RESUMO
Salmonid alphavirus strain 3 is responsible for outbreaks of pancreas disease in salmon and rainbow trout in Norway. Although the extensive amount of research on SAV3 focused mainly on the heart and pancreas (of clinical importance), tropism and pathogenesis studies of the virus in other salmon tissues are limited. Here, we used a combination of RT-qPCR (Q_nsp1 gene) and in situ hybridization (RNAscope®) to demonstrate the tropism of SAV3 in situ in tissues of Atlantic salmon, employing a challenge model (by cohabitation). In addition, as previous results suggested that the pseudobranch may harbor the virus, the change in the expression of different immune genes upon SAV3 infection (RT-qPCR) was focused on the pseudobranch in this study. In situ hybridization detected SAV3 in different tissues of Atlantic salmon during the acute phase of the infection, with the heart ventricle showing the most extensive infection. Furthermore, the detection of the virus in different adipose tissues associated with the internal organs of the salmon suggests a specific affinity of SAV3 to adipocyte components. The inconsistent immune response to SAV3 in the pseudobranch after infection did not mitigate the infection in that tissue and is probably responsible for the persistent low infection at 4 weeks post-challenge. The early detection of SAV3 in the pseudobranch after infection, along with the persistent low infection over the experimental infection course, suggests a pivotal role of the pseudobranch in SAV3 pathogenesis in Atlantic salmon.
Assuntos
Infecções por Alphavirus , Alphavirus , Doenças dos Peixes , Salmo salar , Animais , Alphavirus/genética , CoraçãoRESUMO
Deficiency in thymidine kinase 2 (TK2) causes mitochondrial DNA depletion. Liver mitochondria are severely affected in Tk2 complete knockout models and have been suggested to play a role in the pathogenesis of the Tk2 knockout phenotype, characterized by loss of hypodermal fat tissue, growth retardation and reduced life span. Here we report a liver specific Tk2 knockout (KO) model to further study mechanisms contributing to the phenotypic changes associated with Tk2 deficiency. Interestingly, the liver specific Tk2 KO mice had a normal life span despite a much lower mtDNA level in liver tissue. Mitochondrial DNA encoded peptide COXI did not differ between the Tk2 KO and control mice. However, the relative liver weight was significantly increased in the male Tk2 KO mouse model. Histology analysis indicated an increased lipid accumulation. We conclude that other enzyme activities can partly compensate Tk2 deficiency to maintain mtDNA at a low but stable level throughout the life span of the liver specific Tk2 KO mice. The lower level of mtDNA was sufficient for survival but led to an abnormal lipid accumulation in liver tissue.
Assuntos
DNA Mitocondrial , Timidina Quinase , Camundongos , Masculino , Animais , Timidina Quinase/genética , DNA Mitocondrial/genética , Camundongos Knockout , Fígado , LipídeosRESUMO
Many sialic acid-binding viruses express a receptor-destroying enzyme (RDE) that removes the virus-targeted receptor and limits viral interactions with the host cell surface. Despite a growing appreciation of how the viral RDE promotes viral fitness, little is known about its direct effects on the host. Infectious salmon anemia virus (ISAV) attaches to 4-O-acetylated sialic acids on Atlantic salmon epithelial, endothelial, and red blood cell surfaces. ISAV receptor binding and destruction are effectuated by the same molecule, the haemagglutinin esterase (HE). We recently discovered a global loss of vascular 4-O-acetylated sialic acids in ISAV-infected fish. The loss correlated with the expression of viral proteins, giving rise to the hypothesis that it was mediated by the HE. Here, we report that the ISAV receptor is also progressively lost from circulating erythrocytes in infected fish. Furthermore, salmon erythrocytes exposed to ISAV ex vivo lost their capacity to bind new ISAV particles. The loss of ISAV binding was not associated with receptor saturation. Moreover, upon loss of the ISAV receptor, erythrocyte surfaces became more available to the lectin wheat germ agglutinin, suggesting a potential to alter interactions with endogenous lectins of similar specificity. The pruning of erythrocyte surfaces was inhibited by an antibody that prevented ISAV attachment. Furthermore, recombinant HE, but not an esterase-silenced mutant, was sufficient to induce the observed surface modulation. This links the ISAV-induced erythrocyte modulation to the hydrolytic activity of the HE and shows that the observed effects are not mediated by endogenous esterases. Our findings are the first to directly link a viral RDE to extensive cell surface modulation in infected individuals. This raises the questions of whether other sialic acid-binding viruses that express RDEs affect host cells to a similar extent, and if such RDE-mediated cell surface modulation influences host biological functions with relevance to viral disease.
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Isavirus , Salmo salar , Animais , Isavirus/fisiologia , Ácidos Siálicos , Ácido N-Acetilneuramínico , Esterases , EritrócitosRESUMO
Therapeutic promotion of intestinal regeneration holds great promise, but defining the cellular mechanisms that influence tissue regeneration remains an unmet challenge. To gain insight into the process of mucosal healing, we longitudinally examined the immune cell composition during intestinal damage and regeneration. B cells were the dominant cell type in the healing colon, and single-cell RNA sequencing (scRNA-seq) revealed expansion of an IFN-induced B cell subset during experimental mucosal healing that predominantly located in damaged areas and associated with colitis severity. B cell depletion accelerated recovery upon injury, decreased epithelial ulceration, and enhanced gene expression programs associated with tissue remodeling. scRNA-seq from the epithelial and stromal compartments combined with spatial transcriptomics and multiplex immunostaining showed that B cells decreased interactions between stromal and epithelial cells during mucosal healing. Activated B cells disrupted the epithelial-stromal cross talk required for organoid survival. Thus, B cell expansion during injury impairs epithelial-stromal cell interactions required for mucosal healing, with implications for the treatment of IBD.
Assuntos
Colite , Mucosa Intestinal , Animais , Cicatrização , Células Epiteliais/metabolismo , Epitélio , Modelos Animais de DoençasRESUMO
Thymidine kinase 2 (TK2) deficiency in humans leads to a myopathic form of mitochondrial DNA (mtDNA) deficiency. Here we present a skeletal and cardiac muscle specific TK2 knockout mouse (mTk2 KO). The mice showed dilated hearts and markedly reduced adipose tissue during week 12 to 16. A severe decrease of mtDNA was found only in skeletal muscle and heart tissue in mTk2 KO mice. Expression analysis of key metabolic genes of 16 weeks knockout mice showed significant changes of genes involved in lipid metabolism, with different patterns in heart and skeletal muscle. Our study further suggests that lipoprotein lipase (LPL) from liver supports the metabolism when heart and skeletal muscle were impaired due to mitochondrial dysfunction. The angiotensin-converting enzyme 2 (ACE2), which is involved in glucose homeostasis, was also affected by mtDNA deficiency in our study. Interestingly, both the gene and protein expression of ACE2 were increased in cardiac tissue of mTk2 KO mice. Since ACE2 is a receptor for the SARS-CoV-2 virus, its regulation in relation to mitochondrial function may have important clinical implications.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Transtornos do Metabolismo dos Lipídeos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/genética , COVID-19/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Metabolismo dos Lipídeos , Transtornos do Metabolismo dos Lipídeos/genética , Transtornos do Metabolismo dos Lipídeos/metabolismo , Transtornos do Metabolismo dos Lipídeos/virologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , SARS-CoV-2 , Regulação para CimaRESUMO
We aimed to assess if the discrepant prognostic information between Programmed Death Ligand 1 (PD-L1) protein versus mRNA expression in early breast cancer (BC) could be attributed to heterogeneity in its expression. PD-L1 protein and mRNA expression in BC tissue microarrays from two clinical patient cohorts were evaluated (105 patients; cohort 1: untreated; cohort 2: neoadjuvant chemotherapy-treated). Immunohistochemistry (IHC) with SP142, SP263 was performed. PD-L1 mRNA was evaluated using bulk gene expression and RNA-FISH RNAscope®, the latter scored in a semi-quantitative manner and combined with immunofluorescence (IF) staining for the simultaneous detection of PD-L1 protein expression. PD-L1 expression was assessed in cores as a whole and in two regions of interest (ROI) from the same core. The cell origin of PD-L1 expression was evaluated using multiplex fluorescent IHC. IHC PD-L1 expression between SP142 and SP263 was concordant in 86.7% of cores (p < 0.001). PD-L1 IF/IHC was weakly correlated with spatial mRNA expression (concordance 54.6-71.2%). PD-L1 was mostly expressed by lymphocytes intra-tumorally, while its stromal expression was mostly observed in macrophages. Our results demonstrate only moderate concordance between the various methods of assessing PD-L1 expression at the protein and mRNA levels, which may be attributed to both analytical performance and spatial heterogeneity.
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Hepatic nerves have a complex role in synchronizing liver metabolism. Here, we used three-dimensional (3D) immunoimaging to explore the integrity of the hepatic nervous system in experimental and human nonalcoholic fatty liver disease (NAFLD). We demonstrate parallel signs of mild degeneration and axonal sprouting of sympathetic innervations in early stages of experimental NAFLD and a collapse of sympathetic arborization in steatohepatitis. Human fatty livers display a similar pattern of sympathetic nerve degeneration, correlating with the severity of NAFLD pathology. We show that chronic sympathetic hyperexcitation is a key factor in the axonal degeneration, here genetically phenocopied in mice deficient of the Rac-1 activator Vav3. In experimental steatohepatitis, 3D imaging reveals a severe portal vein contraction, spatially correlated with the extension of the remaining nerves around the portal vein, enlightening a potential intrahepatic neuronal mechanism of portal hypertension. These fundamental alterations in liver innervation and vasculature uncover previously unidentified neuronal components in NAFLD pathomechanisms.
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Deoxyguanosine kinase (DGUOK) deficiency causes mtDNA depletion and mitochondrial dysfunction. We reported long survival of DGUOK knockout (Dguok-/-) mice despite low (<5%) mtDNA content in liver tissue. However, the molecular mechanisms enabling the extended survival remain unknown. Using transcriptomics, proteomics and metabolomics followed by in vitro assays, we aimed to identify the molecular pathways involved in the extended survival of the Dguok-/- mice. At the early stage, the serine synthesis and folate cycle were activated but declined later. Increased activity of the mitochondrial citric acid cycle (TCA cycle) and the urea cycle and degradation of branched chain amino acids were hallmarks of the extended lifespan in DGUOK deficiency. Furthermore, the increased synthesis of TCA cycle intermediates was supported by coordination of two pyruvate kinase genes, PKLR and PKM, indicating a central coordinating role of pyruvate kinases to support the long-term survival in mitochondrial dysfunction.
Assuntos
Adaptação Biológica , Metabolismo Energético , Mitocôndrias/genética , Mitocôndrias/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Aminoácidos/metabolismo , Animais , Sobrevivência Celular/genética , Ciclo do Ácido Cítrico , Ciclo-Oxigenase 1 , DNA Mitocondrial/genética , Metabolismo dos Lipídeos , Fígado/metabolismo , Proteínas de Membrana , Redes e Vias Metabólicas , Camundongos , Camundongos Knockout , Fosforilação OxidativaRESUMO
X-ray fluorescence computed tomography (XFCT) with nanoparticles (NPs) as contrast agents shows potential for molecular biomedical imaging with higher spatial resolution than present methods. To date the technique has been demonstrated on phantoms and mice, however, parameters such as radiation dose, exposure times and sensitivity have not yet allowed for high-spatial-resolution in vivo longitudinal imaging, i.e., imaging of the same animal at different time points. Here we show in vivo XFCT with spatial resolution in the 200- [Formula: see text] range in a proof-of-principle longitudinal study where mice are imaged five times each during an eight-week period following tail-vein injection of NPs. We rely on a 24 keV x-ray pencil-beam-based excitation of in-house-synthesized molybdenum oxide NPs (MoO2) to provide the high signal-to-background x-ray fluorescence detection necessary for XFCT imaging with low radiation dose and short exposure times. We quantify the uptake and clearance of NPs in vivo through imaging, and monitor animal well-being over the course of the study with support from histology and DNA stability analysis to assess the impact of x-ray exposure and NPs on animal welfare. We conclude that the presented imaging arrangement has potential for in vivo longitudinal studies, putting emphasis on designing biocompatible NPs as the future focus for active-targeting preclinical XFCT.
Assuntos
Nanopartículas Metálicas , Nanopartículas , Animais , Fluorescência , Estudos Longitudinais , Camundongos , Molibdênio , Imagens de Fantasmas , Tomografia Computadorizada por Raios X , Raios XRESUMO
Hyperactivated Notch signalling has been implicated in breast cancer, but how elevated levels of Notch signalling contribute to mammary dysplasia and tumorigenesis is not fully understood. In this study, we express an activated form of Notch1 in the mouse mammary luminal lineage and analyse the consequences for tumour formation and the transcriptomic landscape in the luminal lineage. Simultaneous conditional activation of a Notch1 intracellular domain (Notch1 ICD) and EGFP in the luminal lineage was achieved by removal of a stop cassette by CRE-recombinase expression from the whey acidic protein (WAP) promoter. Mice in which Notch1 ICD was activated in the luminal lineage (WAP-CRE;R26-N1ICD mice) exhibit ductal hyperplasia after lactation with an increase in branching frequency and in the number of side-branch ends in the ductal tree. A subset of the mice developed mammary tumours and the majority of the tumour cells expressed EGFP (as a proxy for Notch1 ICD), indicating that the tumours originate from the Notch1 ICD-expressing cells. Single-cell transcriptome analysis of the EGFP-positive mammary cells identified six subtypes of luminal cells. The same six subtypes were found in control mice (WAP-CRE;R26-tdTomato mice expressing the tdTomato reporter from WAP-CRE-mediated activation), but the proportion of cells in the various subtypes differed between the WAP-CRE;R26-N1ICD and control WAP-CRE;R26-tdTomato mice. In conclusion, we show that Notch1 ICD expression in the luminal lineage produces a ductal hyperplasia and branching phenotype accompanied by altered luminal cell subtype partitioning.
Assuntos
Transformação Celular Neoplásica/metabolismo , Células Epiteliais/metabolismo , Hiperplasia/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Regiões Promotoras Genéticas/genética , Animais , Feminino , Glândulas Mamárias Animais/citologia , Neoplasias Mamárias Animais/patologia , Camundongos Transgênicos , Fenobarbital/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Tetradecylthioacetic acid (TTA) is a synthetic fatty acid with a sulfur substitution in the ß-position. This modification renders TTA unable to undergo complete ß-oxidation and increases its biological activity, including activation of peroxisome proliferator activated receptors (PPARs) with preference for PPARα. This study investigated the effects of TTA on lipid and lipoprotein metabolism in the intestine and liver of mice fed a high fat diet (HFD). Mice receiving HFD supplemented with 0.75% (w/w) TTA had significantly lower body weights compared to mice fed the diet without TTA. Plasma triacylglycerol (TAG) was reduced 3-fold with TTA treatment, concurrent with increase in liver TAG. Total cholesterol was unchanged in plasma and liver. However, TTA promoted a shift in the plasma lipoprotein fractions with an increase in larger HDL particles. Histological analysis of the small intestine revealed a reduced size of lipid droplets in enterocytes of TTA treated mice, accompanied by increased mRNA expression of fatty acid transporter genes. Expression of the cholesterol efflux pump Abca1 was induced in the small intestine, but not in the liver. Scd1 displayed markedly increased mRNA and protein expression in the intestine of the TTA group. It is concluded that TTA treatment of HFD fed mice leads to increased expression of genes involved in uptake and transport of fatty acids and HDL cholesterol in the small intestine with concomitant changes in the plasma profile of smaller lipoproteins.
Assuntos
HDL-Colesterol/sangue , Dieta Hiperlipídica/efeitos adversos , Lipoproteínas/metabolismo , PPAR alfa/agonistas , Sulfetos/administração & dosagem , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Sulfetos/farmacologia , Triglicerídeos/sangueRESUMO
Increased expression of GLI1, the main Hedgehog signalling pathway effector, is related to unfavourable prognosis and progressive disease of certain breast cancer subtypes. We used conditional transgenic mice induced to overexpress GLI1 in the mammary epithelium either alone or in combination with deletion of one Trp53 allele to address the role of elevated GLI1 expression in breast tumour initiation and progression. Induced GLI1 expression facilitates mammary gland tumour formation and this was further increased upon heterozygous deletion of Trp53. The GLI1-induced primary tumours were of different murine molecular subtypes, including Normal-likeEx , Class8Ex , Claudin-LowEx and Erbb2-likeEx . The gene expression profiles of some of the tumours correlated well with the PAM50 subtypes for human breast cancer. Whole-exome sequencing revealed somatic mutation profiles with only little overlap between the primary tumours. Orthotopically serially transplanted GLI1-induced tumours maintained the main morphological characteristics of the primary tumours for ≥10 generations. Independent of Trp53 status and molecular subtype, the serially transplanted GLI1-induced tumours were able to grow both in the absence of transgenic GLI1 expression and in the presence of the GLI1 inhibitor GANT61. These data suggest that elevated GLI1 expression has a determinant role in tumour initiation; however, additional genetic events are required for tumour progression.
Assuntos
Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Proteína GLI1 em Dedos de Zinco/genética , Animais , Feminino , Expressão Gênica , Heterogeneidade Genética , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Proteína GLI1 em Dedos de Zinco/biossínteseRESUMO
Overcoming cellular growth restriction, including the evasion of cellular senescence, is a hallmark of cancer. We report that PAK4 is overexpressed in all human breast cancer subtypes and associated with poor patient outcome. In mice, MMTV-PAK4 overexpression promotes spontaneous mammary cancer, while PAK4 gene depletion delays MMTV-PyMT driven tumors. Importantly, PAK4 prevents senescence-like growth arrest in breast cancer cells in vitro, in vivo and ex vivo, but is not needed in non-immortalized cells, while PAK4 overexpression in untransformed human mammary epithelial cells abrogates H-RAS-V12-induced senescence. Mechanistically, a PAK4 - RELB - C/EBPß axis controls the senescence-like growth arrest and a PAK4 phosphorylation residue (RELB-Ser151) is critical for RELB-DNA interaction, transcriptional activity and expression of the senescence regulator C/EBPß. These findings establish PAK4 as a promoter of breast cancer that can overcome oncogene-induced senescence and reveal a selective vulnerability of cancer to PAK4 inhibition.
Assuntos
Neoplasias da Mama/patologia , Fator de Transcrição RelB/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Mama/citologia , Mama/patologia , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Senescência Celular/genética , Células Epiteliais , Feminino , Técnicas de Silenciamento de Genes , Humanos , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Cultura Primária de Células , Prognóstico , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/genéticaRESUMO
BACKGROUND: Claudin-low breast cancer is a molecular subtype associated with poor prognosis and without targeted treatment options. The claudin-low subtype is defined by certain biological characteristics, some of which may be clinically actionable, such as high immunogenicity. In mice, the medroxyprogesterone acetate (MPA) and 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumor model yields a heterogeneous set of tumors, a subset of which display claudin-low features. Neither the genomic characteristics of MPA/DMBA-induced claudin-low tumors nor those of human claudin-low breast tumors have been thoroughly explored. METHODS: The transcriptomic characteristics and subtypes of MPA/DMBA-induced mouse mammary tumors were determined using gene expression microarrays. Somatic mutations and copy number aberrations in MPA/DMBA-induced tumors were identified from whole exome sequencing data. A publicly available dataset was queried to explore the genomic characteristics of human claudin-low breast cancer and to validate findings in the murine tumors. RESULTS: Half of MPA/DMBA-induced tumors showed a claudin-low-like subtype. All tumors carried mutations in known driver genes. While the specific genes carrying mutations varied between tumors, there was a consistent mutational signature with an overweight of T>A transversions in TG dinucleotides. Most tumors carried copy number aberrations with a potential oncogenic driver effect. Overall, several genomic events were observed recurrently; however, none accurately delineated claudin-low-like tumors. Human claudin-low breast cancers carried a distinct set of genomic characteristics, in particular a relatively low burden of mutations and copy number aberrations. The gene expression characteristics of claudin-low-like MPA/DMBA-induced tumors accurately reflected those of human claudin-low tumors, including epithelial-mesenchymal transition phenotype, high level of immune activation, and low degree of differentiation. There was an elevated expression of the immunosuppressive genes PTGS2 (encoding COX-2) and CD274 (encoding PD-L1) in human and murine claudin-low tumors. CONCLUSIONS: Our findings show that the claudin-low breast cancer subtype is not demarcated by specific genomic aberrations, but carries potentially targetable characteristics warranting further research.
Assuntos
Claudinas/metabolismo , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Oncogenes , Transcriptoma , Animais , Biópsia , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , MutaçãoRESUMO
Deoxyguanosine kinase (DGUOK) provides guanosine and adenosine nucleotides for mitochondrial DNA (mtDNA) replication, and its deficiency in humans leads to hepatocerebral mtDNA depletion syndrome or to isolated hepatic disease. There are poor treatment options for DGUOK deficiency and the aim of this study was to generate a model for further studies of the disease that could reveal novel treatment strategies. We report a Dguok-deficient mouse strain that, similar to humans, is most severely affected in the liver. The Dguok complete knockout mice (Dguok-/-) were born normal, but began to lose weight at week 6. A change of fur color from black to blueish grey started at week 16 and was complete at week 20. The movements and behavior were indistinguishable compared to wild-type (wt) mice. A decrease of mtDNA copy number occurred in multiple tissues, with the liver being the most severely affected. The mtDNA-encoded protein cytochrome c oxidase was much lower in Dguok-/- liver tissue than in the wt, whereas the expression of the nuclear-encoded succinate dehydrogenase complex subunit A was unaffected. Histopathology showed severe alterations and immunohistochemistry showed signs of both oxidative stress and regeneration in Dguok-/- liver. The subcutaneous fat layer was undetectable in Dguok-/-, which, in addition to gene expression analysis, indicated an altered lipid metabolism. We conclude that Dguok has a major role for the synthesis of deoxyribonucleotides for mtDNA replication particularly in the liver, similar to the human disorder. Our data also show a catabolic lipid metabolism in liver tissue of Dguok-/-.
Assuntos
DNA Mitocondrial , Metabolismo dos Lipídeos , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Animais , Biomarcadores , Modelos Animais de Doenças , Feminino , Dosagem de Genes , Perfilação da Expressão Gênica , Marcação de Genes , Loci Gênicos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Fenótipo , Gordura Subcutânea/metabolismo , TranscriptomaRESUMO
Obesity triggers the development of non-alcoholic fatty liver disease (NAFLD), which involves alterations of regulatory transcription networks and epigenomes in hepatocytes. Here we demonstrate that G protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor (NCOR) and histone deacetylase 3 (HDAC3) complex, has a central role in these alterations and accelerates the progression of NAFLD towards non-alcoholic steatohepatitis (NASH). Hepatocyte-specific Gps2 knockout in mice alleviates the development of diet-induced steatosis and fibrosis and causes activation of lipid catabolic genes. Integrative cistrome, epigenome and transcriptome analysis identifies the lipid-sensing peroxisome proliferator-activated receptor α (PPARα, NR1C1) as a direct GPS2 target. Liver gene expression data from human patients reveal that Gps2 expression positively correlates with a NASH/fibrosis gene signature. Collectively, our data suggest that the GPS2-PPARα partnership in hepatocytes coordinates the progression of NAFLD in mice and in humans and thus might be of therapeutic interest.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/patologia , PPAR alfa/metabolismo , Animais , Biópsia , Conjuntos de Dados como Assunto , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Progressão da Doença , Epigênese Genética , Fibrose , Células HEK293 , Hepatócitos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , PPAR alfa/genéticaRESUMO
The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeleton in hematopoietic cells and mutated in two severe immunodeficiency diseases with high incidence of cancer. Wiskott-Aldrich syndrome (WAS) is caused by loss-of-function mutations in WASp and most frequently associated with lymphoreticular tumors of poor prognosis. X-linked neuropenia (XLN) is caused by gain-of-function mutations in WASp and associated with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). To understand the role of WASp in tumorigenesis, we bred WASp+, WASp-, and WASp-XLN mice onto tumor susceptible p53+/- background and sub-lethally irradiated them to enhance tumor development. We followed the cohorts for 24 weeks and tumors were characterized by histology and flow cytometry to define the tumor incidence, onset, and cell origin. We found that p53+/-WASp+ mice developed malignancies, including solid tumors and T cell lymphomas with 71.4% of survival 24 weeks after irradiation. p53+/-WASp- mice showed lower survival rate and developed various early onset malignancies. Surprisingly, the p53+/-WASp-XLN mice developed malignancy mostly with late onset, which caused delayed mortality in this colony. This study provides evidence for that loss-of-function and gain-of-function mutations in WASp influence tumor incidence and onset.
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In a large scale screen for skin, hair, and nail abnormalities in null mice generated by The Jackson Laboratory's KOMP center, homozygous mutant Far2tm2b(KOMP)Wtsi/2J (hereafter referrred to as Far2-/-) mice were found to develop focal areas of alopecia as they aged. As sebocytes matured in wildtype C57BL/NJ mice they became pale with fine, uniformly sized clear lipid containing vacuoles that were released when sebocytes disintegrated in the duct. By contrast, the Far2-/- null mice had sebocytes that were similar within the gland but become brightly eosinophilic when the cells entered the sebaceous gland duct. As sebocytes disintegrated, their contents did not readily dissipate. Scattered throughout the dermis, and often at the dermal hypodermal fat junction, were dystrophic hair follicles or ruptured follicles with a foreign body granulomatous reaction surrounding free hair shafts (trichogranuloma). The Meibomian and clitoral glands (modified sebaceous glands) of Far2-/- mice showed ducts dilated to various degrees that were associated with mild changes in the sebocytes as seen in the truncal skin. Skin surface lipidomic analysis revealed a lower level of wax esters, cholesterol esters, ceramides, and diacylglycerols compared to wildtype control mice. Similar changes were described in a number of other mouse mutations that affected the sebaceous glands resulting in primary cicatricial alopecia.
Assuntos
Aldeído Oxirredutases/genética , Alopecia/genética , Cicatriz/genética , Folículo Piloso/patologia , Glândulas Sebáceas/patologia , Alopecia/patologia , Animais , Cicatriz/patologia , Modelos Animais de Doenças , Feminino , Humanos , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glândulas Sebáceas/citologiaRESUMO
BACKGROUND: The WNT pathway regulates intestinal stem cells and is frequently disrupted in intestinal adenomas. The pathway contains several potential biotargets for interference, including the poly-ADP ribosyltransferase enzymes tankyrase1 and 2. LGR5 is a known WNT pathway target gene and marker of intestinal stem cells. The LGR5+ stem cells are located in the crypt base and capable of regenerating all intestinal epithelial cell lineages. RESULTS: We treated Lgr5-EGFP-Ires-CreERT2;R26R-Confetti mice with the tankyrase inhibitor G007-LK for up to 3 weeks to assess the effect on duodenal stem cell homeostasis and on the integrity of intestinal epithelium. At the administered doses, G007-LK treatment inhibited WNT signalling in LGR5+ stem cells and reduced the number and distribution of cells traced from duodenal LGR5+ stem cells. However, the gross morphology of the duodenum remained unaltered and G007-LK-treated mice showed no signs of weight loss or any other visible morphological changes. The inhibitory effect on LGR5+ stem cell proliferation was reversible. CONCLUSION: We show that the tankyrase inhibitor G007-LK is well tolerated by the mice, although proliferation of the LGR5+ intestinal stem cells was inhibited. Our observations suggest the presence of a tankyrase inhibitor-resistant cell population in the duodenum, able to rescue tissue integrity in the presence of G007-LK-mediated inhibition of the WNT signalling dependent LGR5+ intestinal epithelial stem cells.
Assuntos
Proliferação de Células/efeitos dos fármacos , Duodeno/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Sulfonas/farmacologia , Tanquirases/antagonistas & inibidores , Triazóis/farmacologia , Animais , Duodeno/citologia , Imunofluorescência , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Receptores Acoplados a Proteínas G/genética , Sulfonas/farmacocinética , Tanquirases/farmacocinética , Tanquirases/farmacologia , Triazóis/farmacocinéticaRESUMO
Abstract Background The WNT pathway regulates intestinal stem cells and is frequently disrupted in intestinal adenomas. The pathway contains several potential biotargets for interference, including the poly-ADP ribosyltransferase enzymes tankyrase1 and 2. LGR5 is a known WNT pathway target gene and marker of intestinal stem cells. The LGR5+ stem cells are located in the crypt base and capable of regenerating all intestinal epithelial cell lineages. Results We treated Lgr5-EGFP-Ires-CreERT2;R26R-Confetti mice with the tankyrase inhibitor G007-LK for up to 3 weeks to assess the effect on duodenal stem cell homeostasis and on the integrity of intestinal epithelium. At the administered doses, G007-LK treatment inhibited WNT signalling in LGR5+ stem cells and reduced the number and distribution of cells traced from duodenal LGR5+ stem cells. However, the gross morphology of the duodenum remained unaltered and G007-LK-treated mice showed no signs of weight loss or any other visible morphological changes. The inhibitory effect on LGR5+ stem cell proliferation was reversible. Conclusion We show that the tankyrase inhibitor G007-LK is well tolerated by the mice, although proliferation of the LGR5+ intestinal stem cells was inhibited. Our observations suggest the presence of a tankyrase inhibitor-resistant cell population in the duodenum, able to rescue tissue integrity in the presence of G007-LK-mediated inhibition of the WNT signalling dependent LGR5+ intestinal epithelial stem cells.