RESUMEN
INTRODUCTION: WNT1-inducible signalling pathway protein 1 (WISP1) promotes progression of several tumor entities often correlating with worse prognosis. Here its expression regulation and role in the progression of chronic liver diseases (CLD) was investigated. METHODS: WISP1 expression was analyzed in human HCC datasets, in biopsies and serum samples and an HCC patient tissue microarray (TMA) including correlation to clinicopathological parameters. Spatial distribution of WISP1 expression was determined using RNAscope analysis. Regulation of WISP1 expression was investigated in cytokine-stimulated primary mouse hepatocytes (PMH) by array analysis and qRT-PCR. Outcome of WISP1 stimulation was analyzed by IncuCyte S3-live cell imaging, qRT-PCR, and immunoblotting in murine AML12 cells. RESULTS: In a TMA, high WISP1 expression was positively correlated with early HCC stages and male sex. Highest WISP1 expression levels were detected in patients with cirrhosis as compared to healthy individuals, patients with early fibrosis, and non-cirrhotic HCC in liver biopsies, expression datasets and serum samples. WISP1 transcripts were predominantly detected in hepatocytes of cirrhotic rather than tumorous liver tissue. High WISP1 expression was associated with better survival. In PMH, AML12 and HepaRG, WISP1 was identified as a specific TGF-ß1 target gene. Accordingly, expression levels of both cytokines positively correlated in human HCC patient samples. WISP1-stimulation induced the expression of Bcl-xL, PCNA and p21 in AML12 cells. CONCLUSIONS: WISP1 expression is induced by TGF-ß1 in hepatocytes and is associated with cirrhotic liver disease. We propose a crucial role of WISP1 in balancing pro- and anti-tumorigenic effects during premalignant stages of CLD.
RESUMEN
Amyloid beta (Aß) plays a major role in the pathogenesis of Alzheimer's disease and, more recently, has been shown to protect against liver fibrosis. Therefore, we studied Aß-42 levels and the expression of genes involved in the generation, degradation, and transport of Aß proteins in liver samples from patients at different stages of metabolic dysfunction-associated liver disease (MASLD) and under steatotic conditions in vitro/in vivo. Amyloid precursor protein (APP), key Aß-metabolizing proteins, and Aß-42 were analyzed using RT-PCR, Western blotting, Luminex analysis in steatotic in vitro and fatty liver mouse models, and TaqMan qRT-PCR analysis in hepatic samples from patients with MASLD. Hepatocytes loaded with palmitic acid induced APP, presenilin, and neprilysin (NEP) expression, which was reversed by oleic acid. Increased APP and NEP, decreased BACE1, and unchanged Aß-42 protein levels were found in the steatotic mouse liver compared to the normal liver. Aß-42 concentrations were low in MASLD samples of patients with moderate to severe fibrosis compared to the livers of patients with mild or no MASLD. Consistent with the reduced Aß-42 levels, the mRNA expression of proteins involved in APP degradation (ADAM9/10/17, BACE2) and Aß-42 cleavage (MMP2/7/9, ACE) was increased. In the steatotic liver, the expression of APP- and Aß-metabolizing proteins is increased, most likely related to oxidative stress, but does not affect hepatic Aß-42 levels. Consistent with our previous findings, low Aß-42 levels in patients with liver fibrosis appear to be caused by the reduced production and enhanced non-amyloidogenic processing of APP.
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Péptidos beta-Amiloides , Hígado Graso , Hígado , Animales , Humanos , Péptidos beta-Amiloides/metabolismo , Ratones , Hígado Graso/metabolismo , Hígado Graso/patología , Hígado/metabolismo , Hígado/patología , Masculino , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Fragmentos de Péptidos/metabolismo , Ratones Endogámicos C57BL , Hepatocitos/metabolismo , Hepatocitos/patología , Femenino , Modelos Animales de Enfermedad , Neprilisina/metabolismo , Neprilisina/genéticaRESUMEN
Inflammasomes and innate immune cells have been shown to contribute to liver injury, thereby activating Kupffer cells, which release several cytokines, including IL-6, IL-1ß, and TNFα. Augmenter of liver regeneration (ALR) is a hepatotropic co-mitogen that was found to have anti-oxidative and anti-apoptotic properties and to attenuate experimental non-alcoholic fatty liver disease (NAFLD) and cholestasis. Additionally, hepatic ALR expression is diminished in patients with NAFLD or cholestasis, but less is known about the mechanisms of its regulation under these conditions. Therefore, we aimed to investigate the role of IL-1ß in ALR expression and to elucidate the molecular mechanism of this regulation in vitro. We found that ALR promoter activity and mRNA and protein expression were reduced upon treatment with IL-1ß. Early growth response protein-1 (Egr-1), an ALR inducer, was induced by IL-1ß but could not activate ALR expression, which may be attributed to reduced Egr-1 binding to the ALR promoter. The expression and nuclear localization of hepatocyte nuclear factor 4 α (HNF4α), another ALR-inducing transcription factor, was reduced by IL-1ß. Interestingly, c-Jun, a potential regulator of ALR and HNF4α, showed increased nuclear phosphorylation levels upon IL-1ß treatment but did not change the expression of ALR or HNF4α. In conclusion, this study offers evidence regarding the regulation of anti-apoptotic and anti-oxidative ALR by IL-1ß through reduced Egr-1 promoter binding and diminished HNF4α expression independent of c-Jun activation. Low ALR tissue levels in NAFLD and cholestatic liver injury may be caused by IL-1ß and contribute to disease progression.
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Colestasis , Enfermedad del Hígado Graso no Alcohólico , Humanos , Colestasis/metabolismo , Citocinas/metabolismo , Interleucinas/metabolismo , Hígado/metabolismo , Regeneración Hepática , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismoRESUMEN
Metabolic dysfunction-associated steatotic liver disease (MASLD) comprises a spectrum of liver diseases, ranging from liver steatosis to metabolic dysfunction-associated steatohepatitis (MASH), increasing the risk of developing cirrhosis and hepatocellular carcinoma (HCC). Fibrosis within MASLD is critical for disease development; therefore, the identification of fibrosis-driving factors is indispensable. We analyzed the expression of interleukin 32 (IL-32) and chemokine CC ligand 20 (CCL20), which are known to be linked with inflammation and fibrosis, and for their expression in MASLD and hepatoma cells. RT-PCR, ELISA and Western blotting analyses were performed in both human liver samples and an in vitro steatosis model. IL-32 and CCL20 mRNA expression was increased in tissues of patients with NASH compared to normal liver tissue. Stratification for patatin-like phospholipase domain-containing protein 3 (PNPLA3) status revealed significance for IL-32 only in patients with I148M (rs738409, CG/GG) carrier status. Furthermore, a positive correlation was observed between IL-32 expression and steatosis grade, and between IL-32 as well as CCL20 expression and fibrosis grade. Treatment with the saturated fatty acid palmitic acid (PA) induced mRNA and protein expression of IL-32 and CCL20 in hepatoma cells. This induction was mitigated by the substitution of PA with monounsaturated oleic acid (OA), suggesting the involvement of oxidative stress. Consequently, analysis of stress-induced signaling pathways showed the activation of Erk1/2 and p38 MAPK, which led to an enhanced expression of IL-32 and CCL20. In conclusion, cellular stress in liver epithelial cells induced by PA enhances the expression of IL-32 and CCL20, both known to trigger inflammation and fibrosis.
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Hígado Graso , Hepatocitos , Enfermedades Metabólicas , Humanos , Carcinoma Hepatocelular/genética , Quimiocina CCL20/genética , Quimiocinas , Hepatocitos/metabolismo , Ligandos , Cirrosis Hepática/genética , Neoplasias Hepáticas/genética , Ácido Palmítico , Regulación hacia Arriba , Grasas Insaturadas/metabolismoRESUMEN
Non-alcoholic steatohepatitis (NASH) is a rapidly growing liver disease. The chemoattractant chemerin is abundant in hepatocytes, and hepatocyte expressed prochemerin protected from NASH. Prochemerin is inactive and different active isoforms have been described. Here, the effect of hepatocyte expressed muChem-156, a highly active murine chemerin isoform, was studied in the methionine-choline deficient dietary model of NASH. Mice overexpressing muChem-156 had higher hepatic chemerin protein. Serum chemerin levels and the capability of serum to activate the chemerin receptors was unchanged showing that the liver did not release active chemerin. Notably, activation of the chemerin receptors by hepatic vein blood did not increase in parallel to total chemerin protein in patients with liver cirrhosis. In experimental NASH, muChem-156 had no effect on liver lipids. Accordingly, overexpression of active chemerin in hepatocytes or treatment of hepatocytes with recombinant chemerin did not affect cellular triglyceride and cholesterol levels. Importantly, overexpression of muChem-156 in the murine liver did not change the hepatic expression of inflammatory and profibrotic genes. The downstream targets of chemerin such as p38 kinase were neither activated in the liver of muChem-156 producing mice nor in HepG2, Huh7 and Hepa1-6 cells overexpressing this isoform. Recombinant chemerin had no effect on global gene expression of primary human hepatocytes and hepatic stellate cells within 24 h of incubation. Phosphorylation of p38 kinase was, however, increased upon short-time incubation of HepG2 cells with chemerin. These findings show that muChem-156 overexpression in hepatocytes does not protect from liver steatosis and inflammation.
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Enfermedad del Hígado Graso no Alcohólico , Animales , Quimiocinas , Modelos Animales de Enfermedad , Células Estrelladas Hepáticas/metabolismo , Hepatocitos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Hígado/metabolismo , Ratones , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
Bile acid synthesis is restricted to hepatocytes and is rate-limited by CYP7A1 (cholesterol 7α hydroxylase). CYP7A1 expression undergoes tight regulation and is repressed after partial hepatectomy to prevent the accumulation of toxic bile acids. Augmenter of Liver Regeneration (ALR) is a hepatotrophic factor shown to support liver regeneration by augmenting cell proliferation and reducing apoptosis. Nevertheless, less is known about ALR's role in protecting hepatocytes from bile acid accumulation and bile acid-induced apoptosis. Therefore, HepG2 and Huh-7 cells were incubated with recombinant human ALR (rALR) and the expression of CYP7A1, bile acid-induced apoptosis as well as potential molecular mechanisms were analyzed. We found that rALR reduces CYP7A1 expression by increasing nuclear NFκB levels. Moreover, rALR reduced glycochenodeoxycholate (GCDC)-induced-apoptosis by decreased expression of pro-apoptotic Bax and enhanced expression of anti-apoptotic Mcl-1, which is regulated by phosphatidylinositol-3-kinase (PI3K)/Akt activation and glycogen synthase kinase-3ß (GSK3ß) phosphorylation. Inhibitors for PI3K/Akt (GSK690693) and GSK3ß (SB415286) confirmed the specificity of rALR treatment for this pathway. In addition, rALR reduces pro-death signaling by decreasing GCDC-induced JNK phosphorylation. Taken all together, rALR might contribute to protecting hepatocytes from toxic concentrations of bile acids by down-regulating their denovo synthesis, attenuating apoptosis by activation of PI3K/Akt - GSK3ß pathway and inhibition of JNK signaling. Thereby this suggests a new role of ALR in augmenting the process of liver regeneration.
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Apoptosis , Ácidos y Sales Biliares/biosíntesis , Carcinoma Hepatocelular/terapia , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Hepatocitos/citología , Neoplasias Hepáticas/terapia , Regeneración Hepática , Ácidos y Sales Biliares/farmacología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Colesterol 7-alfa-Hidroxilasa , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Hepatocitos/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is of particular importance in cholesterol metabolism with high levels contributing to hypercholesterolemia. Cholesterol and sphingolipids are low in patients with liver cirrhosis. Purpose of this study was to find associations of plasma PCSK9 with circulating cholesterol and sphingolipid species and measures of liver disease severity in patients with liver cirrhosis. METHODS: PCSK9 protein levels were determined by ELISA in systemic vein (SVP), hepatic vein (HVP) and portal vein plasma of patients with mostly alcoholic liver cirrhosis. PCSK9 and LDL-receptor protein expression were analysed in cirrhotic and non-cirrhotic liver tissues. RESULTS: Serum PCSK9 was reduced in patients with liver cirrhosis in comparison to non-cirrhotic patients. In liver cirrhosis, plasma PCSK9 was not correlated with Child-Pugh score, Model for End-Stage Liver Disease score, bilirubin or aminotransferases. A negative association of SVP PCSK9 with albumin existed. PCSK9 protein in the liver did not change with fibrosis stage and was even positively correlated with LDL-receptor protein levels. Ascites volume and variceal size were not related to PCSK9 levels. Along the same line, transjugular intrahepatic shunt to lower portal pressure did not affect PCSK9 concentrations in the three blood compartments. Serum cholesterol, sphingomyelin and ceramide levels did not correlate with PCSK9. Stratifying patients by high versus low PCSK9 levels using the median as cut-off, several cholesteryl ester species were even low in the subgroup with high PCSK9 levels. A few sphingomyelin species were also reduced in the patients with PCSK9 levels above the median. PCSK9 is highly expressed in the liver but systemic, portal and hepatic vein levels were similar. PCSK9 was not correlated with the inflammatory proteins C-reactive protein, IL-6, galectin-3, resistin or pentraxin 3. Of note, HVP PCSK9 was positively associated with HVP chemerin and negatively with HVP adiponectin levels. CONCLUSIONS: In the cohort of patients with liver cirrhosis mostly secondary to alcohol consumption high PCSK9 was associated with low levels of certain cholesteryl ester and sphingomyelin species. Positive correlations of PCSK9 and LDL-receptor protein in the liver of patients with chronic liver injury are consistent with these findings.
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Colesterol/sangre , Cirrosis Hepática/sangre , Cirrosis Hepática/patología , Proproteína Convertasa 9/metabolismo , Índice de Severidad de la Enfermedad , Adipoquinas/sangre , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , LDL-Colesterol/sangre , Enfermedad Crónica , Estudios de Cohortes , Femenino , Humanos , Mediadores de Inflamación/sangre , Riñón/fisiopatología , Hígado/irrigación sanguínea , Hígado/metabolismo , Hígado/fisiopatología , Cirrosis Hepática/complicaciones , Cirrosis Hepática/fisiopatología , Masculino , Persona de Mediana Edad , Proproteína Convertasa 9/sangre , Receptores de LDL/metabolismo , Esfingolípidos/sangre , Esfingomielinas/sangreRESUMEN
Hepatocellular carcinoma (HCC) still remains a difficult to cure malignancy. In recent years, the focus has shifted to lipid metabolism for the treatment of HCC. Very little is known about hepatitis B virus (HBV) and C virus (HCV)-related hepatic lipid disturbances in non-malignant and cancer tissues. The present study showed that triacylglycerol and cholesterol concentrations were similar in tumor adjacent HBV and HCV liver, and were not induced in the HCC tissues. Higher levels of free cholesterol, polyunsaturated phospholipids and diacylglycerol species were noted in non-tumorous HBV compared to HCV liver. Moreover, polyunsaturated phospholipids and diacylglycerols, and ceramides declined in tumors of HBV infected patients. All of these lipids remained unchanged in HCV-related HCC. In HCV tumors, polyunsaturated phosphatidylinositol levels were even induced. There were no associations of these lipid classes in non-tumor tissues with hepatic inflammation and fibrosis scores. Moreover, these lipids did not correlate with tumor grade or T-stage in HCC tissues. Lipid reprogramming of the three analysed HBV/HCV related tumors mostly resembled HBV-HCC. Indeed, lipid composition of non-tumorous HCV tissue, HCV tumors, HBV tumors and HBV/HCV tumors was highly similar. The tumor suppressor protein p53 regulates lipid metabolism. The p53 and p53S392 protein levels were induced in the tumors of HBV, HCV and double infected patients, and this was significant in HBV infection. Negative correlation of tumor p53 protein with free cholesterol indicates a role of p53 in cholesterol metabolism. In summary, the current study suggests that therapeutic strategies to target lipid metabolism in chronic viral hepatitis and associated cancers have to consider disease etiology.
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Carcinoma Hepatocelular/metabolismo , Colesterol/metabolismo , Hígado/metabolismo , Adulto , Anciano , Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/genética , Colesterol/fisiología , Femenino , Alemania/epidemiología , Hepacivirus/metabolismo , Hepatitis B/virología , Virus de la Hepatitis B/metabolismo , Hepatitis C/virología , Humanos , Metabolismo de los Lípidos/fisiología , Lípidos/fisiología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVE: TGF-ß2 (TGF-ß, transforming growth factor beta), the less-investigated sibling of TGF-ß1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-ß2 in biliary-derived liver diseases. DESIGN: As we also found upregulated TGFB2 in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-ß2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of TgfB2 silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels. RESULTS: TgfB2-induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. TgfB2 expression in MDR2-KO mice was blunted using TgfB2-directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced PparG expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on Ccl3, Ccl4, Ccl5, Mki67 and Notch3 expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, TGFB2 and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue. CONCLUSIONS: Taken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-ß2 silencing and provide a direct rationale for TGF-ß2-directed drug development.
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Colangitis Esclerosante , Silenciador del Gen , Cirrosis Hepática Biliar , Cirrosis Hepática , Oligonucleótidos Antisentido , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Colangitis Esclerosante/metabolismo , Colangitis Esclerosante/patología , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Regulación de la Expresión Génica , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Cirrosis Hepática/prevención & control , Cirrosis Hepática Biliar/metabolismo , Cirrosis Hepática Biliar/patología , Ratones , Ratones Noqueados , Regulación hacia Arriba , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Cholestasis represents pathophysiologic syndromes defined as an impaired bile flow from the liver. As an outcome, bile acids accumulate and promote hepatocytes injury followed by liver cirrhosis and liver failure. Bile acids induce apoptosis, ER stress and mitochondrial membrane instability. In this study we aimed to investigate the role of cytosolic short form of ALR (Augmenter of Liver Regeneration) in the synthesis of bile acids and bile acid-induced apoptosis. Human hepatoma cells over-expressing the short form of ALR (sfALR, 15â¯kDa) were incubated with glycochenodeoxycholic acid (GCDCA), and then primary bile acids' production and apoptosis were analyzed. High levels of cytosolic sfALR reduced CYP7A1 mRNA expression and bile acids levels, the rate-limiting enzyme in the classic pathway of bile acid synthesis. This reduction was attributed to STAT3 (signal transducer and activator of transcription 3) activation and reduction of HNF4α (Hepatocyte nuclear factor 4α). Furthermore, apoptosis induction by GCDCA and TRAIL was reduced in cells over-expressing sfALR which was attributed to reduced expression of death receptor 5 (DR5). We found decreased hepatic mRNA levels of ALR and FOXA2 (Forkhead Box A2), an inducer of ALR expression, in human cholestatic liver samples which might explain the increased accumulation of bile acids and bile acid-induced apoptosis in cholestasis patients.
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Apoptosis/efectos de los fármacos , Ácidos y Sales Biliares/biosíntesis , Ácidos y Sales Biliares/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Factor de Transcripción STAT3/metabolismo , Adulto , Anciano , Colestasis/patología , Colesterol 7-alfa-Hidroxilasa/metabolismo , Citosol/metabolismo , Femenino , Células Hep G2 , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Adulto JovenRESUMEN
Inflammation has been recognized as essential for restorative regeneration. Here, we analyzed the sequential processes during onset of liver injury and subsequent regeneration based on time-resolved transcriptional regulatory networks (TRNs) to understand the relationship between inflammation, mature organ function, and regeneration. Genome-wide expression and TRN analysis were performed time dependently in mouse liver after acute injury by CCl4 (2 h, 8 h, 1, 2, 4, 6, 8, 16 days), as well as lipopolysaccharide (LPS, 24 h) and compared to publicly available data after tunicamycin exposure (mouse, 6 h), hepatocellular carcinoma (HCC, mouse), and human chronic liver disease (non-alcoholic fatty liver, HBV infection and HCC). Spatiotemporal investigation differentiated lobular zones for signaling and transcription factor expression. Acute CCl4 intoxication induced expression of gene clusters enriched for inflammation and stress signaling that peaked between 2 and 24 h, accompanied by a decrease of mature liver functions, particularly metabolic genes. Metabolism decreased not only in pericentral hepatocytes that underwent CCl4-induced necrosis, but extended to the surviving periportal hepatocytes. Proliferation and tissue restorative TRNs occurred only later reaching a maximum at 48 h. The same upstream regulators (e.g. inhibited RXR function) were implicated in increased inflammation and suppressed metabolism. The concomitant inflammation/metabolism TRN occurred similarly after acute LPS and tunicamycin challenges, in chronic mouse models and also in human liver diseases. Downregulation of metabolic genes occurs concomitantly to induce inflammation-associated genes as an early response and appears to be initiated by similar upstream regulators in acute and chronic liver diseases in humans and mice. In the acute setting, proliferation and restorative regeneration associated TRNs peak only later when metabolism is already suppressed.
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Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/genética , Redes Reguladoras de Genes , Hepatitis Crónica/genética , Animales , Tetracloruro de Carbono/toxicidad , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Hepatitis B/genética , Hepatitis B/metabolismo , Hepatitis Crónica/fisiopatología , Humanos , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
The chemoattractant adipokine chemerin is related to the metabolic syndrome, which is a risk factor for different cancers. Recent studies provide evidence that chemerin is an important molecule in colorectal cancer (CRC) and hepatocellular carcinoma (HCC). Serum chemerin is high in CRC patients and low in HCC patients and may serve as a differential diagnostic marker for HCC and liver metastases from CRC. To this end, serum chemerin was measured in 36 patients with CRC metastases, 32 patients with HCC and 49 non-tumor patients by ELISA. Chemerin serum protein levels were, however, similar in the three cohorts. Serum chemerin was higher in hypertensive than normotensive tumor patients but not controls. Cancer patients with hypercholesterolemia or hyperuricemia also had increased serum chemerin. When patients with these comorbidities were excluded from the calculation, chemerin was higher in CRC than HCC patients but did not differ from controls. Chemerin did not correlate with the tumor markers carcinoembryonic antigen, carbohydrate antigen 19-9 and alpha-fetoprotein in both cohorts and was not changed with tumor-node-metastasis stage in HCC. Chemerin was not associated with hepatic fat, liver inflammation and fibrosis. To conclude, systemic chemerin did not discriminate between CRC metastases and HCC. Comorbidities among tumor patients were linked with elevated systemic chemerin.
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Carcinoma Hepatocelular/secundario , Quimiocinas/sangre , Neoplasias Colorrectales/patología , Neoplasias Hepáticas/secundario , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/diagnóstico , Neoplasias Colorrectales/sangre , Femenino , Humanos , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/diagnóstico , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Adulto JovenRESUMEN
Bile acids (BA) are signaling molecules that activate nuclear factors and g-protein coupled receptors signaling to maintain metabolic homeostasis. However, accumulation of toxic BA promotes liver injury by initiating inflammation, inducing apoptosis and causing oxidative stress leading to cirrhosis and liver failure. Augmenter of Liver Regeneration (ALR) is a hepatotrophic growth factor with anti-apoptotic and anti-oxidative properties that has been shown to improve mitochondrial and hepatic functions in rats after bile duct ligation. In the current study we aimed to analyze the regulation of the pro-survival protein, ALR, under conditions of cytotoxic concentrations of BA. Promoter studies of ALR (-733/+527â¯bp) revealed potential binding sites for various transcription factors like Egr-1, HNF4α and two bile acid response elements (BARE). Using a full-length and several truncated promoter constructs for ALR we analyzed promoter activity and showed that BA reduce ALR promoter activity whereas Egr-1 transfection induces it. EMSA and supershift analysis confirmed the specific binding of Egr-1 to its response element within ALR promoter and this binding was reduced upon simultaneous stimulation with BA. We also showed that ALR promoter activity and protein expression are induced by HNF4α1 and repressed by SHP. In conclusion, these results indicate that BA negatively regulate ALR expression by SHP activation.
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Ácidos y Sales Biliares/farmacología , Reductasas del Citocromo/biosíntesis , Regulación de la Expresión Génica/fisiología , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Regulación hacia Abajo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Células Hep G2 , Humanos , Oxidorreductasas actuantes sobre Donantes de Grupos SulfuroRESUMEN
The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-ß1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-ß1-induced hepcidin expression are still unclear. Here we show that TGF-ß1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-ß1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-ß1 depends on functional TGF-ß1 type I receptor (ALK5) and TGF-ß1 type II receptor (TßRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-ß1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5-mediated hepcidin response to TGF-ß1. TGF-ß1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-ß1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-ß1 and BMP6 may control the sensing of systemic and/or hepatic iron levels.
Asunto(s)
Hepatocitos/metabolismo , Hepcidinas/genética , Factor de Crecimiento Transformador beta1/fisiología , Animales , Proteína Morfogenética Ósea 6/metabolismo , Células Cultivadas , Femenino , Expresión Génica , Hepcidinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Activación TranscripcionalRESUMEN
Lipid composition affects membrane function, cell proliferation and cell death and is changed in cancer tissues. Hepatocellular carcinoma (HCC) is an aggressive cancer and this study aimed at a comprehensive characterization of hepatic and serum lipids in human HCC. Cholesteryl ester were higher in tumorous tissues (TT) compared to adjacent non-tumorous tissues (NT). Free cholesterol exerting cytotoxic effects was not changed. Phosphatidylethanolamine, -serine (PS) and -inositol but not phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) were reduced in HCC tissues. Saturated species mostly increased and polyunsaturated species were diminished in all of these phospholipids. Ceramide (Cer) was markedly reduced in HCC tissues and higher levels of sphingomyelin suggest impaired sphingomyelinase activity as one of the underlying mechanisms. Importantly, ceramide in NT increased in HCC stage T3. Ceramide released from hepatocytes attracts immune cells and a positive association of the macrophage specific receptor CD163 with NT ceramide was identified. HCC associated lipid changes did not differ in patients suffering from type 2 diabetes. Protein levels of p53 were induced in TT and negatively correlated with Cer d18:1/16:0 and PS 36:1. Of the lipid species changed in HCC tissues only TT Cer d18:1/16:0, Cer d18:1/24:1, PC 38:6 and LPC 22:6 correlated with the respective serum levels. Our study demonstrates a considerably altered hepatic lipidome in HCC tissues. Ceramide was markedly reduced in HCC tissues, and therefore, raising ceramide levels specifically in the tumor represents a reasonable therapeutic approach for the treatment of this malignancy.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Ceramidas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfolípidos/metabolismo , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/sangre , Ceramidas/sangre , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Grasos Insaturados/sangre , Hígado Graso/sangre , Hígado Graso/metabolismo , Humanos , Neoplasias Hepáticas/sangre , Masculino , Persona de Mediana Edad , Mutación/genética , Fosfolípidos/sangre , Análisis de Componente Principal , Esfingomielinas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Chemerin is associated with insulin resistance and is expressed in the liver. Nonalcoholic fatty liver disease (NAFLD) is related to impaired insulin sensitivity, but studies evaluating hepatic and serum chemerin in NAFLD resulted in discordant data. MATERIALS AND METHODS: Chemerin mRNA was determined in the liver tissue obtained from 33 controls and 76 NAFLD patients. Chemerin serum levels were measured in a different cohort of patients with ultrasound-diagnosed NAFLD and the respective controls. Hepatic stellate cells and hepatocytes were exposed to selected metabolites and nuclear receptor agonists to study the regulation of chemerin. Effect of recombinant chemerin on hepatocyte released proteins was analysed. RESULTS: Hepatic chemerin expression was not related to BMI, gender, type 2 diabetes and hypertension. Chemerin mRNA did not correlate with steatosis and was negatively associated with inflammation, fibrosis and nonalcoholic steatohepatitis (NASH) score. Patients with NASH had lower chemerin mRNA compared to those with borderline NASH and controls. Factors with a role in NASH mostly did not regulate chemerin in the liver cells. Of note, liver X receptor agonist reduced chemerin protein. Serum chemerin was not changed in NAFLD. Levels positively correlated with age, waist-to-hip ratio, systolic blood pressure, serum FGF21 and lipocalin 2, and negatively with transferrin saturation. Chemerin induced FGF21 in supernatants of primary human hepatocytes. Hepcidin, a major regulator of iron homoeostasis and lipocalin 2, were not regulated by chemerin. CONCLUSION: Chemerin mRNA is reduced in the liver of NASH patients, and liver X receptor seems to have a role herein.
Asunto(s)
Quimiocinas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/genética , ARN Mensajero/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Índice de Masa Corporal , Estudios de Casos y Controles , Línea Celular , Células Cultivadas , Quimiocinas/sangre , Quimiocinas/farmacología , Comorbilidad , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/epidemiología , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Células Hep G2 , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepcidinas/metabolismo , Humanos , Hidrocarburos Fluorados/farmacología , Hipertensión/epidemiología , Hipoglucemiantes/farmacología , Técnicas In Vitro , Resistencia a la Insulina , Péptidos y Proteínas de Señalización Intercelular/sangre , Péptidos y Proteínas de Señalización Intercelular/farmacología , Leptina/metabolismo , Lipocalina 2/metabolismo , Receptores X del Hígado/agonistas , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/epidemiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Citoplasmáticos y Nucleares/agonistas , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rosiglitazona , Índice de Severidad de la Enfermedad , Sulfonamidas/farmacología , Tiazolidinedionas/farmacología , Relación Cintura-Cadera , Adulto JovenRESUMEN
Tubulin alpha 8 (TUBA8) is highly abundant in murine liver tumors suggesting a role in hepatocellular carcinoma (HCC). Non-alcoholic steatohepatitis (NASH) is a risk factor for HCC. In mice that are fed with a methionine-choline deficient diet for two weeks to induce advanced murine NASH, we do see increased hepatic levels of TUBA8 protein. In animals given a high-fat diet for 14 weeks or an atherogenic diet for 12 weeks, hepatic TUBA8 is unchanged. TUBA8 is highly expressed in human hepatic stellate cells (HSC) and co-localizes with the HSC marker desmin in the murine liver. Inflammatory (TNF, LPS, IL-6) and profibrotic mediators (TGF-beta) do not regulate TUBA8 in HepG2 cells, primary HSC and the HSC cell line LX-2, when stimulated for 24 h. Agonists of the farnesoid X receptor and peroxisome proliferator activated receptor gamma, which are nuclear receptors involved in NASH and HCC pathophysiology, have no effect on TUBA8 in HepG2 and LX-2 cells. In human HCC tissues of 18 patients TUBA8 is significantly upregulated when compared to the corresponding non-tumorous tissues. Compared to non-transformed hepatocytes, TUBA8 protein is strongly expressed in transformed cells. Thus, TUBA8 is a marker of HSC whose cell number is increased in NASH, while higher levels in HCC may be related to induction of TUBA8 in parenchymal cells.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Células Estrelladas Hepáticas/metabolismo , Neoplasias Hepáticas/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Tubulina (Proteína)/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Colina/efectos adversos , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Células Hep G2 , Humanos , Masculino , Metionina/efectos adversos , Ratones , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Células RAW 264.7 , Regulación hacia ArribaRESUMEN
The acute-phase response (APR) is an inflammatory process triggered mainly by IL-6 in response to neoplasm, tissue injury, infection or inflammation. Signaling of IL-6 is transduced by activating STAT3 which rapidly results in production of acute-phase proteins (APPs) such as fibrinogen ß (FGB) and haptoglobin (HP). Augmenter of liver regeneration (ALR), a hepatotrophic factor supporting liver regeneration, was reported to be upregulated after liver damage. In this study we analyzed the role of ALR for IL-6 signaling and APR. Thus, we investigated the expression and release of APPs in human liver cells under conditions of increased exogenous or endogenous ALR. HepG2 cells and ALR-reexpressing HepG2 cells were treated with IL-6 in the presence or absence of exogenous ALR for different time points. The mRNA expression and release of both FGB and HP were measured by RT-PCR and ELISA. We found that exogenously applied ALR attenuated the IL-6-induced mRNA expression and protein secretion of both FGB and HP. In contrast, IL-6 stimulation in HepG2 cells which re-express ALR, revealed elevated APR shown by increased mRNA expression and secretion of FGB and HP. Furthermore, we found that ALR-mediated regulation of IL-6-induced APP production is accompanied by altered STAT3 activity. While exogenous ALR reduced the IL-6-induced phosphorylation of STAT3, endogenous ALR enhanced STAT3 activity in liver cells. In conclusion, ALR, dependent on its localization, changes APR at least in part, by modifying STAT3 activation. This study shows a dual signaling of ALR and suggests that ALR is pivotal for the regulation of APR, a crucial event in liver injury and regeneration.
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Reacción de Fase Aguda/genética , Reductasas del Citocromo/metabolismo , Hepatocitos/metabolismo , Factor de Transcripción STAT3/metabolismo , Reacción de Fase Aguda/patología , Reductasas del Citocromo/genética , Fibrinógeno/genética , Fibrinógeno/metabolismo , Haptoglobinas/genética , Haptoglobinas/metabolismo , Células Hep G2 , Humanos , Interleucina-6/farmacología , Hígado/metabolismo , Regeneración Hepática , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Fosforilación , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Inhibidoras de STAT Activados/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción STAT3/genética , Transducción de Señal , Proteína 3 Supresora de la Señalización de Citocinas/genética , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Regulación hacia ArribaRESUMEN
The adaptor protein alpha-syntrophin (SNTA) is differentially expressed in varying types of cancer and affects triglyceride levels, inflammatory response and cell proliferation. However, little is known about the expression of SNTA in liver diseases. Non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis, inflammation and eventually fibrosis, and may progress to hepatocellular carcinoma (HCC). Here, SNTA mRNA was analyzed in liver tissues from 71 non-alcoholic fatty liver disease patients and 32 controls to assess associations with disease characteristics. SNTA mRNA expression was reduced in NASH liver and negatively correlated with steatosis, inflammation, fibrosis and NASH scores. In the NASH patients, those with type 2 diabetes had a higher fibrosis score, reduced inflammation and increased hepatic SNTA mRNA levels demonstrating a strong association of SNTA mRNA levels with inflammation. Recently, we have shown diminished expression of the high-density lipoprotein scavenger receptor BI (SR-BI) in the liver of syntrophin-deficient mice. Indeed, hepatic SNTA and SR-BI mRNA were positively correlated. SNTA protein was further determined in tumor and non-tumorous tissues of 21 HCC patients. Protein expression was unchanged in the tumor and not related to staging and grading. Present study identified associations of hepatic SNTA mRNA levels with SR-BI and features of NASH assuming a function of this protein in chronic liver disease and cholesterol metabolism.
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Biomarcadores/metabolismo , Proteínas de Unión al Calcio/metabolismo , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Enfermedad del Hígado Graso no Alcohólico/diagnóstico , Receptores Depuradores de Clase B/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Proteínas de Unión al Calcio/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Diagnóstico Diferencial , Femenino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Proteínas Musculares/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Receptores Depuradores de Clase B/genética , Adulto JovenRESUMEN
Chemokine (CC-motif) receptor-like 2 (CCRL2) is a decoy receptor and regulates the local responses of the chemokine chemerin. Recently our group has shown that the functional chemerin receptor, chemokine-like receptor 1 (CMKLR1), correlates with fibrosis and non-alcoholic steatohepatitis (NASH) score in males only. In our current study, we wanted to know whether CCRL2 shows similar correlations as CMKLR1. Therefore, we analyzed the hepatic expression of CCRL2 in murine NASH and in liver tissues obtained from 85 patients with non-alcoholic fatty liver disease (NAFLD) and 33 controls. CCRL2 mRNA was not significantly changed in murine and human NASH liver. CCRL2 mRNA levels were positively correlated with inflammation, fibrosis and NASH scores in the patients. Concordantly, CCRL2 was related to the mRNA levels of F4/80, transforming growth factor beta and alpha smooth muscle actin in murine NASH. In the human cohort, CCRL2 mRNA correlated with fibrosis score and CMKLR1 mRNA in both gender. CCRL2 mRNA was induced in the liver of type 2 diabetes and hypercholesterolemic patients, but still positively correlated with fibrosis score when these patients were excluded from calculations. Human hepatic stellate cells (HSC), hepatic sinusoidal endothelial cells and Kupffer cells (KC) express CCRL2 mRNA. TNF induces CCRL2 expression in HSC and lipopolysaccharide in KC suggesting that correlations identified in NAFLD patients are partly related to the activation of these cells.