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BACKGROUND AND AIMS: Interleukin-22 has beneficial effects on inflammation and impaired hepatic regeneration that characterize alcohol-associated hepatitis (AH). F-652 is a recombinant fusion protein of human interleukin-22 and immunoglobulin G2 fragment crystallizable. This study aims to assess the safety and efficacy signals of F-652 in patients with moderate and severe AH. APPROACH AND RESULTS: A phase-2 dose-escalating study was carried out. F-652 (10 µg/kg, 30 µg/kg, or 45 µg/kg) administered on days 1 and 7 was tested in 3 patients each with moderate (Model for End-Stage Liver Disease [MELD] scores: 11-20) and severe AH (MELD scores: 21-28). Safety was defined by absence of serious adverse events and efficacy was assessed by Lille score, changes in MELD score, and serum bilirubin and aminotransferases at days 28 and 42. Three independent propensity-matched comparator patient cohorts were used. Plasma extracellular vesicles and multiplex serum cytokines were measured to assess inflammation and hepatic regeneration. Eighteen patients (9 moderate and 9 severe AH) were enrolled, 66% were male, and the mean age was 48 years. The half-life of F-652 following the first dose was 61-85 hours. There were no serious adverse events leading to discontinuation. The MELD score and serum aminotransferases decreased significantly at days 28 and 42 from baseline (P < 0.05). Day-7 Lille score was 0.45 or less in 83% patients as compared with 6%, 12%, and 56% among the comparator cohorts. Extracellular vesicle counts decreased significantly at day 28 (P < 0.013). Cytokine inflammatory markers were down-regulated, and regeneration markers were up-regulated at days 28 and 42. CONCLUSIONS: F-652 is safe in doses up to 45 µg/kg and associated with a high rate of improvement as determined by Lille and MELD scores, reductions in markers of inflammation and increases in markers of hepatic regeneration. This study supports the need for randomized placebo-controlled trials to test the efficacy of F-652 in AH.
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Hepatitis Alcohólica/tratamiento farmacológico , Inmunoglobulina G , Interleucinas/agonistas , Proteínas Recombinantes de Fusión/administración & dosificación , Adulto , Cálculo de Dosificación de Drogas , Enfermedad Hepática en Estado Terminal , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Teóricos , Proteínas Recombinantes de Fusión/efectos adversos , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Interleucina-22RESUMEN
BACKGROUND & AIMS: Steatohepatitis drives fibrogenesis in alcohol-related liver disease. Recent studies have suggested that hepatic stellate cells (HSCs) may regulate the parenchymal cell injury and inflammation that precedes liver fibrosis, although the mechanism remains incompletely defined. Neuropilin-1 (NRP-1) and synectin are membrane proteins implicated in HSC activation. In this study, we disrupted NRP-1 and synectin as models to evaluate the role of HSC activation on the development of steatohepatitis in response to alcohol feeding in mice. METHODS: Mice with HSC-selective deletion of NRP (ColCre/Nrp1loxP) or synectin (ColCre/synectinloxP) vs. paired Nrp1loxP or synectinloxP mice were fed a control diet or the chronic/binge alcohol feeding model. Several markers of steatosis and inflammation were evaluated. RESULTS: ColCre/Nrp1loxP mice showed less fibrosis, as expected, but also less inflammation and steatosis, with lower hepatic triglyceride content. Similar results were observed in the synectin model. Hepatocytes treated with supernatant of HSCs from ColCre/Nrp1loxP mice compared to supernatant from Nrp1loxP mice were protected against ethanol-induced lipid droplet formation. An adipokine and inflammatory protein array from the supernatant of HSCs with NRP-1 knockdown showed a significant reduction in Igfbp3 (a major insulin-like growth factor-binding protein with multiple metabolic functions) and an increase in SerpinA12 (a serine-protease inhibitor) secretion compared to wild-type HSCs. Recombinant Igfbp3 induced lipid droplets, triglyceride accumulation, and lipogenic genes in hepatocytes in vitro, while SerpinA12 was protective against ethanol-induced steatosis. Finally, Igfbp3 was increased, and SerpinA12 was decreased in serum and liver tissue from patients with alcoholic hepatitis. CONCLUSION: Selective deletion of NRP-1 from HSCs attenuates alcohol-induced steatohepatitis through regulation of Igfbp3 and SerpinA12 signaling. LAY SUMMARY: Hepatic stellate cells are known for their role in fibrosis (scarring of the liver). In this study, we describe their role in the modulation of fat deposition and inflammation in the liver, which occurs secondary to alcohol damage.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Hígado Graso Alcohólico , Células Estrelladas Hepáticas/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Neuropilina-1/metabolismo , Serpinas/metabolismo , Animales , Modelos Animales de Enfermedad , Hígado Graso Alcohólico/complicaciones , Hígado Graso Alcohólico/metabolismo , Hígado Graso Alcohólico/patología , Fibrosis/etiología , Fibrosis/inmunología , Inflamación/metabolismo , Ratones , Inhibidores de Serina Proteinasa/metabolismo , Transducción de SeñalRESUMEN
Liver fibrosis is characterized by the activation and migration of hepatic stellate cells (HSCs), followed by matrix deposition. Recently, several studies have shown the importance of extracellular vesicles (EVs) derived from liver cells, such as hepatocytes and endothelial cells, in liver pathobiology. While most of the studies describe how liver cells modulate HSC behavior, an important gap exists in the understanding of HSC-derived signals and more specifically HSC-derived EVs in liver fibrosis. Here, we investigated the molecules released through HSC-derived EVs, the mechanism of their release, and the role of these EVs in fibrosis. Mass spectrometric analysis showed that platelet-derived growth factor (PDGF) receptor-alpha (PDGFRα) was enriched in EVs derived from PDGF-BB-treated HSCs. Moreover, patients with liver fibrosis had increased PDGFRα levels in serum EVs compared to healthy individuals. Mechanistically, in vitro tyrosine720-to-phenylalanine mutation on the PDGFRα sequence abolished enrichment of PDGFRα in EVs and redirected the receptor toward degradation. Congruently, the inhibition of Src homology 2 domain tyrosine phosphatase 2, the regulatory binding partner of phosphorylated tyrosine720, also inhibited PDGFRα enrichment in EVs. EVs derived from PDGFRα-overexpressing cells promoted in vitro HSC migration and in vivo liver fibrosis. Finally, administration of Src homology 2 domain tyrosine phosphatase 2inhibitor, SHP099, to carbon tetrachloride-administered mice inhibited PDGFRα enrichment in serum EVs and reduced liver fibrosis. CONCLUSION: PDGFRα is enriched in EVs derived from PDGF-BB-treated HSCs in an Src homology 2 domain tyrosine phosphatase 2-dependent manner and these PDGFRα-enriched EVs participate in development of liver fibrosis. (Hepatology 2018;68:333-348).
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Vesículas Extracelulares/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/etiología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Adulto , Animales , Movimiento Celular , Femenino , Humanos , Cirrosis Hepática/metabolismo , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Adulto JovenRESUMEN
Interleukin-22 (IL-22) is a Th17 cell hepatoprotective cytokine that is undergoing clinical trials to treat patients with alcoholic hepatitis (AH). Lipopolysaccharide (LPS) activation of macrophage is implicated in hepatocyte cell death and pathogenesis of AH. The role of IL-22 production from macrophage, its regulation by LPS, and effects on alcohol-induced hepatocyte cell death are unexplored and were examined in this study. Low levels of IL-22 mRNA/protein were detected in macrophage but were significantly upregulated by 6.5-fold in response to the tissue reparative cytokine IL-10. Conversely, LPS significantly decreased IL-22 mRNA levels in a temporal and concentration-dependent manner with a maximum reduction of 5-fold. LPS downregulation of IL-22 mRNA levels was rescued in the presence of a pharmacological inhibitor of c-Jun NH2-terminal kinase (JNK) and by JNK knockdown. Next, we explored whether macrophage-derived IL-22 regulated ethanol-induced hepatocyte death. Conditioned media from IL-10-stimulated macrophages attenuated ethanol-induced hepatocyte caspase-3/7 activity, and apoptosis as assessed by fluorometric assay and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively. This effect was diminished in conditioned media from macrophages with IL-22 knockdown. Cytokine analysis in sera samples of patients with AH revealed that IL-22 levels were significantly elevated compared with healthy controls and heavy-drinking controls, implying a state of IL-22 resistance in human AH. Macrophage-derived IL-22 protects hepatocytes from ethanol-induced cell death. IL-22 downregulation is a new regulatory target of LPS in the pathogenesis of AH.
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Apoptosis/inmunología , Etanol/toxicidad , Hepatocitos/efectos de los fármacos , Hepatocitos/inmunología , Interleucinas/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas/inmunología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Interleucina-22RESUMEN
BACKGROUND & AIMS: Hepatocyte cellular dysfunction and death induced by lipids and macrophage-associated inflammation are characteristics of nonalcoholic steatohepatitis (NASH). The fatty acid palmitate can activate death receptor 5 (DR5) on hepatocytes, leading to their death, but little is known about how this process contributes to macrophage-associated inflammation. We investigated whether lipid-induced DR5 signaling results in the release of extracellular vesicles (EVs) from hepatocytes, and whether these can induce an inflammatory macrophage phenotype. METHODS: Primary mouse and human hepatocytes and Huh7 cells were incubated with palmitate, its metabolite lysophosphatidylcholine, or diluent (control). The released EV were isolated, characterized, quantified, and applied to macrophages. C57BL/6 mice were placed on chow or a diet high in fat, fructose, and cholesterol to induce NASH. Some mice also were given the ROCK1 inhibitor fasudil; 2 weeks later, serum EVs were isolated and characterized by immunoblot and nanoparticle-tracking analyses. Livers were collected and analyzed by histology, immunohistochemistry, and quantitative polymerase chain reaction. RESULTS: Incubation of primary hepatocytes and Huh7 cells with palmitate or lysophosphatidylcholine increased their release of EVs, compared with control cells. This release was reduced by inactivating mediators of the DR5 signaling pathway or rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) inhibition. Hepatocyte-derived EVs contained tumor necrosis factor-related apoptosis-inducing ligand and induced expression of interleukin 1ß and interleukin 6 messenger RNAs in mouse bone marrow-derived macrophages. Activation of macrophages required DR5 and receptor-interacting protein kinase 1. Administration of the ROCK1 inhibitor fasudil to mice with NASH reduced serum levels of EVs; this reduction was associated with decreased liver injury, inflammation, and fibrosis. CONCLUSIONS: Lipids, which stimulate DR5, induce release of hepatocyte EVs, which activate an inflammatory phenotype in macrophages. Strategies to inhibit ROCK1-dependent release of EVs by hepatocytes might be developed for the treatment of patients with NASH.
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Vesículas Extracelulares/efectos de los fármacos , Hepatitis/metabolismo , Hepatocitos/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Hígado/efectos de los fármacos , Lisofosfatidilcolinas/farmacología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Ácido Palmítico/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Caspasas/metabolismo , Línea Celular Tumoral , Vesículas Extracelulares/metabolismo , Células HEK293 , Hepatitis/tratamiento farmacológico , Hepatitis/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/metabolismo , Cirrosis Hepática Experimental/patología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/patología , Fenotipo , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Ratas Sprague-Dawley , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transfección , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
Extracellular vesicles (EVs) are nanometer-sized, membrane-bound vesicles released by cells into the extracellular milieu. EVs are now recognized to play a critical role in cell-to-cell communication. EVs contain important cargo in the form of proteins, lipids, and nucleic acids and serve as vectors for delivering this cargo from donor to acceptor or target cell. EVs are released under both physiologic and pathologic conditions, including liver diseases, and exert a wide range of effects on target cells. This review provides an overview on EV biogenesis, secretion, cargo, and target cell interactions in the context of select liver diseases. Specifically, the diverse roles of EVs in nonalcoholic steatohepatitis, alcoholic liver disease, viral hepatitis, cholangiopathies, and hepatobiliary malignancies are emphasized. Liver diseases often result in an increased release of EVs and/or in different cargo sorting into these EVs. Either of these alterations can drive disease pathogenesis. Given this fact, EVs represent a potential target for therapeutic intervention in liver disorders. Because altered EV composition may reflect the underlying disease condition, circulating EVs can be exploited for diagnostic and prognostic purposes as a liquid biopsy. Furthermore, ex vivo modified or synthesized EVs can be engineered as therapeutic nano-shuttles. Finally, we highlight areas that merit further investigation relevant to understanding how EVs regulate liver disease pathogenesis. (Hepatology 2016;64:2219-2233).
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Vesículas Extracelulares , Hepatopatías/patología , Animales , Comunicación Celular , Vesículas Extracelulares/fisiología , Hepatitis/etiología , Hepatitis/patología , Humanos , Hepatopatías/etiología , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/patología , Biogénesis de OrganelosRESUMEN
Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.
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Exosomas/metabolismo , Células Estrelladas Hepáticas/citología , Cirrosis Hepática/metabolismo , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Animales , Movimiento Celular , Células Estrelladas Hepáticas/metabolismo , Humanos , Integrinas/genética , Integrinas/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/fisiopatología , Ratones , Ratones Endogámicos C57BL , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/metabolismo , Receptores de Esfingosina-1-FosfatoRESUMEN
BACKGROUND & AIMS: The mechanisms by which hepatocyte exposure to alcohol activates inflammatory cells such as macrophages in alcoholic liver disease (ALD) are unclear. The role of released nano-sized membrane vesicles, termed extracellular vesicles (EV), in cell-to-cell communication has become increasingly recognized. We tested the hypothesis that hepatocytes exposed to alcohol may increase EV release to elicit macrophage activation. METHODS: Primary hepatocytes or HepG2 hepatocyte cell lines overexpressing ethanol-metabolizing enzymes alcohol dehydrogenase (HepG2(ADH)) or cytochrome P450 2E1 (HepG2(Cyp2E1)) were treated with ethanol and EV release was quantified with nanoparticle tracking analysis. EV mediated macrophage activation was monitored by analysing inflammatory cytokines and macrophage associated mRNA expression, immunohistochemistry, biochemical serum alanine aminotransferase and triglycerides analysis in our in vitro macrophage activation and in vivo murine ethanol feeding studies. RESULTS: Ethanol significantly increased EV release by 3.3-fold from HepG2(Cyp2E1) cells and was associated with activation of caspase-3. Blockade of caspase activation with pharmacological or genetic approaches abrogated alcohol-induced EV release. EV stimulated macrophage activation and inflammatory cytokine induction. An unbiased microarray-based approach and antibody neutralization experiments demonstrated a critical role of CD40 ligand (CD40L) in EV mediated macrophage activation. In vivo, wild-type mice receiving a pan-caspase, Rho kinase inhibitor or with genetic deletion of CD40 (CD40(-/-)) or the caspase-activating TRAIL receptor (TR(-/-)), were protected from alcohol-induced injury and associated macrophage infiltration. Moreover, serum from patients with alcoholic hepatitis showed increased levels of CD40L enriched EV. CONCLUSION: In conclusion, hepatocytes release CD40L containing EV in a caspase-dependent manner in response to alcohol exposure which promotes macrophage activation, contributing to inflammation in ALD.
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Ligando de CD40/fisiología , Caspasas/fisiología , Etanol/toxicidad , Vesículas Extracelulares/fisiología , Hepatocitos/metabolismo , Hepatopatías Alcohólicas/etiología , Activación de Macrófagos/efectos de los fármacos , Animales , Apoptosis , Citocromo P-450 CYP2E1/fisiología , Etanol/metabolismo , Femenino , Células Hep G2 , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
UNLABELLED: Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under transforming growth factor beta (TGF-ß) stimulation, hepatic stellate cells (HSCs), which are liver-specific pericytes, transdifferentiate into tumor-associated myofibroblasts that promote tumor implantation (TI) and growth in the liver. However, the regulation of this HSC activation process remains poorly understood. In this study, we tested whether vasodilator-stimulated phosphoprotein (VASP) of HSCs regulated the TGF-ß-mediated HSC activation process and tumor growth. In both an experimental liver metastasis mouse model and cancer patients, colorectal cancer cells reaching liver sinusoids induced up-regulation of VASP and alpha-smooth muscle actin (α-SMA) in adjacent HSCs. VASP knockdown in HSCs inhibited TGF-ß-mediated myofibroblastic activation of HSCs, TI, and growth in mice. Mechanistically, VASP formed protein complexes with TGF-ß receptor II (TßRII) and Rab11, a Ras-like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11-dependent targeting of TßRII to the plasma membrane, thereby desensitizing HSCs to TGF-ß1 stimulation. CONCLUSIONS: Our study demonstrates a requirement of VASP for TGF-ß-mediated HSC activation in the tumor microenvironment by regulating Rab11-dependent recycling of TßRII to the plasma membrane. VASP and its effector, Rab11, in the tumor microenvironment thus present therapeutic targets for reducing TI and metastatic growth in the liver.
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Moléculas de Adhesión Celular/metabolismo , Neoplasias Colorrectales/patología , Células Estrelladas Hepáticas/metabolismo , Neoplasias Hepáticas Experimentales/secundario , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Moléculas de Adhesión Celular/genética , Membrana Celular/metabolismo , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HT29 , Células Estrelladas Hepáticas/patología , Humanos , Neoplasias Hepáticas Experimentales/metabolismo , Ratones , Proteínas de Microfilamentos/genética , Miofibroblastos/patología , Comunicación Paracrina , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Hepatic stellate cells (HSC) and liver endothelial cells (LEC) migrate to sites of injury and perpetuate alcohol-induced liver injury. High-mobility group box 1 (HMGB1) is a protein released from the nucleus of injured cells that has been implicated as a proinflammatory mediator. We hypothesized that HMGB1 may be released from ethanol-stimulated liver parenchymal cells and contribute to HSC and LEC recruitment. Ethanol stimulation of rat hepatocytes and HepG2 cells resulted in translocation of HMGB1 from the nucleus as assessed by Western blot. HMGB1 protein levels were increased in the supernatant of ethanol-treated hepatocytes compared with vehicle-treated cells. Migration of both HSC and LEC was increased in response to conditioned medium for ethanol-stimulated hepatocytes (CMEtOH) compared with vehicle-stimulated hepatocytes (CMVEH) (P < 0.05). However, the effect of CMEtOH on migration was almost entirely reversed by treatment with HMGB1-neutralizing antibody or when HepG2 cells were pretransfected with HMGB1-siRNA compared with control siRNA-transfected HepG2 cells (P < 0.05). Recombinant HMGB1 (100 ng/ml) also stimulated migration of HSC and LEC compared with vehicle stimulation (P < 0.05 for both HSC and LEC). HMGB1 stimulation of HSC increased the phosphorylation of Src and Erk and HMGB1-induced HSC migration was blocked by the Src inhibitor PP2 and the Erk inhibitor U0126. Hepatocytes release HMGB1 in response to ethanol with subsequent recruitment of HSC and LEC. This pathway has implications for HSC and LEC recruitment to sites of ethanol-induced liver injury.
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Células Endoteliales/metabolismo , Etanol/farmacología , Proteína HMGB1/metabolismo , Células Estrelladas Hepáticas/metabolismo , Hepatopatías Alcohólicas/metabolismo , Animales , Butadienos/farmacología , Movimiento Celular , Células Endoteliales/efectos de los fármacos , Etanol/toxicidad , Células Hep G2 , Células Estrelladas Hepáticas/efectos de los fármacos , Humanos , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hepatopatías Alcohólicas/etiología , Ratones , Ratones Endogámicos C57BL , Nitrilos/farmacología , Fosforilación , Cultivo Primario de Células , Transporte de Proteínas , Pirimidinas/farmacología , Ratas , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Alcohol-associated liver disease (ALD) has been recognized as the most common cause of advanced liver disease worldwide, though mechanisms of pathogenesis remain incompletely understood. The X-linked inhibitor of apoptosis (XIAP) protein was originally described as an anti-apoptotic protein that directly binds and inhibits caspases-3, 7, and 9. Here, we investigated the function of XIAP in hepatocytes in vitro using gain and loss-of-function approaches. We noted an XIAP-dependent increase in caspase activation as well as increased inflammatory markers and pro-inflammatory EV release from hepatocytes in vitro. Primary hepatocytes (PMH) from Xiap Alb.Cre and Xiap loxP mice exhibited higher cell death but surprisingly, lower expression of inflammation markers. Conditioned media from these isolated Xiap deleted PMH further decrease inflammation in bone marrow-derived macrophages. Also, interestingly, when administered an ethanol plus Fas-agonist-Jo2 model and an ethanol plus CCl4 model, these animals failed to develop an exacerbated disease phenotype in vivo. Of note, neither Xiap Alb . Cre nor Xiap AAV8.Cre mice presented with aggravated liver injury, hepatocyte apoptosis, liver steatosis, or fibrosis. Since therapeutics targeting XIAP are currently in clinical trials and caspase-induced death is very important for development of ALD, we sought to explore the potential basis of this unexpected lack of effect. We utilized scRNA-seq and spatially reconstructed hepatocyte transcriptome data from human liver tissue and observed that XIAP was significantly zonated, along with its endogenous inhibitor second mitochondria-derived activator of caspases (SMAC) in periportal region. This contrasted with pericentral zonation of other IAPs including cIAP1 and Apollon as well as caspases 3, 7, and 9. Thus providing a potential explanation for compensation of the effect of Xiap deletion by other IAPs. In conclusion, our findings implicate a potential zonallydependent role for SMAC that prevented development of a phenotype in XIAP knockout mice in ALD models. Targeting SMAC may also be important in addition to current efforts of targeting XIAP in treatment of ALD.
RESUMEN
BACKGROUND & AIMS: Transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts is a key event in the pathogenesis of liver fibrosis. Transforming growth factor ß (TGF-ß) and platelet-derived growth factor (PDGF) are canonical HSC activators after liver injury. The aim of this study was to analyze the epigenetic modulators that differentially control TGF-ß and PDGF signaling pathways. METHODS: We performed a transcriptomic comparison of HSCs treated with TGF-ß or PDGF-BB using RNA sequencing. Among the targets that distinguish these 2 pathways, we focused on the histone methyltransferase class of epigenetic modulators. RESULTS: Enhancer of zeste homolog 2 (EZH2) was expressed differentially, showing significant up-regulation in HSCs activated with TGF-ß but not with PDGF-BB. Indeed, EZH2 inhibition using either a pharmacologic (GSK-503) or a genetic (small interfering RNA) approach caused a significant attenuation of TGF-ß-induced fibronectin, collagen 1α1, and α-smooth muscle actin, both at messenger RNA and protein levels. Conversely, adenoviral overexpression of EZH2 in HSCs resulted in a significant stimulation of fibronectin protein and messenger RNA levels in TGF-ß-treated cells. Finally, we conducted in vivo experiments with mice chronically treated with carbon tetrachloride or bile duct ligation. Administration of GSK-503 to mice receiving either carbon tetrachloride or bile duct ligation led to attenuated fibrosis as assessed by Trichrome and Sirius red stains, hydroxyproline, and α-smooth muscle actin/collagen protein assays. CONCLUSIONS: TGF-ß and PDGF share redundant and distinct transcriptomic targets, with the former predominating in HSC activation. The EZH2 histone methyltransferase is preferentially involved in the TGF-ß as opposed to the PDGF signaling pathway. Inhibition of EZH2 attenuates fibrogenic gene transcription in TGF-ß-treated HSCs and reduces liver fibrosis in vivo. The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE119606 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119606).
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Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Conductos Biliares/patología , Tetracloruro de Carbono , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Ligadura , Cirrosis Hepática/genética , Ratones Endogámicos C57BL , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
The scaffold protein synectin plays a critical role in the trafficking and regulation of membrane receptor pathways. As platelet-derived growth factor receptor (PDGFR) is essential for hepatic stellate cell (HSC) activation and liver fibrosis, we sought to determine the role of synectin on the PDGFR pathway and development of liver fibrosis. Mice with deletion of synectin from HSC were found to be protected from liver fibrosis. mRNA sequencing revealed that knockdown of synectin in HSC demonstrated reductions in the fibrosis pathway of genes, including PDGFR-ß. Chromatin IP assay of the PDGFR-ß promoter upon synectin knockdown revealed a pattern of histone marks associated with decreased transcription, dependent on p300 histone acetyltransferase. Synectin knockdown was found to downregulate PDGFR-α protein levels, as well, but through an alternative mechanism: protection from autophagic degradation. Site-directed mutagenesis revealed that ubiquitination of specific PDGFR-α lysine residues was responsible for its autophagic degradation. Furthermore, functional studies showed decreased PDGF-dependent migration and proliferation of HSC after synectin knockdown. Finally, human cirrhotic livers demonstrated increased synectin protein levels. This work provides insight into differential transcriptional and posttranslational mechanisms of synectin regulation of PDGFRs, which are critical to fibrogenesis.