TGF-β1 mediated activation of Rho kinase induces TGF-β2 and endothelin-1 expression in human hepatic stellate cells

Background & Aims: TGF-beta 1 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-beta 1 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. Methods: 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-beta 1 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-beta 2, and thrombospondin-1 were measured by ELISA. Results: TGF-beta 1 activated Rho kinase and Smad pathways in LX-2 cells. The syntheses of endothelin-1 and TGF-beta 2 were significantly inhibited in TGF-beta 1 treated LX-2 cells, by isoform nonselective 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-beta 2. Conclusions: The TGF-beta 1 mediated secretion of endothelin-1 and TGF-beta 2 is mediated by Rho kinase activation in human HSC. (c) 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.