RESUMEN
Chronic liver inflammation (CLI) is a risk factor for development of hepatocellular carcinoma (HCC). Galectin-1 (Gal1) is involved in the regulation of inflammation, angiogenesis, and tumorigenesis, exhibiting multiple anti-inflammatory and protumorigenic activities. We aimed to explore its regulatory role in CLI and HCC progression using an established model of CLI-mediated HCC development, Abcb4 [multidrug-resistance 2 (Mdr2)]-knockout (KO) mice, which express high levels of Gal1 in the liver. We generated double-KO (dKO) Gal1-KO/Mdr2-KO mice on C57BL/6 and FVB/N genetic backgrounds and compared HCC development in the generated strains with their parental Mdr2-KO strains. Loss of Gal1 increased liver injury, inflammation, fibrosis, and ductular reaction in dKO mice of both strains starting from an early age. Aged dKO mutants displayed earlier hepatocarcinogenesis and increased tumor size compared with control Mdr2-KO mice. We found that osteopontin, a well-known modulator of HCC development, and oncogenic proteins Ntrk2 (TrkB) and S100A4 were overexpressed in dKO compared with Mdr2-KO livers. Our results demonstrate that in Mdr2-KO mice, a model of CLI-mediated HCC, Gal1-mediated protection from hepatitis, liver fibrosis, and HCC initiation dominates over its known procarcinogenic activities at later stages of HCC development. These findings suggest that anti-Gal1 treatments may not be applicable at all stages of CLI-mediated HCC.-Potikha, T., Pappo, O., Mizrahi, L., Olam, D., Maller, S. M., Rabinovich, G. A., Galun, E., Goldenberg, D. S. Lack of galectin-1 exacerbates chronic hepatitis, liver fibrosis, and carcinogenesis in murine hepatocellular carcinoma model.
Asunto(s)
Galectina 1/fisiología , Hepatitis/metabolismo , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Proteínas de Neoplasias/metabolismo , Lesiones Precancerosas/metabolismo , Empalme Alternativo , Animales , División Celular , Enfermedad Crónica , Cocarcinogénesis , Femenino , Galectina 1/deficiencia , Galectina 1/genética , Células Hep G2 , Hepatitis/genética , Hepatitis/patología , Hepatocitos/patología , Humanos , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Neoplasias Hepáticas Experimentales/etiología , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Proteínas de Neoplasias/genética , Osteopontina/biosíntesis , Osteopontina/deficiencia , Osteopontina/genética , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/genética , Organismos Libres de Patógenos EspecíficosRESUMEN
Galectin-1 (Gal1) is a known immune/inflammatory regulator which acts both extracellularly and intracellularly, modulating innate and adaptive immune responses. Here, we explored the role of Gal1 in liver regeneration using 70% partial hepatectomy (PHx) of C57BL/6 wild type and Gal1-knockout (Gal1-KO, Lgals1-/-) mice. Gene or protein expression, in liver samples collected at time intervals from 2 to 168 hours post-operation, was tested by either RT-PCR or by immunoblotting and immunohistochemistry, respectively. We demonstrated that Gal1 transcript and protein expression was induced in the liver tissue of wild type mice upon PHx. Liver regeneration following PHx was significantly delayed in the Gal1-KO compared to the control liver. This delay was accompanied by a decreased Akt phosphorylation, and accumulation of the hepatocyte nuclear p21 protein in the Gal1-KO versus control livers at 24 and 48 hours following PHx. Transcripts of several known regulators of inflammation, cell cycle and cell signaling, including some known PHx-induced genes, were aberrantly expressed (mainly down-regulated) in Gal1-KO compared to control livers at 2, 6 and 24 hours post-PHx. Transient steatosis, which is imperative for liver regeneration following PHx, was significantly delayed and decreased in the Gal1-KO compared to the control liver and was accompanied by a significantly decreased expression in the mutant liver of several genes encoding lipid metabolism regulators. Our results demonstrate that Gal1 protein is essential for efficient liver regeneration following PHx through the regulation of liver inflammation, hepatic cell proliferation, and the control of lipid storage in the regenerating liver.
Asunto(s)
Proliferación Celular , Galectina 1/metabolismo , Hepatectomía , Regeneración Hepática , Hígado/cirugía , Animales , Ciclo Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Hígado Graso/genética , Hígado Graso/metabolismo , Hígado Graso/patología , Galectina 1/deficiencia , Galectina 1/genética , Genotipo , Hepatitis/genética , Hepatitis/metabolismo , Hepatitis/patología , Hígado/metabolismo , Hígado/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
UNLABELLED: Chronic inflammation is strongly associated with an increased risk for hepatocellular carcinoma (HCC) development. The multidrug resistance 2 (Mdr2)-knockout (KO) mouse (adenosine triphosphate-binding cassette b4(-/-) ), a model of inflammation-mediated HCC, develops chronic cholestatic hepatitis at an early age and HCC at an adult age. To delineate factors contributing to hepatocarcinogenesis, we compared the severity of early chronic hepatitis and late HCC development in two Mdr2-KO strains: Friend virus B-type/N (FVB) and C57 black 6 (B6). We demonstrated that hepatocarcinogenesis was significantly less efficient in the Mdr2-KO/B6 mice versus the Mdr2-KO/FVB mice; this difference was more prominent in males. Chronic hepatitis in the Mdr2-KO/B6 males was more severe at 1 month of age but was less severe at 3 months of age in comparison with age-matched Mdr2-KO/FVB males. A comparative genome-scale gene expression analysis of male livers of both strains at 3 months of age revealed both common and strain-specific aberrantly expressed genes, including genes associated with the regulation of inflammation, the response to oxidative stress, and lipid metabolism. One of these regulators, galectin-1 (Gal-1), possesses both anti-inflammatory and protumorigenic activities. To study its regulatory role in the liver, we transferred the Gal-1-KO mutation (lectin galactoside-binding soluble 1(-/-) ) from the B6 strain to the FVB strain, and we demonstrated that endogenous Gal-1 protected the liver against concanavalin A-induced hepatitis with the B6 genetic background but not the FVB genetic background. CONCLUSION: Decreased chronic hepatitis in Mdr2-KO/B6 mice at the age of 3 months correlated with a significant retardation of liver tumor development in this strain versus the Mdr2-KO/FVB strain. We found candidate factors that may determine strain-specific differences in the course of chronic hepatitis and HCC development in the Mdr2-KO model, including inefficient anti-inflammatory activity of the endogenous lectin Gal-1 in the FVB strain.
Asunto(s)
Carcinoma Hepatocelular/patología , Transformación Celular Neoplásica/patología , Galectina 1/fisiología , Hepatitis Crónica/patología , Neoplasias Hepáticas/patología , Ratones Endogámicos/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Carcinoma Hepatocelular/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Concanavalina A , Hepatitis Crónica/complicaciones , Hepatitis Crónica/etiología , Hígado/metabolismo , Neoplasias Hepáticas/etiología , Masculino , Metionina Adenosiltransferasa/biosíntesis , Ratones , Ratones Noqueados , Miembro 4 de la Subfamilia B de Casete de Unión a ATPAsunto(s)
Proteínas de la Matriz Extracelular/genética , Mutación del Sistema de Lectura , Proteinosis Lipoidea de Urbach y Wiethe/genética , Eliminación de Secuencia , Adulto , Sustitución de Aminoácidos , Biopsia , Codón de Terminación , Análisis Mutacional de ADN , Exones , Predisposición Genética a la Enfermedad , Homocigoto , Humanos , Proteinosis Lipoidea de Urbach y Wiethe/patología , Masculino , Persona de Mediana Edad , Fenotipo , Piel/patologíaRESUMEN
BACKGROUND: Mutations in lipase H (LIPH) are a rare cause of autosomal recessive hypotrichosis (HT) simplex. OBJECTIVE: In this study, we investigated the clinical and molecular basis of HT simplex with woolly hair in 3 nonrelated families. METHODS: Three families of Jewish, Arab Muslim, and Italian origin that presented with HT with woolly hair were studied. The phenotype was confirmed by clinical, microscopic, and histologic examination. Polymorphic microsatellite genotyping and direct automated DNA sequencing of the LIPH gene were used to identify the mutations in our probands. RESULTS: All patients had woolly hair since birth. At presentation, scalp hair density was reduced or normal. Sequencing of the LIPH gene revealed two homozygous mutations: a large recurrent 90-base pair duplication mutation in exon 2 in the Jewish and Arab families, and a novel deletion/insertion mutation in exon 4 in the Italian family. LIMITATIONS: Only 3 families were studied. CONCLUSION: Mutations in LIPH result in variable degrees of HT. Woolly hair is an essential component of the clinical spectrum. A hot spot in the LIPH gene may be c.280_369dup in exon 2.
Asunto(s)
Folículo Piloso/patología , Cabello/patología , Hipotricosis/genética , Hipotricosis/patología , Lipasa/genética , Adolescente , Árabes/genética , Secuencia de Bases , Niño , Preescolar , Exones/genética , Femenino , Duplicación de Gen , Genes Recesivos , Humanos , Italia , Judíos/genética , Masculino , Datos de Secuencia Molecular , Mutación , Linaje , Fenotipo , Adulto JovenRESUMEN
BACKGROUND: The hepatitis C virus (HCV) is one of the major risk factors for the development of hepatocellular carcinoma (HCC). Nevertheless, transgenic mice which express the whole HCV polyprotein (HCV-Tg) do not develop HCC. Whereas chronic HCV infection causes inflammation in patients, in HCV-Tg mice, the host immune reaction against viral proteins is lacking. We aimed to test the role of HCV proteins in HCC development on the background of chronic inflammation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We crossed HCV-Tg mice that do not develop HCC with the Mdr2-knockout (Mdr2-KO) mice which develop inflammation-associated HCC, to generate Mdr2-KO/HCV-Tg mice. We studied the effect of the HCV transgene on tumor incidence, hepatocyte mitosis and apoptosis, and investigated the potential contributing factors for the generated phenotype by gene expression and protein analyses. The Mdr2-KO/HCV-Tg females from the N2 generation of this breeding (having 75% of the FVB/N genome and 25% of the C57BL/6 genome) produced significantly larger tumors in comparison with Mdr2-KO mice. In parallel, the Mdr2-KO/HCV-Tg females had an enhanced inflammatory gene expression signature. However, in the N7 generation (having 99.2% of the FVB/N genome and 0.8% of the C57BL/6 genome) there was no difference in tumor development between Mdr2-KO/HCV-Tg and Mdr2-KO animals of both sexes. The HCV transgene was similarly expressed in the livers of Mdr2-KO/HCV-Tg females of both generations, as revealed by detection of the HCV transcript and the core protein. CONCLUSION: These findings suggest that the HCV transgene accelerated inflammation-associated hepatocarcinogenesis in a host genetic background-dependent manner.
Asunto(s)
Hepacivirus/fisiología , Neoplasias Hepáticas Experimentales/virología , Animales , Secuencia de Bases , Cartilla de ADN , Femenino , Perfilación de la Expresión Génica , Hepacivirus/genética , Inmunohistoquímica , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransgenesRESUMEN
Lipoid proteinosis (LP) (OMIM 247100) is a rare, autosomal recessive disorder. Recent studies have shown that LP is the result of reduced expression of the extracellular matrix protein gene (ECM-1), in which loss-of-function mutations have been described. In the present report, we describe a large consanguineous family with LP. We identified a homozygous splice-site mutation in intron 1 (IVS1 + 1G-->C) in three clinically affected patients. This is the first splice-site mutation reported in LP and is the most 5' of all ECM-1 mutations described thus far. It is predicted to result in the removal of the translation initiation site, thus ablating all three known ECM-1 isoforms (ECM-1a, ECM-1b, and ECM-1c). In addition, we found a novel splicing variant that is not associated with the disease (DQ010946) and results in the generation of a short, prematurely terminating transcript. This case further emphasizes the role of ECM-1 in LP and highlights the unresolved genotype-phenotype correlation in this disease.
Asunto(s)
Consanguinidad , ADN Recombinante , Proteínas de la Matriz Extracelular/genética , Proteinosis Lipoidea de Urbach y Wiethe/genética , Mutación , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Citosina , Femenino , Guanina , Homocigoto , Humanos , Lactante , Intrones , Proteinosis Lipoidea de Urbach y Wiethe/patología , Masculino , Datos de Secuencia Molecular , Linaje , ARNRESUMEN
The cyclin-dependent kinase (CDK) inhibitor p57Kip2 (CDKN1C) is a negative regulator of cell proliferation, binding to a variety of cyclin-CDK complexes and inhibiting their kinase activities in vitro. The p57Kip2 gene is imprinted and the maternal allele is expressed in terminally differentiated cells, including human beta-cells. Somatic loss of p57Kip2 expression is associated with increased beta-cell proliferation in the focal form of Hyperinsulinism of Infancy. We cloned and sequenced the rat ortholog of p57Kip2, and demonstrate that it is highly homologous to the mouse gene. However, the human and rodent genes are quite divergent. Despite having highly homologous C- and N-terminal domains, the mid-portion of the human gene is entirely different from that of its rodent counterparts. Expression of p57Kip2 was evaluated during fetal and postnatal development, and a highly cell-specific, temporal and spatial expression profile was found. In contrast to other tissues, the expression pattern in rat pancreas was entirely opposite from that previously reported in man, with high levels of expression in rodent exocrine cells, but no expression in beta-cells during any stage of development. These findings demonstrate that p57Kip2 expression is highly regulated. In the pancreas, the functional significance of this gene appears to be quite different in humans when compared with rodents, suggesting that a better understanding of the function of this protein may provide new insights into the mechanisms involved in the control of human beta-cell mass.
Asunto(s)
Proteínas Nucleares/genética , Páncreas/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Inhibidor p57 de las Quinasas Dependientes de la Ciclina , Desarrollo Fetal , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/biosíntesis , Especificidad de Órganos , Páncreas/embriología , Ratas , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Long-chain neurotoxins derived from the venom of the Buthidae scorpions, which affect voltage-gated sodium channels (VGSCs) can be subdivided according to their toxicity to insects into insect-selective excitatory and depressant toxins (beta-toxins) and the alpha-like toxins which affect both mammals and insects. In the present study by the aid of reverse-phase HPLC column chromatography, RT-PCR, cloning and various toxicity assays, a new insect selective toxin designated as BjalphaIT was isolated from the venom of the Judean Black Scorpion (Buthotus judaicus), and its full primary sequence was determined: MNYLVVICFALLLMTVVESGRDAYIADNLNCAYTCGSNSYCNTECTKNGAVSGYCQWLGKYGNACWCINLPDKVPIRIPGACR (leader sequence is underlined). Despite its lack of toxicity to mammals and potent toxicity to insects, BjalphaIT reveals an amino acid sequence and an inferred spatial arrangement that is characteristic of the well-known scorpion alpha-toxins highly toxic to mammals. BjalphaITs sharp distinction between insects and mammals was also revealed by its effect on sodium conductance of two cloned neuronal VGSCs heterloguously expressed in Xenopus laevis oocytes and assayed with the two-electrode voltage-clamp technique. BjalphaIT completely inhibits the inactivation process of the insect para/tipE VGSC at a concentration of 100 nM, in contrast to the rat brain Na(v)1.2/beta1 which is resistant to the toxin. The above categorical distinction between mammal and insect VGSCs exhibited by BjalphaIT enables its employment in the clarification of the molecular basis of the animal group specificity of scorpion venom derived neurotoxic polypeptides and voltage-gated sodium channels.
Asunto(s)
Venenos de Escorpión/química , Venenos de Escorpión/aislamiento & purificación , Escorpiones/química , Secuencia de Aminoácidos , Animales , Insectos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Venenos de Escorpión/genética , Escorpiones/genética , Especificidad de la EspecieRESUMEN
Hyperinsulinism of infancy is a genetically heterogeneous disease characterized by dysregulation of insulin secretion resulting in severe hypoglycemia. To date, mutations in five different genes, the sulfonylurea receptor (SUR1, ABCC8), the inward rectifying potassium channel (K(IR)6.2, KCNJ11), glucokinase (GCK), glutamate dehydrogenase (GLUD1), and short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD), have been implicated. Previous reports suggest that, in 40% of patients, no mutation can be identified in any of these genes, suggesting additional locus heterogeneity. However, previous studies did not screen all five genes using direct sequencing, the most sensitive technique available for mutation detection. We selected 15 hyperinsulinism of infancy patients and systematically sequenced the promoter and all coding exons and intron/exon boundaries of ABCC8 and KCNJ11. If no mutation was identified, the coding sequence and intron/exon boundaries of GCK, GLUD1, and SCHAD were sequenced. Seven novel mutations were found in the ABCC8 coding region, one mutation was found in the KCNJ11 coding region, and one novel mutation was found in each of the two promoter regions screened. Functional studies on beta-cells from six patients showed abnormal ATP-sensitive K+ channel function in five of the patients; the sixth had normal channel activity, and no mutations were found. Photolabeling studies using a reconstituted system showed that all missense mutations altered intracellular trafficking. Each of the promoter mutations decreased expression of a reporter gene by about 60% in a heterologous expression system. In four patients (27%), no mutations were identified. Thus, further genetic heterogeneity is suggested in this disorder. These patients represent a cohort that can be used for searching for mutations in other candidate genes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Mapeo Cromosómico , Heterogeneidad Genética , Hiperinsulinismo/genética , Mutación , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio/genética , Receptores de Droga/genética , Adenina , Estudios de Cohortes , Electrofisiología , Guanina , Humanos , Hiperinsulinismo/fisiopatología , Recién Nacido , Islotes Pancreáticos/fisiopatología , Mutación Missense , Linaje , Regiones Promotoras Genéticas/genética , Receptores de SulfonilureasRESUMEN
Hereditary inclusion body myopathy (HIBM) is a neuromuscular disorder, caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, the key enzyme of sialic acid biosynthesis. In Middle Eastern patients a single homozygous mutation occurs, converting methionine-712 to threonine. Recombinant expression of the mutated enzyme revealed slightly reduced N-acetylmannosamine kinase activity, in agreement with the localization of the mutation within the kinase domain. B lymphoblastoid cell lines derived from patients expressing the mutated enzyme also display reduced UDP-N-acetylglucosamine 2-epimerase activity. Nevertheless, no reduced cellular sialylation was found in those cells by colorimetric assays and lectin analysis, indicating that HIBM is not directly caused by an altered overall expression of sialic acids.