Employment of gene expression profiling to identify transcriptional regulators of hepatic stellate cells.

Activated hepatic stellate cells (HSC) play a central role in scar formation that leads to liver fibrosis. The molecular mechanisms underlying this process are not fully understood. Microarray and bioinformatics analyses have proven to be useful in identifying transcription factors that regulate cellular processes such as cell differentiation. Using oligonucleotide microarrays, we performed transcriptional analyses of activated human HSC cultured on Matrigel-coated tissue culture dishes. Examination of microarray data following Matrigel-induced deactivation of HSC revealed a significant down-regulation of myocardin, an important transcriptional regulator in smooth and cardiac muscle development. Thus, gene expression profiling as well as functional assays of activated HSC have provided the first evidence of the involvement of myocardin in HSC activation.

TGF-ß1 mediated activation of Rho kinase induces TGF-ß2 and endothelin-1 expression in human hepatic stellate cells.

BACKGROUND & AIMS: TGF-ß1 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-ß1 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-ß1 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-ß2, and thrombospondin-1 were measured by ELISA. RESULTS: TGF-ß1 activated Rho kinase and Smad pathways in LX-2 cells. The syntheses of endothelin-1 and TGF-ß2 were significantly inhibited in TGF-ß1 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-ß2. CONCLUSIONS: The TGF-ß1 mediated secretion of endothelin-1 and TGF-ß2 is mediated by Rho kinase activation in human HSC.

Rho-kinase mediates lysophosphatidic acid-induced IL-8 and MCP-1 production via p38 and JNK pathways in human endothelial cells.

Lysophosphatidic acid (LPA), an inflammatory mediator that is elevated in multiple inflammatory diseases, is a potent activator of Rho kinase (ROCK) signaling and of chemokine production in endothelial cells. In this study, LPA activated ROCK, p38, JNK and NF-kappaB pathways and induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) mRNA and protein expression in human endothelial cells. We mapped signaling events downstream of ROCK, driving chemokine production. In summary, MCP-1 production was partly regulated by ROCK acting upstream of p38 and JNK and mediated downstream by NF-kappaB. IL-8 production was largely driven by ROCK through p38 and JNK activation, but with no involvement of NF-kappaB.

Biochemical, cellular, and anti-inflammatory properties of a potent, selective, orally bioavailable benzamide inhibitor of Rho kinase activity.

Rho kinase, is the most widely studied downstream effector of the small Rho GTPase RhoA. Two Rho kinase isoforms have been described and are frequently referred to in the literature as ROCK1 and ROCK2. The RhoA-Rho kinase pathway has been implicated in the recruitment of cellular infiltrates to disease loci in a number of preclinical animal models of inflammatory disease. In this study, we used biochemical enzyme assays and a cellular target biomarker assay to define PF-4950834 [N-methyl-3-{[(4-pyridin-4-ylbenzoyl)amino]methyl}benzamide] as an ATP-competitive, selective Rho kinase inhibitor. We further used PF-4950834 to study the role of Rho kinase activation in lymphocyte and neutrophil migration in addition to the endothelial cell-mediated expression of adhesion molecules and chemokines, which are essential for leukocyte recruitment. The inhibitor blocked stromal cell-derived factor-1alpha-mediated chemotaxis of T lymphocytes in vitro and the synthesis of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in activated human endothelial cells in vitro. The secretion of chemokines interleukin-8 and monocyte chemoattractant protein-1 was also inhibited in activated endothelial cells. In addition, when dosed orally, the compound potently inhibited neutrophil migration in a carrageenan-induced acute inflammation model. In summary, we have used a pharmacologic approach to link Rho kinase activation to multiple phenotypes that can contribute to leukocyte infiltration. Inhibition of this pathway therefore could be strongly anti-inflammatory and provide therapeutic benefit in chronic inflammatory diseases.

Rho kinase-2 activation in human endothelial cells drives lysophosphatidic acid-mediated expression of cell adhesion molecules via NF-kappaB p65.

Endothelial cells play an important role in the recruitment of immune cells to a disease locus through the induced expression of chemokines and cell adhesion molecules (CAMs). The proinflammatory lysophospholipid, lysophosphatidic acid (LPA), which is elevated in multiple inflammatory diseases, is a potent activator of the RhoA/Rho kinase signaling pathway and has been shown to induce the expression of CAMs in endothelial cells. The present study was undertaken to map signal transduction downstream of LPA and to investigate the contributions of the Rho kinase isoforms ROCK1 and ROCK2 to adhesion molecule expression in human umbilical vein endothelial cells. LPA activated Rho kinase within minutes and subsequently the NF-kappaB pathway through phosphorylation of the p65 subunit. The lipid also induced the late expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Pharmacologic inhibition of Rho kinase signaling blocked LPA-induced p65 phosphorylation and suppressed ICAM-1 and VCAM-1 expression. Inhibition of the NF-kappaB pathway had no impact on LPA-induced Rho kinase activation, but inhibited adhesion molecule expression. Small interfering RNA-facilitated knockdown of each isoform identified ROCK2 as the mediator of LPA-driven phosphorylation of NF-kappaB p65 and of ICAM-1 and VCAM-1 mRNA and protein induction. Taken collectively, our data are consistent with Rho kinase being upstream of NF-kappaB in driving LPA-mediated adhesion molecule expression. This study also provides the first evidence of the critical involvement of ROCK2 in LPA-induced CAM expression through activation of the NF-kappaB pathway in human endothelial cells.