CGMP-dependent protein kinase 1 (cGK1) modulates human hepatic stellate cell activation
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ABSTRACT: The activation of hepatic stellate cells (HSC) plays a crucial role in non-alcoholic fatty liver disease (NAFLD), which could further develop to non-alcoholic steatohepatitis (NASH) and liver fibrosis/cirrhosis. Since cGMP-dependent protein kinase 1 (cGK1) deficient (cGK1-KO) mice displayed hepatic insulin resistance we hypothesized that cGK1 modulates HSC activation and its metabolic consequences. First, retinol storage and gene expression were studied in cGK1-KO mice. Second, we investigated the effects of cGK1-silencing on gene expression in the human stellate cell line LX2. Finally, cGK1 expression was investigated in human liver biopsies covering a wide range of liver fat content. Retinyl-ester level in the liver of cGK1-KO mice was lower compared to wild-type animals, which was associated with increased inflammatory gene expression. mRNA regulation in cGK1-silenced LX2 cells showed stronger stellate cell activation profile, altered matrix degradation and elevated chemokine level. On the other hand, activation of LX2 cells suppressed cGK1 expression, which was associated with human data, showing a negative correlation between cGK1 mRNA and liver fat content in liver biopsies. These results suggest that the lack of cGK1 could possibly lead to stellate cell activation, which elevates chemokine expression and inflammatory processes, which in turn disturbs hepatic insulin sensitivity.
Project description:Aim of the study was to characterize at a molecular level (changes in transcriptomes) the crosstalk between tumor hepatocytes and activated hepatic stellate cells (HSC) in liver cancer. This was adressed by using a coculture model system of HepaRG cell line (tumor hepatocytes, human), and LX2 cell line (HSC, human). By using genome-wide expression profiling, we demonstrated that hepatocyte-HSC crosstalk is bidirectional and results in the deregulation of functionally relevant gene networks. HepaRG and LX2 cells were cultured alone in serum- and DMSO-free William's E medium or together using 1 M-BM-5m pore size transwell inserts which allow diffusion of media components but prevent cell migration (BD Biosciences, San Jose, CA). Triplicate experiments were performed: HepaRG (culture versus coculture), LX2 (culture versus coculture).
Project description:Analysis of human hepatic stellate cell line LX2 stimulated for 24h in serum-free DMEM medium containing 0 or 50 ng/ml recombinant human GDF2 protein. Results provide insight into the activation effects of GDF2 on human hepatic stellate cell. We used microarrays to detail the global programme of gene expression underlying activation of hepatic stellate cells and identified liver-fibrosis-related genes genes during this process.
Project description:Suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) are both histone deacetylases inhibitor (HDACi), and are able to attenuate the activation of hepatic stelllate cells. To explore the underlying molecular mechanisms, we performed gene expression profile analyses of human hepatic stellate cell line LX2 treated with SAHA or VPA for 24 hours. Duplicate experiments were performed: Untreated LX2, SAHA treated LX2 and VPA treated LX2.
Project description:Suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA ) are both histone deacetylases inhibitor (HDACi), and are able to attenuate the activation of hepatic stelllate cells. To explore the underlying molecular mechanisms, we performed miRNA expression profile analyses of human hepatic stellate cell line LX2 treated with SAHA or VPA for 24 hours. Duplicate experiments were performed: Untreated LX2, SAHA treated LX2 and VPA treated LX2.
Project description:Hepatic fibrosis, a common pathological manifestation of chronic liver injury, is generally considered to be the end result of an increase in extracellular matrix produced by activated hepatic stellate cells (HSCs). Targeting the mechanisms underlying HSC activation may provide a powerful therapeutic strategy for the prevention and treatment of liver fibrosis. A high-throughput screening assay was established, and the histone deacetylase inhibitor givinostat was identified as a potent inhibitor of HSC activation in vitro. Givinostat significantly inhibited HSC activation in vivo, ameliorated carbon tetrachloride-induced mouse liver fibrosis and lowered plasma aminotransferases. Transcriptomic analysis revealed the most significantly regulated genes in the givinostat treatment group in comparison with those in the solvent group, among which, Dmkn, Msln and Upk3b were identified as potential regulators of HSC activation. Givinostat significantly reduced the mRNA expression of Dmkn, Msln and Upk3b in both a mouse liver fibrosis model and in HSC-LX2 cells. Knocking down any of the aforementioned genes inhibited the TGF-β1-induced expression of α-smooth muscle actin and collagen type I, indicating that they are crucial for HSC activation. In summary, using a novel strategy targeting HSC activation, the present study identified a potential epigenetic drug for the treatment of hepatic fibrosis and revealed novel regulators of HSC activation.
Project description:Aim of the study was to characterize at a molecular level (changes in transcriptomes) the crosstalk between tumor hepatocytes and activated hepatic stellate cells (HSC) in liver cancer. This was adressed by using a coculture model system of HepaRG cell line (tumor hepatocytes, human), and LX2 cell line (HSC, human). By using genome-wide expression profiling, we demonstrated that hepatocyte-HSC crosstalk is bidirectional and results in the deregulation of functionally relevant gene networks.
Project description:Metabolomic analysis on hepatic stellate cells isolated from PBS- or thioacetamide (TAA)-treated wild-type and Cyp1b1 knockout mice was performed to determine the metabolic basis by which CYP1B1 ablation inhibits HSC activation and liver fibrosis.
Project description:Activation and migration of hepatic stellate cells (HSCs) followed by matrix deposition are characteristics of liver fibrosis. Several studies have shown the importance of hepatocyte and endothelial cell-derived extracellular vesicles (EVs) in liver pathobiology. However, less is known about the role of HSC-derived EVs in liver diseases. In this study, we investigated the molecules released through HSC-derived EVs and whether these can promote fibrosis.
Project description:The communacation between the Hepatocellular carcinoma (HCC) and hepatic stellate cells(HSC) is not completely understood. Then a tumor-on-a-chip model was used to evaluate the crosstalk between the HCC cells (HCCLM3) and HSCs (LX2). After 5 days co-culture, we degraded the collagen hydrogel to isolate HCCLM3 cells and LX2 cells for RNA-seq.
Project description:The human hepatic stellate cell line LX2 was treated with 8 Gy X-ray irradiation and/or 2ng/ml recombinant human TGF-β1. The iTRAQ-based high throughput quantitative proteomic approach was used to obtain a comprehensive view of the protein ensembles affected by irradiation and/or TGF-β1 treatment on LX2. This study provides clues for further investigation of the mechanisms behind radiation-induced liver fibrosis.