The diffuse stellate cell system

Hepatic stellate cells (HSCs) play an important role in liver fibrogenesis. Morphologically similar cells have been found at extrahepatic sites such as pancreas, kidney and colon. The true phenotypic relationship between these cells has not been fully established. We carried out immunohistochemical staining in normal tissues from liver, kidney, colon, pancreas, lung and heart, obtained from a range of species. Immunoreactivity to antibodies directed to synemin, glial fibrillary acidic protein (GFAP), nestin, neurofilament-L, β-tubulin, protein gene product 9.5 (PGP9.5), S100, desmin, alpha-smooth muscle actin (α-SMA) and vimentin was examined. Synemin was identified in HSCs, pancreatic stellate cells, mesangial cells and in peribronchiolar stellate-shaped fibroblasts. GFAP positivity was detected in HSCs and peribronchiolar stellate-shaped fibroblasts. Desmin immunoreactivity was detected in HSCs, pancreatic stellate cells, mesangial cells, periglomerular and peritubular fibroblasts, subepithelial fibroblasts, as well as in peribronchiolar stellate-shaped fibroblasts. Vimentin expression was evident in HSCs, periductal fibroblasts, pancreatic stellate cells, fibroblasts within the fibroconnective tissue capsule, mesangial cells, subepithelial fibroblasts and the interstitial cells of Cajal, as well as in peribronchiolar fibroblasts. Mesangial cells and peritubular fibroblasts showed nestin immunoreactivity. Our data indicates that mesenchymal cells at extrahepatic sites express many of the neural and muscle-associated proteins seen in HSCs; there are however species differences in the expression pattern of these proteins. The findings support the concept of a diffuse stellate cell system in mammals.     

Modulation of cytokeratin expression during in vitro cultivation of human hepatic stellate cells: evidence of transdifferentiation from epithelial to mesenchymal phenotype

The embryonal origin of hepatic stellate cells (HSCs), the principal cells in hepatic fibrogenesis, is still intriguing. To explore the origin and the differentiation of HSCs, we studied the expression of cytokeratin 18 (CK18) and 19 (CK19), the standard markers of simple epithelial cells, in cultured human HSCs. Hepatic stellate cells were isolated from five normal human livers. In immunofluorescence staining, both clone C-51 anti-CK18 antibody and clone RCK108 anti-CK19 antibody labeled almost all stellate cells in the primary culture. Double immunofluorescence staining for cytokeratin/vimentin and cytokeratin/alpha-smooth muscle actin detected by confocal laser scanning microscopy clearly demonstrated the localization of cytokeratin immunoreactivity in human HSCs. During subsequent cultivation of human HSCs to the tenth passage, immunocytochemical staining and western blot analysis demonstrated gradually decreasing profiles of CK18 and CK19 expression. The progressive reduction of cytokeratin expression was further confirmed in a culture of clone cells originated from a single HSC. In conclusion, both CK18 and CK19 are expressed in cultured human HSCs, and the extent of their expression decreases gradually during prolonged cultivation. Our results suggest that HSCs may be of epithelial origin, and that they undergo the transdifferentiation from epithelial to mesenchymal phenotype during an activation process in vitro.     

Regulator of G-Protein Signaling-5 Is a Marker of Hepatic Stellate Cells and Expression Mediates Response to Liver Injury

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 Rgs5^LacZ/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.     

Syncoilin is an intermediate filament protein in activated hepatic stellate cells

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl₄- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, β-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.     

Soluble factors derived from human amniotic epithelial cells suppress collagen production in human hepatic stellate cells

BackgroundIntravenous infusion of human amniotic epithelial cells (hAECs) has been shown to ameliorate hepatic fibrosis in murine models. Hepatic stellate cells (HSCs) are the principal collagen-secreting cells in the liver. The aim of this study was to investigate whether factors secreted by hAECs and present in hAEC-conditioned medium (CM) have anti-fibrotic effects on activated human HSCs.MethodsHuman AECs were isolated from the placenta and cultured. Human hepatic stellate cells were exposed to hAEC CM to determine potential anti-fibrotic effects.ResultsHSCs treated for 48 h with hAEC CM displayed a significant reduction in the expression of the myofibroblast markers α-smooth muscle actin and platelet-derived growth factor. Expression of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1) and intracellular collagen were reduced by 45% and 46%, respectively. Human AEC CM induced HSC apoptosis in 11.8% of treated cells and reduced HSC proliferation. Soluble human leukocyte antigen–G1, a hAEC-derived factor, significantly decreased TGF-β1 and collagen production in activated HSCs, although the effect on collagen production was less than that of hAEC CM. The reduction in collagen and TGF-B1 could not be attributed to PGE2, relaxin, IL-10, TGF-B3, FasL or TRAIL.ConclusionsHuman AEC CM treatment suppresses markers of activation, proliferation and fibrosis in human HSCs as well as inducing apoptosis and reducing proliferation. Human AEC CM treatment may be effective in ameliorating liver fibrosis and warrants further study.     

A novel synthetic oleanolic acid derivative (CPU-II<Subscript>2</Subscript>) attenuates liver fibrosis in mice through regulating the function of hepatic stellate cells

Regulation on the function of the hepatic stellate cells (HSCs) is one of the proposed therapeutic approaches to liver fibrosis. In the present study, we examined the in vitro and in vivo effects of CPU-II₂, a novel synthetic oleanolic acid (OLA) derivative with nitrate, on hepatic fibrosis. This compound alleviated CCl₄-induced hepatic fibrosis in mice with a decrease in hepatic hydroxyproline (Hyp) content and histological changes. CPU-II₂ also attenuated the mRNA expression of α-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinase type 1 (TIMP-1) induced by CCl₄ in mice and reduced both mRNA and protein levels of α-SMA in HSC-T6 cells. Interestingly, CPU-II₂ did not affect the survival of HSC-T6 cells but decreased the expression of procollagen-α1 (I) in HSC-T6 cells through down-regulating the phosphorylation of p38 MAPK. Conclusion: CPU-II₂ attenuates the development of liver fibrosis rather by regulating the function of HSCs through p38 MAPK pathway than by damaging the stellate cells.     

A simple method for the simultaneous isolation of stellate cells and hepatocytes from rat liver tissue

Hepatic stellate cells (HSCs), also referred to as Ito cells, perisinusiodal cells and fat-storing cells, have numerous vital functions. They are the main extracellular matrix-producing cells within the liver and are involved in the storage of retinol. HSCs are also known to secrete a number of liver mitogens. Current isolation techniques are cumbersome and most require a pronase digestion step, which destroys any hepatocytes present. We present a simple method for isolation and culture of hepatic stellate cells from the normally discarded washings from a two-step collagenase hepatocyte isolation, which has shown a yield of more than 1.5 × 10⁶ viable HSCs after 5 days in culture. The cells were positively identified as HSCs by staining for two intermediate filaments (desmin and GFAP) and observing their distinct morphology from other liver cell types. This efficient method allows rapid and consistent isolation of stellate cells to give a culture that may be passaged several times.     

Brivanib Attenuates Hepatic Fibrosis In Vivo and Stellate Cell Activation In Vitro by Inhibition of FGF,VEGF and PDGF Signaling

MethodsIn vivo, we induced liver fibrosis by bile duct ligation (BDL), chronic carbon tetrachloride (CCl₄), and chronic thioacetamide (TAA) administration. Liver fibrosis was examined by immunohistochemistry and Western immunoblotting. In vitro, we used LX-2 human hepatic stellate cells (HSCs) to assess the effect of brivanib on stellate cell proliferation and activation.ResultsAfter in vivo induction with BDL, CCl₄, and TAA, mice treated with brivanib showed reduced liver fibrosis and decreased expression of collagen Iα1 and α-smooth muscle actin in the liver. In vitro, brivanib decreased proliferation of HSCs induced by platelet-derived growth factor (PDGF), VEGF, and FGF. Brivanib also decreased stellate cell viability and inhibited PDGFBB-induced phosphorylation of its cognate receptor.ConclusionBrivanib reduces liver fibrosis in three different animal models and decreases human hepatic stellate cell activation. Brivanib may represent a novel therapeutic approach to treatment of liver fibrosis and prevention of liver cancer.     

Alteration of protein glycosylation in human hepatic stellate cells activated with transforming growth factor-β1

Although aberrant glycosylation of human glycoproteins is related to liver fibrosis that results from chronic damage to the liver in conjunction with the activation of hepatic stellate cells (HSCs), little is known about the precision alteration of protein glycosylation referred to the activation of HSCs by transforming growth factor-β1 (TGF-β1). The human HSCs, LX-2 were activated by TGF-β1. The lectin microarrays were used to probe the alteration of protein glycosylation in the activated HSCs compared with the quiescent HSCs. Lectin histochemistry was used to further validate the lectin binding profiles and assess the distribution of glycosidic residues in cells. As a result, 14 lectins (e. g. AAL, PHA-E, ECA and ConA) showed increased signal while 7 lectins (e. g. UEA-I and GNA) showed decreased signal in the activated LX-2 compared with the quiescent LX-2. Meanwhile, AAL, PHA-E and ECA staining showed moderate binding to the cytoplasma membrane in the quiescent LX-2, and the binding intensified in the same regions of the activated LX-2. In conclusion, the precision alteration of protein glycosylation related to the activation of the HSCs may provide useful information to find new molecular mechanism of HSC activation and antifibrotic therapeutic strategies.     

Adherens junction-associated protein distribution differs in smooth muscle tissue and acutely isolated cells

This study was designed to examine how smooth muscle (SM) cell (SMC) isolation affects the distribution of some adherens junction (AJ) complex-associated proteins. Immunofluorescence procedures for identifying protein distribution were used on gastrointestinal and tracheal SM tissues and freshly isolated SMCs from dogs and rabbits. As confirmed by force measurements, relaxation, Ca(2+) depletion, and cholinergic activation of SM tissues do not cause significant redistribution of the AJ-associated proteins vinculin, talin, or fibronectin away from the plasma membrane. Unlike SMCs in tissue, freshly isolated SMCs show a variable peripheral/cytoplasmic vinculin and talin distribution that is not altered by activation. Enzymatic treatment of SM tissues (as done for the first step of SMC isolation) results in loss of fibronectin immunoreactivity in SMCs still in the tissue but fails to cause redistribution of vinculin, talin, or caveolin away from the periphery. The loss of fibronectin immunofluorescence with enzymatic digestion correlates significantly with loss of tissue force production. These results confirm that the AJ-associated proteins vinculin and talin do not redistribute throughout SMCs in tissues when relaxed, when generating force, or after enzymatic digestion. In addition, in freshly isolated SMCs, the distribution of these proteins is significantly altered in approximately 50% of the SMCs. The cause of this redistribution is currently unknown, as is the impact on intracellular signaling and mechanics of these cells. Use of these two systems (SMCs in tissues vs. freshly isolated SMCs) provides an ideal situation for studying the role of the AJ in SMC signaling and mechanics.     

Regulatory role of vHL/HIF-1alpha in hypoxia-induced VEGF production in hepatic stellate cells

Activated hepatic stellate cells (HSCs) produce cyclooxygenase-2 (COX-2) protein to induce vascular endothelial growth factor (VEGF) production that participates in angiogenesis in injured liver. To reveal the unknown regulatory mechanism, we used hypoxic atmosphere mimicking injured-tissue microenvironment to induce VEGF expression in a rat hepatic stellate cell line (T6-HSCs). The present study showed that hypoxia up-regulated the protein levels of COX-2 and hypoxia-inducible factor-1-alpha (HIF-1alpha), but rapidly effected degradation of von Hippel-Lindau (vHL) protein. As a result, expression of VEGF in HSCs was markedly elevated; and pretreatment with COX-2 inhibitors (nimesulide or indomethacin) could significantly ameliorate the angiogenic event. Collectively, hypoxic HSCs increased accumulation of HIF-1alpha protein and induced VEGF expression in a time-dependent manner. Inhibition of COX-2 activities would prevent vHL protein from degradation and suppress HIF-1alpha up-regulation. Thus, vHL/HIF-1alpha has a regulatory role in COX-2-mediated VEGF production in hypoxic stellate cells in injured liver.     

X4 Human immunodeficiency virus type 1 gp120 promotes human hepatic stellate cell activation and collagen I expression through interactions with CXCR4

Background & Aims

Patients coinfected with HIV-1 and HCV develop more rapid liver fibrosis than patients monoinfected with HCV. HIV RNA levels correlate with fibrosis progression implicating HIV directly in the fibrotic process. While activated hepatic stellate cells (HSCs) express the 2 major HIV chemokine coreceptors, CXCR4 and CCR5, little is known about the pro-fibrogenic effects of the HIV-1 envelope protein, gp120, on HSCs. We therefore examined the in vitro impact of X4 gp120 on HSC activation, collagen I expression, and underlying signaling pathways and examined the in vivo expression of gp120 in HIV/HCV coinfected livers.

Methods

Primary human HSCs and LX-2 cells, a human HSC line, were challenged with X4 gp120 and expression of fibrogenic markers assessed by qRT-PCR and Western blot +/− either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Downstream intracellular signaling pathways were evaluated with Western blot and pre-treatment with specific pathway inhibitors. Gp120 immunostaining was performed on HIV/HCV coinfected liver biopsies.

Results

X4 gp 120 significantly increased expression of alpha-smooth muscle actin (a-SMA) and collagen I in HSCs which was blocked by pre-incubation with either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Furthermore, X4 gp120 promoted Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and pretreatment with an ERK inhibitor attenuated HSC activation and collagen I expression. Sinusoidal staining for gp120 was evident in HIV/HCV coinfected livers.

Conclusions

X4 HIV-1 gp120 is pro-fibrogenic through its interactions with CXCR4 on activated HSCs. The availability of small molecule inhibitors to CXCR4 make this a potential anti-fibrotic target in HIV/HCV coinfected patients.     

Apoptosis of T cells in the hepatic fibrotic tissue of the rat: a possible inducing role of hepatic myofibroblast-like cells

Apoptosis of T cells contributes to the immune homeostasis in inflamed organs. A prominent T-cell infiltration is usually seen in human chronic active hepatitis, being associated with liver fibrosis. In order to demonstrate T-cell apoptosis in the hepatic fibrotic tissue, we induced T-cell infiltration in the fibrotic liver of the rat by injecting concanavalin A (Con A), a T-cell mitogen. Lymphocytes increased in number with a peak at 1 day, preferentially distributing in the fibrotic tissue rather than the parenchyma. They consisted of CD4-positive and CD8-positive cells, and gave the feature of lymphoblasts. Double staining for CD3 and TUNEL demonstrated that T cells underwent apoptosis. Apoptotic cells were more frequent in the fibrotic livers than the normal livers, and were spatially associated with alpha-smooth muscle actin-positive myofibroblast-like cells that possibly derived from hepatic stellate cells (HSCs) and portal fibroblasts through activation. In vitro experiments demonstrated that lymphocyte apoptosis was more frequently induced in the co-culture of Con A-activated splenic T cells/activated HSCs compared to that induced in activated T cells/quiescent HSCs or resting T cells/activated HSCs. The present results indicate that T cells which have extravasated and infiltrated the hepatic fibrotic tissue undergo apoptosis probably through an interaction with myofibroblast-like cells, suggesting the regulatory role of the latter cells in T-cell accumulation in the fibrotic liver.     

Neuregulin-1 suppresses cardiomyocyte apoptosis by activating PI3K/Akt and inhibiting mitochondrial permeability transition pore

In order to explore the effects of fat-specific protein 27 (Fsp27) on regulation of hepatic stellate cell (HSC) activation and liver fibrosis. HSCs were isolated from rat liver tissues and cultivated in vitro for gene expression and lentivirus infection. CCK-8 cell viability assay, immunofluorescence staining, qRT-PCR, and western blot assays were used to assess phenotypic changes and gene expression in HSCs. The rat liver fibrosis model was produced by intraperitoneal injection of carbon tetrachloride for assessing the effects of Fsp27 in the rat liver. Gene expression was then detected by immunohistochemistry and ELISA assays. The results of the study showed that Fsp27 was constitutively expressed in primary quiescent HSCs, but was absent in activated HSCs. Ectopic expression of Fsp27 significantly inhibited HSC proliferation and activation, as well as expression of α-smooth muscle actin. Fsp27 expression also significantly reduced collagen I production and matrix metalloproteinases 2 protein levels, and to a lesser degree, reduced tissue inhibitors of metalloproteinases 1 expression. In vivo data showed that ectopic expression of Fsp27 protein significantly reduced levels of hydroxyproline in liver tissue, and decreased serum levels of collagen III and hyaluronic acid, which in turn, suppressed liver fibrosis in rats. From these findings, it can be concluded that Fsp27 expression suppressed HSC activation in vitro and liver fibrogenesis in vivo. Further studies are needed to explore whether expression of Fsp27 can be selected as a potential novel strategy for anti-fibrotic therapy against liver fibrosis.     

Endoplasmic reticulum stress and autophagy are involved in adipocyte-induced fibrosis in hepatic stellate cells

Liver fibrosis, with the characterization of progressive accumulation of extracellular matrix (ECM), is the common pathologic feature in the process of chronic liver disease. Hepatic stellate cells (HSCs) which are activated and differentiate into proliferative and contractile myofibroblasts are recognized as the main drivers of fibrosis. Obesity-related adipocytokine dysregulation is known to accelerate liver fibrosis progression, but the direct fibrogenic effect of mature adipocytes on HSCs has been rarely reported. Therefore, the purpose of this study was to explore the fibrogenic effect of adipocyte 3T3-L1 cells on hepatic stellate LX-2 cells. The results showed that incubating LX-2 cells with the supernatant of 3T3-L1 adipocytes triggered the expression of ECM related proteins, such as α-smooth muscle actin (α-SMA), type I collagen (CO-I), and activated TGF β/Smad2/3 signaling pathway in LX-2 cells. In addition, 3T3-L1 cells inhibited insulin sensitivity, activated endoplasmic reticulum stress and autophagy to promote the development of fibrosis. These results supported the notion that mature adipocytes can directly activate hepatic stellate cells, and the establishment of an in vitro model of adipocytes on HSCs provides an insight into screening of drugs for liver diseases, such as nonalcoholic fatty liver disease.

     

Ascorbic acid deficiency accelerates aging of hepatic stellate cells with up-regulation of PPARγ

Senescent cells have been observed in certain aged or damaged tissues. However, the information about the effects of aging on liver cells is limited. In the present study, we have examined age-related histological changes in the livers of senescence marker protein knockout (SMP30-/-) mice, which are considered as a murine aging model due to the more sensitive response to apoptotic reagents and due to their shorter life span. In livers of old SMP30-/- mice, numerous hepatic stellate cells (HSCs) were hypertrophic and contained abundant microvesicular lipid droplets in cytoplasm. We have found that the expression of peroxisome proliferators-activated receptor γ (PPARγ), which is a protein related to lipid metabolism and HSC quiescence, was increased in hypertrophic HSCs by aging and vitamin C (VC) deficiency, whereas these phenomena were dramatically reduced by antioxidant treatment. Therefore, these prominent phenotypic changes can be considered as aging markers in the livers of animals which are subjected to antioxidant property evaluation.     

The plasticity of p19 ARF null hepatic stellate cells and the dynamics of activation

In the healthy adult liver, quiescent hepatic stellate cells (HSCs) present the major site for vitamin A storage in cytoplasmic lipid droplets. During liver injury due to viral infection or alcohol intoxication, HSCs get activated and produce high amounts of extracellular matrix components for tissue repair and fibrogenesis. Employing p19 ARF deficiency, we established a non-transformed murine HSC model to investigate their plasticity and the dynamics of HSC activation. Primary HSCs isolated from livers of adult p19 ARF null mice underwent spontaneous activation through long-term passaging without an obvious replicative limit. The immortalized cell line, referred to as M1-4HSC, showed stellate cell characteristics including the expression of desmin, glial fibrillary acidic protein, alpha-smooth muscle actin and pro-collagen I. Treatment of these non-tumorigenic M1-4HSC with pro-fibrogenic TGF-beta1 provoked a morphological transition to a myofibroblastoid cell type which was accompanied by enhanced cellular turnover and impaired migration. In addition, M1-4HSCs expressed constituents of cell adhesion complexes such as p120(ctn) and beta-catenin at cell borders, which dislocalized in the cytoplasm during stimulation to myofibroblasts, pointing to the epitheloid characteristics of HSCs. By virtue of its non-transformed phenotype and unlimited availability of cells, the p19(ARF) deficient model of activated HSCs and corresponding myofibroblasts render this system a highly valuable tool for studying the cellular and molecular basis of hepatic fibrogenesis.     

FBXO31 modulates activation of hepatic stellate cells and liver fibrogenesis by promoting ubiquitination of Smad7

Liver fibrosis is a critical pathological process in the early stage of many liver diseases, including hepatic cirrhosis and liver cancer. However, the molecular mechanism is not fully revealed. In this study, we investigated the role of F-box protein 31 (FBXO31) in liver fibrosis. We found FBXO31 upregulated in carbon tetrachloride (CCl₄) induced liver fibrosis and in activated hepatic stellate cells, induced by transforming growth factor-β (TGF-β). The enforced expression of FBXO31 caused enhanced proliferation and increased expression of α-smooth muscle actin (α-SMA) and Col-1 in HSC-T6 cells. Conversely, suppression of FBXO31 resulted in inhibition of proliferation and decreased accumulation of α-SMA and Col-1 in HSC-T6 cells. In addition, upregulation of FBXO31 in HSC-T6 cells decreased accumulation of Smad7, the negative regulator of the TGF-β/smad signaling pathway, and suppression of the FBXO31 increased accumulation of Smad7. Immunofluorescence staining showed FBXO31 colocalized with Smad7 in HSC-T6 cells and in liver tissues of BALB/c mice treated with CCl₄. Immunoprecipitation demonstrated FBXO31 interacted with Smad7. Moreover, FBXO31 enhanced ubiquitination of Smad7. In conclusion, FBXO31 modulates activation of HSCs and liver fibrogenesis by promoting ubiquitination of Smad7.