Gas6/Axl pathway is activated in chronic liver disease and its targeting reduces fibrosis via hepatic stellate cell inactivation.
ABSTRACT: Liver fibrosis, an important health concern associated to chronic liver injury that provides a permissive environment for cancer development, is characterized by accumulation of extracellular matrix components mainly derived from activated hepatic stellate cells (HSCs). Axl, a receptor tyrosine kinase and its ligand Gas6, are involved in cell differentiation, immune response and carcinogenesis.HSCs were obtained from WT and Axl(-/-) mice, treated with recombinant Gas6 protein (rGas6), Axl siRNAs or the Axl inhibitor BGB324, and analyzed by western blot and real-time PCR. Experimental fibrosis was studied in CCl4-treated WT and Axl(-/-) mice, and in combination with Axl inhibitor. Gas6 and Axl serum levels were measured in alcoholic liver disease (ALD) and hepatitis C virus (HCV) patients.In primary mouse HSCs, Gas6 and Axl levels paralleled HSC activation. rGas6 phosphorylated Axl and AKT prior to HSC phenotypic changes, while Axl siRNA silencing reduced HSC activation. Moreover, BGB324 blocked Axl/AKT phosphorylation and diminished HSC activation. In addition, Axl(-/-) mice displayed decreased HSC activation in vitro and liver fibrogenesis after chronic damage by CCl4 administration. Similarly, BGB324 reduced collagen deposition and CCl4-induced liver fibrosis in mice. Importantly, Gas6 and Axl serum levels increased in ALD and HCV patients, inversely correlating with liver functionality.The Gas6/Axl axis is required for full HSC activation. Gas6 and Axl serum levels increase in parallel to chronic liver disease progression. Axl targeting may be a therapeutic strategy for liver fibrosis management.
Project description:Alcoholic liver disease (ALD) is a leading cause of cirrhosis in the United States, which is characterized by extensive deposition of extracellular matrix proteins and formation of a fibrous scar. Hepatic stellate cells (HSCs) are the major source of collagen type 1 producing myofibroblasts in ALD fibrosis. However, the mechanism of alcohol-induced activation of human and mouse HSCs is not fully understood. We compared the gene-expression profiles of primary cultured human HSCs (hHSCs) isolated from patients with ALD (n = 3) or without underlying liver disease (n = 4) using RNA-sequencing analysis. Furthermore, the gene-expression profile of ALD hHSCs was compared with that of alcohol-activated mHSCs (isolated from intragastric alcohol-fed mice) or CCl4-activated mouse HSCs (mHSCs). Comparative transcriptome analysis revealed that ALD hHSCs, in addition to alcohol-activated and CCl4-activated mHSCs, share the expression of common HSC activation (Col1a1 [collagen type I alpha 1 chain], Acta1 [actin alpha 1, skeletal muscle], PAI1 [plasminogen activator inhibitor-1], TIMP1 [tissue inhibitor of metalloproteinase 1], and LOXL2 [lysyl oxidase homolog 2]), indicating that a common mechanism underlies the activation of human and mouse HSCs. Furthermore, alcohol-activated mHSCs most closely recapitulate the gene-expression profile of ALD hHSCs. We identified the genes that are similarly and uniquely up-regulated in primary cultured alcohol-activated hHSCs and freshly isolated mHSCs, which include CSF1R (macrophage colony-stimulating factor 1 receptor), PLEK (pleckstrin), LAPTM5 (lysosmal-associated transmembrane protein 5), CD74 (class I transactivator, the invariant chain), CD53, MMP9 (matrix metallopeptidase 9), CD14, CTSS (cathepsin S), TYROBP (TYRO protein tyrosine kinase-binding protein), and ITGB2 (integrin beta-2), and other genes (compared with CCl4-activated mHSCs). Conclusion: We identified genes in alcohol-activated mHSCs from intragastric alcohol-fed mice that are largely consistent with the gene-expression profile of primary cultured hHSCs from patients with ALD. These genes are unique to alcohol-induced HSC activation in two species, and therefore may become targets or readout for antifibrotic therapy in experimental models of ALD.
Project description:Recent studies suggest that Src family kinase (SFK) plays important roles in systemic sclerosis and pulmonary fibrosis. However, how SFKs contributed to the pathogenesis of liver fibrosis remains largely unknown. Here, we investigated the role of Fyn, a member of SFK, in hepatic stellate cell (HSC) activation and liver fibrosis, and evaluated the anti-fibrotic effects of Saracatinib, a clinically proven safe Fyn inhibitor. Fyn activation was examined in human normal and fibrotic liver tissues. The roles of Fyn in HSC activation and liver fibrosis were evaluated in HSC cell lines by using Fyn siRNA and in Fyn knockout mice. The effects of Saracatinib on HSC activation and liver fibrosis were determined in primary HSCs and CCl4 induced liver fibrosis model. We showed that the Fyn was activated in the liver of human fibrosis patients. TGF-? induced the activation of Fyn in HSC cell lines. Knockdown of Fyn significantly blocked HSC activation, proliferation, and migration. Fyn deficient mice were resistant to CCl4 induced liver fibrosis. Saracatinib treatment abolished the activation of Fyn, downregulated the Fyn/FAK/N-WASP signaling in HSCs, and subsequently prevented the activation of HSCs. Saracatinib treatment significantly reduced the severity liver fibrosis induced by CCl4 in mice. In conclusions, our findings supported the critical role of Fyn in HSC activation and development of liver fibrosis. Fyn could serve as a promising drug target for liver fibrosis treatment. Fyn inhibitor Saracatinib significantly inhibited HSC activation and attenuated liver fibrosis in mouse model.
Project description:BACKGROUND & AIMS:The role of aryl hydrocarbon receptor (AhR) in liver fibrosis is controversial because loss and gain of AhR activity both lead to liver fibrosis. The goal of this study was to investigate how the expression of AhR by different liver cell types, hepatic stellate cells (HSCs) in particular, affects liver fibrosis in mice. METHODS:We studied the effects of AhR on primary mouse and human HSCs, measuring their activation and stimulation of fibrogenesis using RNA-sequencing analysis. C57BL/6J mice were given the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE); were given carbon tetrachloride (CCl4); or underwent bile duct ligation. We also performed studies in mice with disruption of Ahr specifically in HSCs, hepatocytes, or Kupffer cells. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, and immunoblotting. RESULTS:AhR was expressed at high levels in quiescent HSCs, but the expression decreased with HSC activation. Activation of HSCs from AhR-knockout mice was accelerated compared with HSCs from wild-type mice. In contrast, TCDD or ITE inhibited spontaneous and transforming growth factor ?-induced activation of HSCs. Mice with disruption of Ahr in HSCs, but not hepatocytes or Kupffer cells, developed more severe fibrosis after administration of CCl4 or bile duct ligation. C57BL/6J mice given ITE did not develop CCl4-induced liver fibrosis, whereas mice without HSC AhR given ITE did develop CCl4-induced liver fibrosis. In studies of mouse and human HSCs, we found that AhR prevents transforming growth factor ?-induced fibrogenesis by disrupting the interaction of Smad3 with ?-catenin, which prevents the expression of genes that mediate fibrogenesis. CONCLUSIONS:In studies of human and mouse HSCs, we found that AhR prevents HSC activation and expression of genes required for liver fibrogenesis. Development of nontoxic AhR agonists or strategies to activate AhR signaling in HSCs might be developed to prevent or treat liver fibrosis.
Project description:Reversal of activated hepatic stellate cells (HSCs) to a quiescent state and apoptosis of activated HSCs are key elements in the reversion of hepatic fibrosis. CCAAT/enhancer binding protein ? (C/EBP-?) has been shown to inhibit HSC activation and promote its apoptosis. This study aims to investigate how C/EBP-? acetylation affects the fate of activated HSCs. Effects of a histone deacetylation inhibitor trichostatin A (TSA) on HSC activation were evaluated in a mouse model of liver fibrosis caused by carbon tetrachloride (CCl4) intoxication. TSA was found to ameliorate CCl4-induced hepatic fibrosis and improve liver function through increasing the protein level and enhancing C/EBP-? acetylation in the mouse liver. C/EBP-? acetylation was determined in HSC lines in the presence or absence of TSA, and the lysine residue K276 was identified as a main acetylation site in C/EBP-? protein. C/EBP-? acetylation increased its stability and protein level, and inhibited HSC activation. The present study demonstrated that C/EBP-? acetylation increases the protein level by inhibiting its ubiquitination-mediated degradation, and may be involved in the fate of activated HSCs. Use of TSA may confer an option in minimizing hepatic fibrosis by suppressing HSC activation, a key process in the initiation and progression of hepatic fibrosis.
Project description:Hepatic stellate cell (HSC) activation on liver injury facilitates fibrosis. Hepatokines affecting HSCs are largely unknown. Here we show that hepcidin inhibits HSC activation and ameliorates liver fibrosis. We observe that hepcidin levels are inversely correlated with exacerbation of fibrosis in patients, and also confirm the relationship in animal models. Adenoviral delivery of hepcidin to mice attenuates liver fibrosis induced by CCl4 treatment or bile duct ligation. In cell-based assays, either hepcidin from hepatocytes or exogenous hepcidin suppresses HSC activation by inhibiting TGF?1-mediated Smad3 phosphorylation via Akt. In activated HSCs, ferroportin is upregulated, which can be prevented by hepcidin treatment. Similarly, ferroportin knockdown in HSCs prohibits TGF?1-inducible Smad3 phosphorylation and increases Akt phosphorylation, whereas ferroportin over-expression has the opposite effect. HSC-specific ferroportin deletion also ameliorates liver fibrosis. In summary, hepcidin suppresses liver fibrosis by impeding TGF?1-induced Smad3 phosphorylation in HSCs, which depends on Akt activated by a deficiency of ferroportin.
Project description:Liver fibrosis is mediated by hepatic stellate cells (HSCs), which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR)-mediated signaling, via endothelin-1 (ET-1) and angiotensin II (AngII), increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5), an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs). Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl4)-induced acute and chronic liver injury in Rgs5LacZ/LacZ reporter mice. Furthermore, CCl4 treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl4 and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.
Project description:Alcoholic liver disease (ALD) is a leading cause of cirrhosis in the United States, which is characterized by extensive deposition of extracellular matrix proteins (ECM) and formation of a fibrous scar. Hepatic Stellate Cells (HSCs) are the major source of Collagen Type I producing myofibroblasts in ALD fibrosis. However, the mechanism of alcohol-induced activation of human and mouse HSCs is not fully understood. We compared the gene expression profiles of primary cultured human HSCs (hHSCs) isolated from patients with ALD (n=3) or without underlying liver disease (n=4) using RNA-Seq analysis. Furthermore, the gene expression profile of ALD hHSCs was compared to that of alcohol-activated mHSCs (isolated from intragastric (IG) alcohol-fed mice), or carbon-tetrachloride (CCl4)-activated mouse HSCs (mHSCs). Comparative transcriptome analysis revealed that ALD hHSCs, and alcohol- and CCl4-activated mHSCs share the expression of common HSC activation (Col1a1, Acta1, PAI-1, TIMP1, LOXL2), indicating that a common mechanism underlies activation of human and mouse HSCs. Furthermore, alcohol-activated mHSCs most closely recapitulate the gene expression profile of ALD hHSCs. We identified the genes that are similarly and uniquely upregulated in primary cultured alcohol-activated hHSCs and freshly isolated mHSCs, which include CSF1R, PLEK, LAPTM5, CD74, CD53, MMP9, CD14, CTSS, TYROBP, ITGB2, and other genes (compared to CCl4-activated mHSCs). We identified genes in alcohol-activated mHSCs from IG alcohol-fed mice that are largely consistent with the gene expression profile of primary cultured hHSCs from ALD patients. These genes are unique to alcohol-induced HSC activation in two species, and therefore, may become new targets or readout for anti-fibrotic therapy in experimental models of ALD. Overall design: Using RNA-seq characterize of human hepatic stellate cells.
Project description:The activation of hepatic stellate cells (HSCs) participates in liver fibrosis, and emerging evidences indicate that microRNAs (miRNAs) are abnormally expressed during HSC activation. However, the potential roles of miRNAs in liver fibrosis still remain elusive. Therefore, this study aimed to investigate the role of miR-199a-3p in liver fibrosis and its underlying mechanism. We found that miR-199a-3p expression was dramatically upregulated during HSC activation in vitro, and during liver fibrogenesis in CCl4-treated rats, and its liver expression was increased in the patients with cirrhosis. By the luciferase assay and RT-qPCR, we revealed that the expression of miR-199a-3p in HSCs was driven by the transcription factor Twist1 which could be further induced by TGF-? treatment. Functional studies showed that inhibition of miR-199a-3p in both human LX2 cells and rat HSCs significantly decreased the expression of fibrotic markers, such as fibronectin and connective tissue growth factor (CTGF), whereas the forced expression of miR-199a-3p exhibited opposite effects, demonstrating the role of miR-199a-3p in promoting HSC activation. Mechanistically, miR-199a-3p plays an important role in TGF-? signalling pathway activation through targeting CAV2 that negatively regulates the expression of transforming growth factor-beta receptor type I (TGF?RI). Importantly, administration of antagomiR-199a-3p in the CCl4-treated mice significantly ameliorated hepatic fibrosis. In conclusion, Twist1-induced miR-199a-3p mediates the activation of HSCs by suppressing CAV2 expression and subsequently increasing TGF?RI expression to promote TGF-? pathway. Our findings highlight the therapeutic potential of miR-199a-3p for hepatic fibrosis.
Project description:Liver fibrosis, a common outcome of chronic liver disease characterized by excessive accumulation of extracellular matrix (ECM), is a leading cause of mortality worldwide. The tyrosine kinase inhibitor neratinib is a human epidermal growth factor receptor 2 (HER2) inhibitor approved by the FDA for HER2-positive breast cancer treatment; however, it has not yet been evaluated for liver fibrosis treatment. We elucidated the anti-fibrotic effects of neratinib in hepatic stellate cells (HSCs) and in vivo models of CCl4-induced liver fibrosis. HSC activation is a key step in liver fibrogenesis and has a crucial role in collagen deposition, as it is primarily responsible for excessive ECM production. The effect of neratinib on HSC was evaluated in transforming growth factor (TGF-?)-incubated LX-2 cells and culture-activated primary human HSCs. In vivo study results indicated that neratinib inhibited the inflammatory response, HSC differentiation, and collagen accumulation induced by CCl4. Moreover, the anti-fibrotic effects of neratinib were not associated with the HER2 signaling pathways. Neratinib inhibited FGF2 expression in activated HSCs and serum FGF2 level in the model, suggesting that neratinib possessed therapeutic potency against liver fibrosis and the potential for application against other fibrotic diseases.
Project description:The non-selectivity of tyrosine kinase inhibitors is the leading cause of drug withdrawals, and limits their application in anti-fibrosis. The role of Src tyrosine kinase Lyn in hepatic fibrosis remains elusive. In this study, we aimed to elucidate the role of Lyn kinase in the pathogenesis of hepatic fibrosis. Through examining Lyn-transgenic (Lyn TG) mice treated with CCl4 (carbon tetrachloride), we determined whether Lyn kinase is involved in the pathogenesis of hepatic fibrosis. On top of that, we also investigated the role of Lyn in the activation of hepatic stellate cells (HSCs) in vitro. Here, we showed that Lyn kinase was highly expressed in liver fibrosis upon CCl4 treatment. Meanwhile, Lyn TG mice showed that perivascular infiltration of mononuclear cells, and the markers of liver injury and hepatocytes apoptosis were significantly increased in liver tissue after CCl4 treatment. In comparison with wild-type (WT) mice after CCl4 treatment, we found that the ?brotic score in liver tissues of Lyn TG mice with the same treatment went up dramatically, so did the gene expression of fibrotic markers. In addition, over-expression of Lyn kinase drastically promoted the expression of HSCs activation markers in vivo or in vitro. Additionally, the Src-specific inhibitor PP2 significantly suppressed the increased expression of integrin ?v?3 in TGF-?1-induced HSCs, and PP2 further induced HSC apoptosis in TGF-?1-treated cells. These results collectively indicated that Lyn kinase is implicated in the pathogenesis of hepatic fibrosis through the modulating of HSC activation.