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1.
J Med Genet ; 2024 May 30.
Article in English | MEDLINE | ID: mdl-38816193

ABSTRACT

BACKGROUND AND AIMS: Variants in ZFYVE19 underlie a disorder characterised by progressive portal fibrosis, portal hypertension and eventual liver decompensation. We aim to create an animal model to elucidate the pathogenic mechanism. METHODS: Zfyve19 knockout (Zfyve19-/- ) mice were generated and exposed to different liver toxins. Their livers were characterised at the tissue, cellular and molecular levels. Findings were compared with those in wild-type mice and in ZFYVE19-deficient patients. ZFYVE19 knockout and knockdown retinal pigment epithelial-1 cells and mouse embryonic fibroblasts were generated to study cell division and cell death. RESULTS: The Zfyve19-/- mice were normal overall, particularly with respect to hepatobiliary features. However, when challenged with α-naphthyl isothiocyanate, Zfyve19-/- mice developed changes resembling those in ZFYVE19-deficient patients, including elevated serum liver injury markers, increased numbers of bile duct profiles with abnormal cholangiocyte polarity and biliary fibrosis. Failure of cell division, centriole and cilia abnormalities, and increased cell death were observed in knockdown/knockout cells. Increased cell death and altered mRNA expression of cell death-related signalling pathways was demonstrated in livers from Zfyve19-/- mice and patients. Transforming growth factor-ß (TGF-ß) and Janus kinase-Signal Transducer and Activator of Transcription 3 (JAK-STAT3) signalling pathways were upregulated in vivo, as were chemokines such as C-X-C motif ligands 1, 10 and 12. CONCLUSIONS: Our findings demonstrated that ZFYVE19 deficiency is a ciliopathy with novel histological features. Failure of cell division with ciliary abnormalities and cell death activates macrophages and may thus lead to biliary fibrosis via TGF-ß pathway in the disease.

2.
J Cell Physiol ; 237(6): 2713-2723, 2022 06.
Article in English | MEDLINE | ID: mdl-35621037

ABSTRACT

TMEM67 (mecklin or MKS3) locates in the transition zone of cilia. Dysfunction of TMEM67 disrupts cilia-related signaling and leads to developmental defects of multiple organs in humans. Typical autosomal recessive TMEM67 defects cause partial overlapping phenotypes, including abnormalities in the brain, eyes, liver, kidneys, bones, and so forth. However, emerging reports of isolated nephronophthisis suggest the possibility of a broader phenotype spectrum. In this study, we analyzed the genetic data of cholestasis patients with no obvious extrahepatic involvement but with an unexplained high level of gamma-glutamyl transpeptidase (GGT). We identified five Han Chinese patients from three unrelated families with biallelic nonnull low-frequency TMEM67 variants. All variants were predicted pathogenic in silico, of which p. Arg820Ile and p. Leu144del were previously unreported. In vitro studies revealed that the protein levels of the TMEM67 variants were significantly decreased; however, their interaction with MKS1 remained unaffected. All the patients, aged 7-39 years old, had silently progressive cholestasis with elevated GGT but had normal bilirubin levels. Histological studies of liver biopsy of patients 1, 3, and 5 showed the presence of congenital hepatic fibrosis. We conclude that variants in TMEM67 are associated with a mild phenotype of unexplained, persistent, anicteric, and high GGT cholestasis without typical symptoms of TMEM67 defects; this possibility should be considered by physicians in gastroenterology and hepatology.


Subject(s)
Cholestasis , gamma-Glutamyltransferase , Cholestasis/genetics , Genetic Diseases, Inborn , Humans , Liver Cirrhosis/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Phenotype , gamma-Glutamyltransferase/genetics
3.
Liver Int ; 42(8): 1836-1848, 2022 08.
Article in English | MEDLINE | ID: mdl-35567760

ABSTRACT

BACKGROUND AND AIMS: Alagille syndrome (ALGS) type 2 caused by mutations in NOTCH2 has genotypic and phenotypic heterogeneity. Diagnosis in some atypical patients with isolated hepatic presentation could be missed. METHODS: Using 2087 patients with paediatric liver manifestations, NOTCH2 allele frequencies, in-silico prediction, protein domains and clinical features were analysed to define the pathogenicity of NOTCH2 variants for diagnosis of ALGS type 2. RESULTS: Among 2087 patients with paediatric liver manifestations, significantly more NOTCH2 variants were absent in gnomAD in patients with elevated γ-glutamyltransferase (GGT) (p = .041). Significantly more NOTCH2 variants which were absent in gnomAD were located in protein functional domains (p = .038). When missense variants were absent in gnomAD and predicted to be pathogenic by at least three out of seven in-silico tools, they were found to be significantly associated with liver manifestations with elevated GGT (p = .003). Comparing this to patients with likely benign (LB) variants, the patients with likely-pathogenic (LP) variants have significantly more liver manifestations with elevated GGT (p = .0001). Significantly more patients with LP variants had extra-hepatic phenotypes of ALGS compared with those patients with LB variants (p = .0004). CONCLUSION: When NOTCH2 variants are absent in gnomAD, null variants and missense variants which were predicted to be pathogenic by at least three in-silico tools could be considered pathogenic in patients with high GGT chronic liver diseases.


Subject(s)
Alagille Syndrome , Receptor, Notch2 , Alagille Syndrome/diagnosis , Alagille Syndrome/genetics , Humans , Jagged-1 Protein/genetics , Jagged-1 Protein/metabolism , Mutation , Phenotype , Receptor, Notch2/genetics , Receptor, Notch2/metabolism , Virulence
4.
J Med Genet ; 58(8): 514-525, 2021 08.
Article in English | MEDLINE | ID: mdl-32737136

ABSTRACT

BACKGROUND: For many children with intrahepatic cholestasis and high-serum gamma-glutamyl transferase (GGT) activity, a genetic aetiology of hepatobiliary disease remains undefined. We sought to identify novel genes mutated in children with idiopathic high-GGT intrahepatic cholestasis, with clinical, histopathological and functional correlations. METHODS: We assembled a cohort of 25 children with undiagnosed high-GGT cholestasis and without clinical features of biliary-tract infection or radiological features of choledochal malformation, sclerosing cholangitis or cholelithiasis. Mutations were identified through whole-exome sequencing and targeted Sanger sequencing. We reviewed histopathological findings and assessed phenotypical effects of ZFYVE19 deficiency in cultured cells by immunofluorescence microscopy. RESULTS: Nine Han Chinese children harboured biallelic, predictedly complete loss-of-function pathogenic mutations in ZFYVE19 (c.314C>G, p.S105X; c.379C>T, p.Q127X; c.514C>T, p.R172X; c.547C>T, p.R183X; c.226A>G, p.M76V). All had portal hypertension and, at liver biopsy, histopathological features of the ductal plate malformation (DPM)/congenital hepatic fibrosis (CHF). Four children required liver transplantation for recurrent gastrointestinal haemorrhage. DPM/CHF was confirmed at hepatectomy, with sclerosing small-duct cholangitis. Immunostaining for two primary-cilium axonemal proteins found expression that was deficient intraluminally and ectopic within cholangiocyte cytoplasm. ZFYVE19 depletion in cultured cells yielded abnormalities of centriole and axoneme. CONCLUSION: Biallelic ZFYVE19 mutations can lead to high-GGT cholestasis and DPM/CHF in vivo. In vitro, they can lead to centriolar and axonemal abnormalities. These observations indicate that mutation in ZFYVE19 results, through as yet undefined mechanisms, in a ciliopathy.


Subject(s)
Cholangitis, Sclerosing/genetics , Cholestasis, Intrahepatic/genetics , Mutation/genetics , Oncogene Proteins/genetics , Alleles , Amino Acid Sequence , Cell Line, Tumor , Genetic Diseases, Inborn , HeLa Cells , Humans , Liver Cirrhosis , Exome Sequencing/methods
5.
Hum Mutat ; 41(2): 502-511, 2020 02.
Article in English | MEDLINE | ID: mdl-31696999

ABSTRACT

To assess the spectrum of pediatric clinical phenotypes in TJP2 disease, we reviewed records of our seven patients in whom intrahepatic cholestasis was associated with biallelic TJP2 variants (13; 12 novel) and correlated clinical manifestations with mutation type. The effect of a splicing variant was analyzed with a minigene assay. The effects of three missense variants were analyzed with protein expression in vitro. Our patients had both remitting and persistent cholestasis. Three exhibited growth retardation. Six responded to treatment with cholestyramine, ursodeoxycholic acid, or both. Two had cholecystolithiasis. None required liver transplantation or developed hepatocellular or cholangiocellular malignancy. None manifested extrahepatic disease not attributable to effects of cholestasis. The variant c.2180-5T>G resulted in exon 15 skipping with in-frame deletion of 32 amino acid residues in TJP2. The three missense variants decreased but did not abolish TJP2 expression. Patients with truncating or canonical splice-site variants had clinically more severe disease. TJP2 disease in children includes a full clinical spectrum of severity, with mild or intermittent forms as well as the severe and minimal forms hitherto described. Biallelic TJP2 variants must be considered in children with clinically intermittent or resolved intrahepatic cholestasis.


Subject(s)
Cholestasis, Intrahepatic/diagnosis , Cholestasis, Intrahepatic/genetics , Genetic Predisposition to Disease , Genetic Variation , Zonula Occludens-2 Protein/genetics , Age of Onset , Alleles , Amino Acid Substitution , Biopsy , Computational Biology/methods , Female , Humans , Infant , Infant, Newborn , Male , Mutation , Pedigree , RNA Splicing , Exome Sequencing
6.
Hum Mutat ; 40(12): 2247-2257, 2019 12.
Article in English | MEDLINE | ID: mdl-31479177

ABSTRACT

The typical phenotype of arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome involves three cardinal symptoms as the name describes, harboring biallelic mutations on VPS33B or VIPAS39. Except for ARC syndrome, low gamma-glutamyltransferase (GGT) cholestasis often implies hereditary hepatopathy of different severity; however, some remain undiagnosed. Several monogenic defects typically with multiorgan manifestations may only present liver dysfunction at times, such as DGUOK defect and AGL defect. Previously, four VPS33B mutated cases were reported without arthrogryposis, or with less severe symptoms and longer lifespan, indicating the possibility of incomplete ARC phenotype of isolated hepatopathy. So we retrospectively reviewed all patients with confirmed VPS33B/VIPARS39 defect in our center and identified three presenting isolated low-GGT cholestasis with intractable pruritus. Distinguished from others with typical ARC phenotype, these patients did not suffer the other two typical characteristics, survived much longer, and shared a novel missense VPS33B variation c.1726T>C, p.Cys576Arg, causing declined protein expression and abolished interaction with VIPAS39 in-vitro. Serum bile acid profiles of our VPS33B/VIPAS39 mutated patients revealed similar changes to primary defect of bile salt export pump, among which those with isolated cholestasis phenotype had a higher level of total secondary bile acids than that with typical ARC phenotype, indicating the partial residual function of VPS33B.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/deficiency , Cholestasis, Intrahepatic/genetics , Mutation, Missense , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Bile Acids and Salts/blood , Case-Control Studies , Child , Child, Preschool , Cholestasis, Intrahepatic/metabolism , Down-Regulation , Female , HEK293 Cells , Humans , Male , Pedigree , Retrospective Studies
7.
FASEB J ; 33(1): 327-338, 2019 01.
Article in English | MEDLINE | ID: mdl-30001167

ABSTRACT

Liver regeneration is a complicated pathophysiologic process that is regulated by a myriad of signaling pathways and transcription factors. The interaction among these pathways and factors, either cooperatively or antagonistically, may ultimately lead to recovery and restoration of liver function or permanent loss of liver function and liver failure. In the present study, we investigated the mechanism whereby the chromatin remodeling protein brahma related gene 1 (Brg1) regulates liver regeneration in mice. The Smarca4-Flox strain of mice was crossbred with the Alb-Cre strain to generate hepatocyte-specific Brg1 knockout mice. Liver injury was induced by partial hepatectomy (PHx). We report that Brg1 deletion in hepatocyte compromised liver regeneration and dampened survival after PHx in mice. Brg1 interacted with ß-catenin to potentiate Wnt signaling and promote hepatocyte proliferation. Mechanistically, Brg1 recruited lysine demethylase 4 (KDM4) to activate ß-catenin target genes. Our data suggest that Brg1 might play an essential role maintaining hepatic homeostasis and contributing to liver repair.-Li, N., Kong, M., Zeng, S., Hao, C., Li, M., Li, L., Xu, Z., Zhu, M., Xu, Y. Brahma related gene 1 (Brg1) contributes to liver regeneration by epigenetically activating the Wnt/ß-catenin pathway in mice.


Subject(s)
Chromatin/genetics , DNA Helicases/physiology , Epigenesis, Genetic , Hepatocytes/cytology , Liver Regeneration , Nuclear Proteins/physiology , Transcription Factors/physiology , Wnt1 Protein/genetics , beta Catenin/genetics , Animals , Cell Proliferation , Cells, Cultured , Chromatin/metabolism , Gene Expression Regulation , Hepatectomy , Hepatocytes/metabolism , Mice , Mice, Knockout , Wnt1 Protein/metabolism , beta Catenin/metabolism
8.
Sci Rep ; 8(1): 16730, 2018 11 13.
Article in English | MEDLINE | ID: mdl-30425268

ABSTRACT

Serum Golgi protein 73 (GP73) is a promising marker for significant fibrosis in adults. However, current diagnostic value of serum GP73 for liver fibrosis in children is unknown. To investigate the relationship between levels of serum GP73 and liver fibrosis in children, we measured serum GP73 in 86 healthy controls and 183 patients with liver diseases using commercially available double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit. The value of serum GP73 in fibrosis stage assessment was compared with aspartate transaminase to platelet ratio index (APRI). We found that serum GP73 was decreasing with age in healthy controls, while it was increasing with the extent of inflammation and fibrosis in patients with liver diseases. Though area under the receiver operating curve (AUROC) of serum GP73 for diagnosing significant fibrosis was nearly equal to APRI (0.62 vs 0.64) in patients aged 3 years or older, AUROC for serum GP73 was superior to APRI (0.76 vs 0.67) in patients aged below 3 years, indicating that serum GP73 is comparable to APRI for diagnosing significant fibrosis in children.


Subject(s)
Aspartate Aminotransferases/metabolism , Blood Platelets/metabolism , Liver Cirrhosis/blood , Liver Cirrhosis/diagnosis , Membrane Proteins/blood , Biomarkers/blood , Case-Control Studies , Child, Preschool , Female , Humans , Infant , Liver Cirrhosis/metabolism , Male
9.
Liver Int ; 38(9): 1676-1685, 2018 09.
Article in English | MEDLINE | ID: mdl-29412511

ABSTRACT

BACKGROUND & AIMS: Genetic defects causing dysfunction in bile salt export pump (BSEP/ABCB11) lead to liver diseases. ABCB11 mutations alter the bile acid metabolome. We asked whether profiling plasma bile acids could reveal compensatory mechanisms and track genetic and clinical status. METHODS: We compared plasma bile acids in 17 ABCB11-mutated patients, 35 healthy controls and 12 genetically undiagnosed cholestasis patients by ultra-high-performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC/MRM-MS). We developed an index to rank bile acid hydrophobicity, and thus toxicity, based on LC retention times. We recruited 42 genetically diagnosed hereditary cholestasis patients, of whom 12 were presumed to have impaired BSEP function but carried mutations in genes other than ABCB11, and 8 healthy controls, for further verification. RESULTS: The overall hydrophobicity indices of total bile acids in both the ABCB11-mutated group (11.89 ± 1.07 min) and the undiagnosed cholestasis group (11.46 ± 1.07 min) were lower than those of healthy controls (13.69 ± 0.77 min) (both p < 0.005). This was owing to increased bile acid modifications. Secondary bile acids were detected in patients without BSEP expression, suggesting biliary bile acid secretion through alternative routes. A diagnostic panel comprising lithocholic acid (LCA), tauro-LCA, glyco-LCA and hyocholic acid was identified that could differentiate the ABCB11-mutated cohort from healthy controls and undiagnosed cholestasis patients (AUC=0.946, p < 0.0001) and, in non-ABCB11-mutated cholestasis patients, could distinguish BSEP dysfunction from normal BSEP function (9/12 vs 0/38, p < 0.0000001). CONCLUSIONS: Profiling of plasma bile acids has provided insights into cholestasis alleviation and may be useful for the clinical management of cholestatic diseases.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 11/genetics , Bile Acids and Salts/blood , Cholestasis, Intrahepatic/blood , Cholestasis, Intrahepatic/genetics , Case-Control Studies , Child, Preschool , China , Chromatography, High Pressure Liquid , Female , Humans , Infant , Male , Mutation
10.
FASEB J ; 32(1): 500-511, 2018 01.
Article in English | MEDLINE | ID: mdl-28970250

ABSTRACT

Hepatic stellate cells (HSCs) are a major source of fibrogenesis in the liver, contributing to cirrhosis. When activated, HSCs transdifferentiate into myofibroblasts and undergo profound functional alterations paralleling an overhaul of the transcriptome, the mechanism of which remains largely undefined. We investigated the involvement of the class III deacetylase sirtuin [silent information regulator 1 (SIRT1)] in HSC activation and liver fibrosis. SIRT1 levels were down-regulated in the livers in mouse models of liver fibrosis, in patients with cirrhosis, and in activated HSCs as opposed to quiescent HSCs. SIRT1 activation halted, whereas SIRT1 inhibition promoted, HSC transdifferentiation into myofibroblasts. Liver fibrosis was exacerbated in mice with HSC-specific deletion of SIRT1 [conditional knockout (cKO)], receiving CCl4 (1 mg/kg) injection or subjected to bile duct ligation, compared to wild-type littermates. SIRT1 regulated peroxisome proliferator activated receptor γ (PPARγ) transcription by deacetylating enhancer of zeste homolog 2 (EZH2) in quiescent HSCs. Finally, EZH2 inhibition or PPARγ activation ameliorated fibrogenesis in cKO mice. In summary, our data suggest that SIRT1 plays an essential role guiding the transition of HSC phenotypes.-Li, M., Hong, W., Hao, C., Li, L., Wu, D., Shen, A., Lu, J., Zheng, Y., Li, P., Xu, Y. SIRT1 antagonizes liver fibrosis by blocking hepatic stellate cell activation in mice.


Subject(s)
Hepatic Stellate Cells/physiology , Liver Cirrhosis, Experimental/prevention & control , Sirtuin 1/physiology , Animals , Cell Transdifferentiation/genetics , Cell Transdifferentiation/physiology , Cells, Cultured , Down-Regulation , Enhancer of Zeste Homolog 2 Protein/metabolism , Hepatic Stellate Cells/pathology , Humans , Liver Cirrhosis/pathology , Liver Cirrhosis/physiopathology , Liver Cirrhosis/prevention & control , Liver Cirrhosis, Experimental/pathology , Liver Cirrhosis, Experimental/physiopathology , Male , Mice , Mice, Knockout , Myofibroblasts/pathology , Myofibroblasts/physiology , PPAR gamma/genetics , Phenotype , Rats , Sirtuin 1/antagonists & inhibitors , Sirtuin 1/deficiency , Sirtuin 1/genetics
11.
Biochim Biophys Acta Mol Basis Dis ; 1863(12): 3202-3211, 2017 12.
Article in English | MEDLINE | ID: mdl-28919365

ABSTRACT

Liver fibrosis is widely perceived as a host defense mechanism that aids tissue repair following liver injury. Excessive fibrogenesis, however, serves to disrupt normal liver structure and precedes such irrevocable human pathologies as cirrhosis and hepatocellular carcinoma. Activation of hepatic stellate cells (HSCs) is a hallmark event during liver fibrosis. In the present study we investigated the mechanism by which the lysine deacetylase SIRT1 regulates HSC activation. We report here that SIRT1 levels were decreased in the liver in different mouse models and in cultured HSCs undergoing activation. SIRT1 down-regulation paralleled HDAC4 up-regulation. HDAC4 was recruited to the SIRT1 promoter during HSC activation and removed acetylated histones H3 and H4 from the SIRT1 promoter leading to SIRT1 trans-repression. HDAC4 silencing restored SIRT1 expression and attenuated HSC activation in SIRT1-dependent manner. More important, selective deletion of SIRT1 in HSCs exacerbated CCl4-induced liver fibrosis in mice. Mechanistically, SIRT1 deacetylated PPARγ to block HSC activation. Together, our data reveal an HDAC4-SIRT1-PPARγ axis that contributes to the regulation of HSC activation and liver fibrosis.


Subject(s)
Hepatic Stellate Cells/enzymology , Liver Cirrhosis/enzymology , Sirtuin 1/metabolism , Animals , Down-Regulation , Gene Expression Profiling , Hepatic Stellate Cells/pathology , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Histones/metabolism , Liver/enzymology , Liver/pathology , Liver Cirrhosis/genetics , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Male , Mice , Mice, Knockout , PPAR gamma/metabolism , Promoter Regions, Genetic , Sirtuin 1/deficiency , Sirtuin 1/genetics , Up-Regulation
12.
J Pediatr Gastroenterol Nutr ; 65(5): 561-568, 2017 11.
Article in English | MEDLINE | ID: mdl-28937538

ABSTRACT

OBJECTIVES: Cerebrotendinous xanthomatosis (CTX) is caused by defects in sterol 27-hydroxylase (CYP27A1, encoded by CYP27A1), a key enzyme in the bile acid synthesis pathway. CTX usually presents as neurologic disease in adults or older children. The rare reports of CTX manifest as neonatal cholestasis assess the cholestasis as transient, with patient survival. Our experience differs. METHODS: Homozygous or compound heterozygous CYP27A1 mutations were detected in 8 neonatal cholestasis patients by whole exome sequencing, panel sequencing, or Sanger sequencing. Their clinical and biochemical data were retrospectively reviewed. Immunostaining for CYP27A1 was conducted in liver of 4 patients. Mass spectrometry was used to analyze patients' urine samples. RESULTS: All 8 infants had severe cholestasis. Five died from, or were transplanted for, liver failure; 3 cleared their jaundice eventually. Marking for CYP27A1 was weak or absent in 3 of the 4 patient specimens. Mass spectrometry of urine revealed a predominance of sulfated and doubly conjugated (sulfated-glucuronidated) bile alcohols. No patient harbored a putatively pathogenic mutation in genes other than CYP27A1 that have been implicated in cholestatic liver disease. CONCLUSIONS: CTX manifest as neonatal cholestasis has a bile acid profile different from CTX manifest in later life, and thus may be overlooked. Immunostaining, mass spectrometry of urine, and genetic studies can support one another in making the diagnosis. A substantial proportion of CTX patients with severe neonatal cholestasis may die or need liver transplantation. CTX manifest in infancy as severe cholestasis warrants further investigation of biochemical diagnostic criteria and best management.


Subject(s)
Cholestasis/etiology , Xanthomatosis, Cerebrotendinous/diagnosis , Cholestanetriol 26-Monooxygenase/genetics , Cholestanetriol 26-Monooxygenase/metabolism , Cholestasis/diagnosis , Cholestasis/mortality , Cholestasis/surgery , Female , Genetic Markers , Humans , Infant, Newborn , Liver/metabolism , Liver Transplantation , Male , Mass Spectrometry , Mutation , Retrospective Studies , Sequence Analysis, DNA/methods , Severity of Illness Index , Xanthomatosis, Cerebrotendinous/complications , Xanthomatosis, Cerebrotendinous/genetics , Xanthomatosis, Cerebrotendinous/metabolism
13.
Oncotarget ; 8(29): 47586-47594, 2017 Jul 18.
Article in English | MEDLINE | ID: mdl-28548935

ABSTRACT

Activation of hepatic stellate cells (HSCs) is a hallmark event during liver fibrogenesis. We have previously shown that the transcriptional modulator MRTF-A contributes to liver fibrosis by programming epigenetic activation of HSCs. In the present study we investigated the mechanism whereby MRTF-A expression is regulated in this process. We report here that MRTF-A protein levels, but not mRNA levels, were up-regulated in vivo in the livers of mice induced to develop hepatic fibrosis. Pro-fibrogenic stimuli (TGF-ß and PDGF-BB) also activated MRTF-A expression post-transcriptionally in vitro in cultured HSCs. miR-206 bound to the 3'-UTR of MRTF-A presumably to inhibit translation. miR-206 levels were down-regulated in response to pro-fibrogenic stimuli in vivo and in vitro allowing MRTF-A proteins to accumulate. Mechanistically, histone deacetylase 4 (HDAC4) was induced by pro-fibrogenic stimuli and recruited to the miR-206 promoter to repress miR-206 transcription. HDAC4 stimulated MRTF-A expression and drove fibrogenesis in HSCs in a miR-206 dependent manner. Therefore, our data reveal an HDAC4-miR-206-MRTF-A axis that can play a potentially important role in HSC activation and liver fibrosis.


Subject(s)
Gene Expression Regulation , Hepatic Stellate Cells/metabolism , Histone Deacetylases/metabolism , Liver Cirrhosis/genetics , Liver Cirrhosis/metabolism , MicroRNAs/genetics , Trans-Activators/genetics , 3' Untranslated Regions , Animals , Cell Line , Liver Cirrhosis/pathology , Male , Mice , RNA Interference , Rats , Transcription, Genetic
14.
Hepatology ; 65(6): 1904-1919, 2017 06.
Article in English | MEDLINE | ID: mdl-28244120

ABSTRACT

Uncontrolled inflammatory response highlights the central theme of nonalcoholic steatohepatitis (NASH), a growing global pandemic. Hepatocytes and macrophages represent two major sources of hepatic inflammation during NASH pathogenesis, contributing to excessive synthesis of proinflammatory mediators. The epigenetic mechanism that accounts for the activation of hepatocytes and macrophages in this process remains obscure. Here, we report that compared to wild-type littermates, mice with a deficiency in the histone H3K9 methyltransferase suppressor of variegation 39 homolog 2 (Suv39h2, knockout) exhibited a less severe form of NASH induced by feeding with a high-fat, high-carbohydrate diet. Pro-NASH stimuli increased Suv39h2 expression in cell culture, in mice, and in human livers. In hepatocytes, Suv39h2 bound to the Sirt1 gene promoter and repressed Sirt1 transcription. Suv39h2 deficiency normalized Sirt1 expression, allowing nuclear factor kappa B/p65 to become hypoacetylated and thus dampening nuclear factor kappa B-dependent transcription of proinflammatory mediators. In macrophages, Suv39h2-mediated repression of peroxisome proliferator-activated receptor gamma transcription favored a proinflammatory M1 phenotype over an anti-inflammatory M2 phenotype, thereby elevating hepatic inflammation. CONCLUSION: Suv39h2 plays a pivotal role in the regulation of inflammatory response in hepatocytes and macrophages, contributing to NASH pathogenesis. (Hepatology 2017;65:1904-1919).


Subject(s)
Diet, High-Fat , Histone-Lysine N-Methyltransferase/metabolism , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Sirtuin 1/metabolism , Analysis of Variance , Animals , Biomarkers/metabolism , Biopsy, Needle , Blotting, Western , Carcinoma, Hepatocellular/parasitology , Carcinoma, Hepatocellular/physiopathology , Cells, Cultured , Disease Models, Animal , Disease Progression , Flow Cytometry , Hepatocytes/metabolism , Histone Methyltransferases , Humans , Immunohistochemistry , Liver Neoplasms/pathology , Liver Neoplasms/physiopathology , Male , Mice , Mice, Inbred C57BL , Random Allocation , Real-Time Polymerase Chain Reaction/methods , Reference Values
15.
Int J Biochem Cell Biol ; 71: 35-43, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26693892

ABSTRACT

Fibrosis following injury is a common adaptive response in the liver, which can lead to irreparable and life-threatening cirrhosis and hepatocellular carcinoma without effectual intervention. The molecular mechanisms underlying fibrogenic response in the liver remains poorly understood. Here we report that mice with deficiency in myocardin-related transcription factor A (MRTF-A) showed resistance to thioacetamide (TAA)-induced liver fibrosis with significantly reduced expression of pro-fibrogenic genes when compared to wild type littermates. Over-expression of MRTF-A enhanced whereas depletion of MRTF-A alleviated pro-fibrogenic transcription induced by TGF-ß, a major pro-fibrogenic factor in hepatic stellate cells (HSCs). Mechanistically, MRTF-A silencing in HSCs impacted the chromatin structure by reducing the deposition of methylated histone H3K4 on the promoters of pro-fibrogenic genes. Further analyses revealed that MRTF-A interacted with and recruited several key epigenetic factors involved in H3K4 methylation, including ASH2, WDR5, and SET1, to the promoters of pro-fibrogenic genes in response to TGF-ß treatment. Over-expression of ASH2, WDR5, or SET1 enhanced the transactivation of pro-fibrogenic gene promoters by TGF-ß in an MRTF-A-dependent manner. In conclusion, MRTF-A regulates liver fibrosis by epigenetically tuning the TGF-ß signaling pathway in HSCs.


Subject(s)
Epigenesis, Genetic , Hepatic Stellate Cells/cytology , Hepatic Stellate Cells/metabolism , Signal Transduction , Trans-Activators/metabolism , Transforming Growth Factor beta/metabolism , Animals , Cell Line , Epigenesis, Genetic/drug effects , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/pathology , Histones/chemistry , Histones/metabolism , Liver Cirrhosis/genetics , Lysine/metabolism , Male , Methylation/drug effects , Mice , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/genetics , Rats , Signal Transduction/drug effects , Transcriptional Activation/drug effects , Transforming Growth Factor beta/pharmacology
16.
Biochim Biophys Acta ; 1849(9): 1219-28, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26241940

ABSTRACT

Transforming growth factor (TGF-ß) induced activation of portal fibroblast cells serves as a primary cause for liver fibrosis following cholestatic injury. The underlying epigenetic mechanism is not clear. We studied the role of a transcriptional modulator, megakaryoblastic leukemia 1 (MKL1) in this process. We report here that MKL1 deficiency ameliorated BDL-induced liver fibrosis in mice as assessed by histological stainings and expression levels of pro-fibrogenic genes. MKL1 silencing by small interfering RNA (siRNA) abrogated TGF-ß induced transactivation of pro-fibrogenic genes in portal fibroblast cells. TGF-ß stimulated the binding of MKL1 on the promoters of pro-fibrogenic genes and promoted the interaction between MKL1 and SMAD3. While SMAD3 was necessary for MKL1 occupancy on the gene promoters, MKL1 depletion impaired SMAD3 binding reciprocally. TGF-ß treatment induced the accumulation of trimethylated histone H3K4 on the gene promoters by recruiting a methyltransferase complex. Knockdown of individual members of this complex significantly weakened the binding of SMAD3 and down-regulated the activation of portal fibroblast cells. In conclusion, we have identified an epigenetic pathway that dictates TGF-ß induced pro-fibrogenic transcription in portal fibroblast thereby providing novel insights for the development of therapeutic solutions to treat liver fibrosis.


Subject(s)
Epigenesis, Genetic , Liver Cirrhosis/physiopathology , MAP Kinase Kinase Kinases/physiology , Transforming Growth Factor beta/physiology , Animals , Bile Ducts/surgery , Cells, Cultured , MAP Kinase Kinase Kinases/metabolism , Male , Mice , Mice, Knockout , Protein Binding , Rats , Smad3 Protein/metabolism
17.
J Hepatol ; 62(1): 165-74, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25109772

ABSTRACT

BACKGROUND & AIMS: Activation of hepatic stellate cells (HSCs) represents a key process in liver injury and, in the absence of intervention, leads to irreversible cirrhosis contributing significantly to the mortality of patients with liver disease. A missing link in the current understanding of HSC activation is the involvement of the epigenetic machinery. We investigated the role of the myocardin related transcription factor A (MRTF-A) in HSC activation. METHODS: Liver fibrosis was induced in wild type (WT) and MRTF-A deficient (KO) mice by CCl4 injection. Expression of mRNA and protein was measured by real-time PCR, Western blotting, and immunohistochemistry. Protein binding to DNA was assayed by chromatin immunoprecipitation (ChIP). Knockdown of endogenous proteins was mediated by either small interfering RNA (siRNA) or short hairpin RNA (shRNA), carried by lentiviral particles. RESULTS: KO mice exhibited resistance to CCl4-induced liver fibrosis compared to WT littermates. The expression of activated HSC signature genes was suppressed in the absence of MRTF-A. ChIP assays revealed that MRTF-A deficiency led to the erasure of key histone modifications, associated with transcriptional activation, such as H3K4 di- and tri-methylation, on the promoter regions of fibrogenic genes. Mechanistically, MRTF-A recruited a histone methyltransferase complex (COMPASS) to the promoters of fibrogenic genes to activate transcription. Silencing of individual COMPASS components dampened transactivation of fibrogenic genes in vitro and blocked liver fibrosis in mice. Oestradiol suppressed HSC activation by dampening the expression and binding activity of COMPASS. CONCLUSIONS: Our data illustrate a novel mechanism that connects MRTF-A dependent histone H3K4 methylation to HSC activation.


Subject(s)
Gene Expression Regulation , Hepatic Stellate Cells/metabolism , Liver Cirrhosis, Experimental/genetics , Nuclear Proteins/genetics , RNA, Messenger/genetics , Trans-Activators/genetics , Animals , Blotting, Western , Cells, Cultured , Hepatic Stellate Cells/pathology , Immunohistochemistry , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/pathology , Mice , Mice, Knockout , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Real-Time Polymerase Chain Reaction , Trans-Activators/metabolism , Transcriptional Activation
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