenuity Pathway Analysis 软件进行数据分析。用 10ng/ml 的 TGF-β1 激活 LX-2 细胞 24 小时。用磷酸化特异性靶标生物标志物抗体通过 Western blot 检测下游途径的激活。用 RNAi 实现 Rho 激酶同工型 1 和 2 的靶向敲低。通过 ELISA 测量内皮素-1、TGF-β2 和血小板反应蛋白-1 的分泌水平。
TGF-β1 激活了 LX-2 细胞中的 Rho 激酶和 Smad 途径。在 TGF-β1 处理的 LX-2 细胞中，内皮素-1 和 TGF-β2 的合成明显受到同工型非选择性 Rho 激酶抑制剂的抑制。每种同工型的 siRNA 敲低表明，内皮素-1 的合成主要由 Rho 激酶-1 同工型介导，而两种同工型都有助于 TGF-β2 的合成。
TGF-β1 介导的内皮素-1 和 TGF-β2 的分泌是通过人 HSC 中 Rho 激酶的激活介导的。
