RESUMO
DNA methylation is a fundamental epigenetic modification, important across biological processes. The maintenance methyltransferase DNMT1 is essential for lineage differentiation during development, but its functions in tissue homeostasis are incompletely understood. We show that epidermis-specific DNMT1 deletion severely disrupts epidermal structure and homeostasis, initiating a massive innate immune response and infiltration of immune cells. Mechanistically, DNA hypomethylation in keratinocytes triggered transposon derepression, mitotic defects, and formation of micronuclei. DNA release into the cytosol of DNMT1-deficient keratinocytes activated signaling through cGAS and STING, thus triggering inflammation. Our findings show that disruption of a key epigenetic mark directly impacts immune and tissue homeostasis, and potentially impacts our understanding of autoinflammatory diseases and cancer immunotherapy.
Assuntos
Metilação de DNA , Dermatite/genética , Epiderme/fisiopatologia , Nucleotidiltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Aberrações Cromossômicas , Citosol/fisiologia , DNA (Citosina-5-)-Metiltransferase 1/genética , Dermatite/imunologia , Dermatite/patologia , Humanos , Imunidade Inata/genética , Helicase IFIH1 Induzida por Interferon/metabolismo , Queratinócitos/imunologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Transgênicos , Nucleotidiltransferases/genéticaRESUMO
Brown adipose tissue (BAT) is a highly specialized adipose tissue in its immobile location and size during the entire adulthood. In response to cold exposure and other ß3-adrenoreceptor stimuli, BAT commits energy consumption by nonshivering thermogenesis (NST). However, the molecular machinery in controlling the BAT mass in adults is unknown. Here, we show our surprising findings that the BAT mass and functions can be manipulated in adult animals by controlling BAT adipocyte differentiation in vivo. Platelet-derived growth factor receptor α (PDGFα) expressed in BAT progenitor cells served a signaling function to avert adipose progenitor differentiation. Genetic and pharmacological loss-of-function of PDGFRα eliminated the differentiation barrier and permitted progenitor cell differentiation to mature and functional BAT adipocytes. Consequently, an enlarged BAT mass (megaBAT) was created by PDGFRα inhibition owing to increases of brown adipocyte numbers. Under cold exposure, a microRNA-485 (miR-485) was identified as a master suppressor of the PDGFRα signaling, and delivery of miR-485 also produced megaBAT in adult animals. Noticeably, megaBAT markedly improved global metabolism, insulin sensitivity, high-fat-diet (HFD)-induced obesity, and diabetes by enhancing NST. Together, our findings demonstrate that the adult BAT mass can be increased by blocking the previously unprecedented inhibitory signaling for BAT progenitor cell differentiation. Thus, blocking the PDGFRα for the generation of megaBAT provides an attractive strategy for treating obesity and type 2 diabetes mellitus (T2DM).
Assuntos
Adipócitos Marrons , Adipócitos , Adipogenia , Tecido Adiposo Marrom , MicroRNAs , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Adipócitos/citologia , Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Animais , Diabetes Mellitus Tipo 2/terapia , Metabolismo Energético , MicroRNAs/genética , MicroRNAs/metabolismo , Obesidade/terapia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Termogênese/genéticaRESUMO
Vascular pericytes, an important cellular component in the tumor microenvironment, are often associated with tumor vasculatures, and their functions in cancer invasion and metastasis are poorly understood. Here we show that PDGF-BB induces pericyte-fibroblast transition (PFT), which significantly contributes to tumor invasion and metastasis. Gain- and loss-of-function experiments demonstrate that PDGF-BB-PDGFRß signaling promotes PFT both in vitro and in in vivo tumors. Genome-wide expression analysis indicates that PDGF-BB-activated pericytes acquire mesenchymal progenitor features. Pharmacological inhibition and genetic deletion of PDGFRß ablate the PDGF-BB-induced PFT. Genetic tracing of pericytes with two independent mouse strains, TN-AP-CreERT2:R26R-tdTomato and NG2-CreERT2:R26R-tdTomato, shows that PFT cells gain stromal fibroblast and myofibroblast markers in tumors. Importantly, coimplantation of PFT cells with less-invasive tumor cells in mice markedly promotes tumor dissemination and invasion, leading to an increased number of circulating tumor cells and metastasis. Our findings reveal a mechanism of vascular pericytes in PDGF-BB-promoted cancer invasion and metastasis by inducing PFT, and thus targeting PFT may offer a new treatment option of cancer metastasis.
Assuntos
Carcinoma de Células Renais/genética , Pericitos/metabolismo , Proteínas Proto-Oncogênicas c-sis/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Becaplermina , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Camundongos , Camundongos Knockout , Metástase Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Pericitos/patologia , Proteínas Proto-Oncogênicas c-sis/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The histone deacetylases HDAC1 and HDAC2 remove acetyl moieties from lysine residues of histones and other proteins and are important regulators of gene expression. By deleting different combinations of Hdac1 and Hdac2 alleles in the epidermis, we reveal a dosage-dependent effect of HDAC1/HDAC2 activity on epidermal proliferation and differentiation. Conditional ablation of either HDAC1 or HDAC2 in the epidermis leads to no obvious phenotype due to compensation by the upregulated paralogue. Strikingly, deletion of a single Hdac2 allele in HDAC1 knockout mice results in severe epidermal defects, including alopecia, hyperkeratosis, hyperproliferation and spontaneous tumour formation. These mice display impaired Sin3A co-repressor complex function, increased levels of c-Myc protein, p53 expression and apoptosis in hair follicles (HFs) and misregulation of HF bulge stem cells. Surprisingly, ablation of HDAC1 but not HDAC2 in a skin tumour model leads to accelerated tumour development. Our data reveal a crucial function of HDAC1/HDAC2 in the control of lineage specificity and a novel role of HDAC1 as a tumour suppressor in the epidermis.
Assuntos
Epiderme/crescimento & desenvolvimento , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Neoplasias Cutâneas/genética , Alopecia/genética , Animais , Apoptose/genética , Linhagem da Célula , Proteínas Correpressoras , Modelos Animais de Doenças , Epiderme/enzimologia , Epiderme/patologia , Regulação da Expressão Gênica , Genes Supressores de Tumor , Genes p53 , Folículo Piloso/patologia , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Ceratose/genética , Ceratose/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
Mechanisms underlying age-related obesity and insulin resistance are generally unknown. Here, we report age-related adipose vascular changes markedly modulated fat mass, adipocyte functions, blood lipid composition, and insulin sensitivity. Notably, VEGF expression levels in various white adipose tissues (WATs) underwent changes uninterruptedly in different age populations. Anti-VEGF and anti- VEGF receptor 2 treatment in different age populations showed marked variations of vascular regression, with midaged mice exhibiting modest sensitivity. Interestingly, anti-VEGF treatment produced opposing effects on WAT adipocyte sizes in different age populations and affected vascular density and adipocyte sizes in brown adipose tissue. Consistent with changes of vasculatures and adipocyte sizes, anti-VEGF treatment increased insulin sensitivity in young and old mice but had no effects in the midaged group. Surprisingly, anti-VEGF treatment significantly improved insulin sensitivity in midaged obese mice fed a high-fat diet. Our findings demonstrate that adipose vasculatures show differential responses to anti-VEGF treatment in various age populations and have therapeutic implications for treatment of obesity and diabetes with anti-VEGF-based antiangiogenic drugs.
Assuntos
Tecido Adiposo Marrom/irrigação sanguínea , Tecido Adiposo Marrom/fisiopatologia , Tecido Adiposo Branco/irrigação sanguínea , Tecido Adiposo Branco/fisiopatologia , Envelhecimento/patologia , Resistência à Insulina , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adipócitos/patologia , Tecido Adiposo Marrom/patologia , Tecido Adiposo Branco/patologia , Animais , Tamanho Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Lipídeos/sangue , Camundongos Endogâmicos C57BL , Obesidade/sangue , Obesidade/patologia , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Lung cancer is one of the most frequent cancer types and the leading cause of cancer death worldwide. Cisplatin is a widely used chemotherapeutic for non-small cell lung carcinoma (NSCLC), however, its positive effects are diminished under hypoxia. We wanted to determine if co-treatment with cisplatin and histone deacetalyse (HDAC) inhibitor panobinostat can reduce hypoxia-induced cisplatin resistance in NSCLC cells, and to elucidate mechanism involved. METHODS: Expression status of different HDACS was determined in two cell lines and in tumor tissue from 20 patients. Cells were treated with cisplatin, panobinostat, or with combination of both under normoxic and hypoxic (1% O(2)) conditions. Cell cycle, viability, acetylation of histones, and activation of apoptosis were determined. HIF-1α stability and its interaction with HDAC4 were analyzed. RESULTS: Most class I and II HDACs were expressed in NSCLC cells and tumor samples. Co-treatment of tumor cells with cisplatin and panobinostat decreased cell viability and increased apoptosis more efficiently than in primary, non-malignant bronchial epithelial cells. Co-treatment induced apoptosis by causing chromatin fragmentation, activation of caspases-3 and 7 and PARP cleavage. Toxic effects were more pronounced under hypoxic conditions. Co-treatment resulted in destabilization and degradation of HIF-1α and HDAC4, a protein responsible for acetylation and de/stabilization of HIF-1α. Direct interaction between HDAC4 and HIF-1α proteins in H23 cells was detected. CONCLUSIONS: Here we show that hypoxia-induced cisplatin resistance can be overcome by combining cisplatin with panobinostat, a potent HDAC inhibitor. These findings may contribute to the development of a new therapeutic strategy for NSCLC.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ácidos Hidroxâmicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Indóis/farmacologia , Neoplasias Pulmonares/metabolismo , Acetilação , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/genética , Panobinostat , Esferoides Celulares/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The distal hereditary motor neuropathies (dHMNs) are a heterogeneous group of neurodegenerative disorders affecting the lower motoneuron. In a family with both autosomal-dominant dHMN and dHMN type V (dHMN/dHMN-V) present in three generations, we excluded mutations in all genes known to be associated with a dHMN phenotype through Sanger sequencing and defined three potential loci through linkage analysis. Whole-exome sequencing of two affected individuals revealed a single candidate variant within the linking regions, i.e., a splice-site alteration in REEP1 (c.304-2A>G). A minigene assay confirmed complete loss of splice-acceptor functionality and skipping of the in-frame exon 5. The resulting mRNA is predicted to be expressed at normal levels and to encode an internally shortened protein (p.102_139del). Loss-of-function REEP1 mutations have previously been identified in dominant hereditary spastic paraplegia (HSP), a disease associated with upper-motoneuron pathology. Consistent with our clinical-genetic data, we show that REEP1 is strongly expressed in the lower motoneurons as well. Upon exogeneous overexpression in cell lines we observe a subcellular localization defect for p.102_139del that differs from that observed for the known HSP-associated missense mutation c.59C>A (p.Ala20Glu). Moreover, we show that p.102_139del, but not p.Ala20Glu, recruits atlastin-1, i.e., one of the REEP1 binding partners, to the altered sites of localization. These data corroborate the loss-of-function nature of REEP1 mutations in HSP and suggest that a different mechanism applies in REEP1-associated dHMN.
Assuntos
Proteínas de Membrana Transportadoras/genética , Mutação , Doenças do Sistema Nervoso Periférico/genética , Linhagem Celular , Exoma , Feminino , Humanos , Masculino , Linhagem , Análise de Sequência de DNARESUMO
BACKGROUND: Reports on common mutations in neuroendocrine tumors (NET) are rare and clonality of NET metastases has not been investigated in this tumor entity yet. We selected one NET and the corresponding lymph node and liver metastases as well as the derivative cell lines to screen for somatic mutations in the primary NET and to track the fate of genetic changes during metastasis and in vitro progression. RESULTS: Applying microarray based sequence capture resequencing including 4,935 Exons from of 203 cancer-associated genes and high-resolution copy number and genotype analysis identified multiple somatic mutations in the primary NET, affecting BRCA2, CTNNB1, ERCC5, HNF1A, KIT, MLL, RB1, ROS1, SMAD4, and TP53. All mutations were confirmed in the patients' lymph node and liver metastasis tissue as well as early cell line passages. In contrast to the tumor derived cell line, higher passages of the metastases derived cell lines lacked somatic mutations and chromosomal alterations, while expression of the classical NET marker serotonin was maintained. CONCLUSION: Our study reveals that both metastases have evolved from the same pair of genetically differing NET cell clones. In both metastases, the in vivo dominating "mutant" tumor cell clone has undergone negative selection in vitro being replaced by the "non-mutant" tumor cell population. This is the first report of a bi-clonal origin of NET derived metastases, indicating selective advantage of interclonal cooperation during metastasis. In addition, this study underscores the importance to monitor cell line integrity using high-resolution genome analysis tools.
Assuntos
Neoplasias Hepáticas/genética , Metástase Linfática/genética , Tumores Neuroendócrinos/genética , Linhagem Celular Tumoral , Cromossomos/genética , Cromossomos/metabolismo , Variações do Número de Cópias de DNA , Éxons , Genótipo , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Mutação , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Serotonina/genética , Serotonina/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
UNLABELLED: Fibroblast growth factors (FGFs) and their high-affinity receptors [fibroblast growth factor receptors (FGFRs)] contribute to autocrine and paracrine growth stimulation in several non-liver cancer entities. Here we report that at least one member of the FGF8 subfamily (FGF8, FGF17, and FGF18) was up-regulated in 59% of 34 human hepatocellular carcinoma (HCC) samples that we investigated. The levels of the corresponding receptors (FGFR2, FGFR3, and FGFR4) were also elevated in the great majority of the HCC cases. Overall, 82% of the HCC cases showed overexpression of at least one FGF and/or FGFR. The functional implications of the deregulated FGF/FGFR system were investigated by the simulation of an insufficient blood supply. When HCC-1.2, HepG2, or Hep3B cells were subjected to serum withdrawal or the hypoxia-mimetic drug deferoxamine mesylate, the expression of FGF8 subfamily members increased dramatically. In the serum-starved cells, the incidence of apoptosis was elevated, whereas the addition of FGF8, FGF17, or FGF18 impaired apoptosis, which was associated with phosphorylation of extracellular signal-regulated kinase 1/2 and ribosomal protein S6. In contrast, down-modulation of FGF18 by small interfering RNA (siRNA) significantly reduced the viability of the hepatocarcinoma cells. siRNA targeting FGF18 also impaired the cells' potential to form clones at a low cell density or in soft agar. With respect to the tumor microenvironment, FGF17 and FGF18 stimulated the growth of HCC-derived myofibroblasts, and FGF8, FGF17, and FGF18 induced the proliferation and tube formation of hepatic endothelial cells. CONCLUSION: FGF8, FGF17, and FGF18 are involved in autocrine and paracrine signaling in HCC and enhance the survival of tumor cells under stress conditions, malignant behavior, and neoangiogenesis. Thus, the FGF8 subfamily supports the development and progression of hepatocellular malignancy.
Assuntos
Carcinoma Hepatocelular/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Neoplasias Hepáticas/genética , Neovascularização Patológica/genética , Receptores de Fatores de Crescimento de Fibroblastos/biossíntese , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Humanos , Hipóxia/fisiopatologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Ratos , Microambiente Tumoral , Regulação para CimaRESUMO
OBJECTIVE: To test the hypothesis that monogenic neuropathies such as Charcot-Marie-Tooth disease (CMT) contribute to frequent but often unexplained neuropathies in the elderly, we performed genetic analysis of 230 patients with unexplained axonal neuropathies and disease onset ≥35 years. METHODS: We recruited patients, collected clinical data, and conducted whole-exome sequencing (WES; n = 126) and MME single-gene sequencing (n = 104). We further queried WES repositories for MME variants and measured blood levels of the MME-encoded protein neprilysin. RESULTS: In the WES cohort, the overall detection rate for assumed disease-causing variants in genes for CMT or other conditions associated with neuropathies was 18.3% (familial cases 26.4%, apparently sporadic cases 12.3%). MME was most frequently involved and accounted for 34.8% of genetically solved cases. The relevance of MME for late-onset neuropathies was further supported by detection of a comparable proportion of cases in an independent patient sample, preponderance of MME variants among patients compared to population frequencies, retrieval of additional late-onset neuropathy patients with MME variants from WES repositories, and low neprilysin levels in patients' blood samples. Transmission of MME variants was often consistent with an incompletely penetrant autosomal-dominant trait and less frequently with autosomal-recessive inheritance. CONCLUSIONS: A detectable fraction of unexplained late-onset axonal neuropathies is genetically determined, by variants in either CMT genes or genes involved in other conditions that affect the peripheral nerves and can mimic a CMT phenotype. MME variants can act as completely penetrant recessive alleles but also confer dominantly inherited susceptibility to axonal neuropathies in an aging population.
Assuntos
Envelhecimento , Neuropatia Hereditária Motora e Sensorial/genética , Neprilisina/genética , Idade de Início , Idoso , Envelhecimento/sangue , Doença de Charcot-Marie-Tooth/sangue , Doença de Charcot-Marie-Tooth/genética , Feminino , Predisposição Genética para Doença/genética , Neuropatia Hereditária Motora e Sensorial/sangue , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Neprilisina/sangue , Sequenciamento do ExomaRESUMO
Ebola virus (EBOV) infection can cause severe and frequently fatal disease in human patients. The EBOV glycoprotein (GP) mediates viral entry into host cells. For this, GP depends on priming by the pH-dependent endolysosomal cysteine proteases cathepsin B (CatB) and, to a lesser degree, cathepsin L (CatL), at least in most cell culture systems. However, there is limited information on whether and how EBOV-GP can acquire resistance to CatB/L inhibitors. Here, we addressed this question using replication-competent vesicular stomatitis virus bearing EBOV-GP. Five passages of this virus in the presence of the CatB/CatL inhibitor MDL28170 were sufficient to select resistant viral variants and sequencing revealed that all GP sequences contained a V37A mutation, which, in the context of native GP, is located in the base of the GP surface unit. In addition, some GP sequences harbored mutation S195R in the receptor-binding domain. Finally, mutational analysis demonstrated that V37A but not S195R conferred resistance against MDL28170 and other CatB/CatL inhibitors. Collectively, a single amino acid substitution in GP is sufficient to confer resistance against CatB/CatL inhibitors, suggesting that usage of CatB/CatL inhibitors for antiviral therapy may rapidly select for resistant viral variants.
RESUMO
Melanoma is a leading cause of high mortality that frequently spreads to the brain and is associated with deterioration in quality and quantity of life. Treatment opportunities have been restricted until now and new therapy options are urgently required. Our focus was to reveal the potential heterogeneity of melanoma brain metastasis. We succeeded to establish a brain melanoma metastasis cell line, namely MUG-Mel1 and two resulting clones D5 and C8 by morphological variety, differences in lipidome, growth behavior, surface, and stem cell markers. Mutation analysis by next-generation sequencing, copy number profiling, and cytogenetics demonstrated the different genetic profile of MUG-Mel1 and clones. Tumorigenicity was unsuccessfully tested in various mouse systems and finally established in a zebra fish model. As innovative treatment option, with high potential to pass the blood-brain barrier a peptide isolated from lactoferricin was studied in potential toxicity. Brain metastases are a major clinical challenge, therefore the development of relevant in vitro and in vivo models derived from brain melanoma metastases provides valuable information about tumor biology and offers great potential to screen for new innovative therapies.
Assuntos
Neoplasias Encefálicas/secundário , Células Clonais/patologia , Melanoma/patologia , Animais , Neoplasias Encefálicas/ultraestrutura , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Feminino , Dosagem de Genes , Humanos , Concentração Inibidora 50 , Lipídeos/análise , Masculino , Melanoma/ultraestrutura , Camundongos Nus , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Peptídeos/farmacologia , Peixe-ZebraRESUMO
Molecular mechanisms underlying the cancer stroma in metastasis need further exploration. Here, we discovered that cancer-associated fibroblasts (CAFs) produced high levels of IL-33 that acted on tumor-associated macrophages (TAMs), causing them to undergo the M1 to M2 transition. Genomic profiling of metastasis-related genes in the IL-33-stimulated TAMs showed a >200-fold increase of MMP9. Signaling analysis demonstrated the IL-33-ST2-NF-κB-MMP9-laminin pathway that governed tumor stroma-mediated metastasis. In mouse and human fibroblast-rich pancreatic cancers, genetic deletion of IL-33, ST2, or MMP9 markedly blocked metastasis. Pharmacological inhibition of NF-κB and MMP9 also blocked cancer metastasis. Deletion of IL-33, ST2, or MMP9 restored laminin, a key basement membrane component associated with tumor microvessels. Together, our data provide mechanistic insights on the IL-33-NF-κB-MMP9-laminin axis that mediates the CAF-TAM-committed cancer metastasis. Thus, targeting the CAF-TAM-vessel axis provides an outstanding therapeutic opportunity for cancer treatment.
Assuntos
Interleucina-33/metabolismo , Neoplasias/imunologia , Microambiente Tumoral/imunologia , Animais , Fibroblastos Associados a Câncer/imunologia , Fibroblastos Associados a Câncer/metabolismo , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/genética , Interleucina-33/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Camundongos , Camundongos Knockout , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Neoplasias/patologia , Transdução de Sinais/imunologia , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Angiogenesis plays an instrumental role in the modulation of adipose tissue mass and metabolism. Targeting adipose vasculature provides an outstanding opportunity for treatment of obesity and metabolic disorders. Here, we report the physiological functions of VEGFR1 in the modulation of adipose angiogenesis, obesity, and global metabolism. Pharmacological inhibition and genetic deletion of endothelial VEGFR1 augmented adipose angiogenesis and browning of subcutaneous white adipose tissue, leading to elevated thermogenesis. In a diet-induced obesity model, endothelial-VEGFR1 deficiency demonstrated a potent anti-obesity effect by improving global metabolism. Along with metabolic changes, fatty liver and insulin sensitivity were also markedly improved in VEGFR1-deficient high fat diet (HFD)-fed mice. Together, our data indicate that targeting of VEGFR1 provides an exciting new opportunity for treatment of obesity and metabolic diseases, such as liver steatosis and type 2 diabetes.
Assuntos
Tecido Adiposo/irrigação sanguínea , Tecido Adiposo/metabolismo , Endotélio Vascular/metabolismo , Doenças Metabólicas/terapia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/deficiência , Tecido Adiposo Marrom/irrigação sanguínea , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/irrigação sanguínea , Tecido Adiposo Branco/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Doenças Metabólicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/terapia , Termogênese , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Distal hereditary motor neuropathy type V (dHMN-V) and Charcot-Marie-Tooth syndrome (CMT) type 2 presenting with predominant hand involvement, also known as CMT2D and Silver syndrome (SS) are rare phenotypically overlapping diseases which can be caused by mutations in the Berardinelli-Seip Congenital Lipodystrophy 2 (BSCL2) and in the glycyl-tRNA synthetase encoding (GARS) genes. Mutations in the heat-shock proteins HSPB1 and HSPB8 can cause related distal hereditary motor neuropathies (dHMN) and are considered candidates for dHMN-V, CMT2, and SS. DESIGN: To define the frequency and distribution of mutations in the GARS, BSCL2, HSPB1 and HSPB8 genes we screened 33 unrelated sporadic and familial patients diagnosed as either dHMN-V, CMT2D or SS. Exon 3 of the BSCL2 gene was screened in further 69 individuals with an unclassified dHMN phenotype or diagnosed as hereditary spastic paraplegia (HSP) complicated by pure motor neuropathy. RESULTS: Four patients diagnosed with dHMN-V or SS carried known heterozygous BSCL2 mutations (N88S and S90L). In one dHMN-V patient we detected a putative GARS mutation (A57V). No mutations were detected in HSPB1 and HSPB8. The diagnostic yield gained in the series of 33 probands was 12% for BSCL2 mutations and 3% for GARS mutations. In the series of unclassified dHMN and complicated HSP cases no mutations were found. CONCLUSIONS: Our data confirm that most likely only two mutations (N88S, S90L) in exon 3 of BSCL2 may lead to dHMN-V or SS phenotypes. Mutations in GARS, HSPB1 and HSPB8. are not a common cause of dHMN-V, SS and CMT2D. We would therefore suggest that a genetic testing of dHMN-V and SS patients should begin with screening of exon 3 of the BSCL2 gene. Screening of the GARS gene is useful in patients with CMT2 with predominant hand involvement and dHMN-V. The rather low frequencies of BSCL2, GARS, HSPB1 and HSPB8 mutations in dHMN-V, CMT2D and SS patients strongly point to further genetic heterogeneity of these related disorders.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Heterogeneidade Genética , Mãos/fisiopatologia , Neuropatia Hereditária Motora e Sensorial/genética , Proteínas Nucleares/genética , Adolescente , Adulto , Idoso , Análise Mutacional de DNA , Saúde da Família , Feminino , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/genética , Neuropatia Hereditária Motora e Sensorial/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares , Mutação/genética , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Paraplegia Espástica Hereditária/complicações , Paraplegia Espástica Hereditária/genética , SíndromeRESUMO
The Human Major Histocompatibility Complex (MHC) is a highly polymorphic region full of immunoregulatory genes. The MHC codes for the human leukocyte antigens (HLA), proteins that present on the cellular surface and that are involved in self-non-self recognition. For matching donors and recipients for organ and stem-cell transplants it is important to know an individual's HLA haplotype determinable in this region. Now, as next-generation sequencing (NGS) platforms mature and become more and more accepted as a standard method, NGS applications have spread from research laboratories to the clinic, where they provide valid genetic insights. Here, we describe a cost-effective microarray-based sequence capture, enrichment, and NGS sequencing approach to characterize MHC haplotypes. Using this approach, ~4 MB of MHC sequence for four DNA samples (donor, recipient and the parents of the recipient) were sequenced in parallel in one NGS instrument run. We complemented this approach using microarray-based genome-wide SNP analysis. Taken together, the use of recently developed tools and protocols for sequence capture and massively parallel sequencing allows for detailed MHC analysis and donor-recipient matching.
Assuntos
Complexo Principal de Histocompatibilidade/genética , Genótipo , Antígenos HLA/genética , Haplótipos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/métodosRESUMO
Visceral fat is considered the genuine and harmful white adipose tissue (WAT) that is associated to development of metabolic disorders, cardiovascular disease, and cancer. Here, we present a new concept to turn the harmful visceral fat into a beneficial energy consumption depot, which is beneficial for improvement of metabolic dysfunctions in obese mice. We show that low temperature-dependent browning of visceral fat caused decreased adipose weight, total body weight, and body mass index, despite increased food intake. In high-fat diet-fed mice, low temperature exposure improved browning of visceral fat, global metabolism via nonshivering thermogenesis, insulin sensitivity, and hepatic steatosis. Genome-wide expression profiling showed upregulation of WAT browning-related genes including Cidea and Dio2. Conversely, Prdm16 was unchanged in healthy mice or was downregulated in obese mice. Surgical removal of visceral fat and genetic knockdown of UCP1 in epididymal fat largely ablated low temperature-increased global thermogenesis and resulted in the death of most mice. Thus, browning of visceral fat may be a compensatory heating mechanism that could provide a novel therapeutic strategy for treating visceral fat-associated obesity and diabetes.
Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Temperatura Baixa , Metabolismo Energético , Gordura Intra-Abdominal/metabolismo , Termogênese , Adiponectina/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Proteínas Reguladoras de Apoptose/genética , Temperatura Corporal , Peso Corporal , Proteínas de Ligação a DNA/genética , Dieta Hiperlipídica , Ingestão de Alimentos , Fígado Gorduroso , Técnicas de Silenciamento de Genes , Resistência à Insulina , Iodeto Peroxidase/genética , Leptina/metabolismo , Camundongos , Camundongos Obesos , Tamanho do Órgão , Fatores de Transcrição/genética , Proteína Desacopladora 1/genética , Regulação para Cima , Iodotironina Desiodinase Tipo IIRESUMO
Understanding the molecular mechanisms regulating beige adipocyte formation may lead to the development of new therapies to combat obesity. Here, we report a miRNA-based autocrine regulatory pathway that controls differentiation of preadipocytes into beige adipocytes. We identify miR-327 as one of the most downregulated miRNAs targeting growth factors in the stromal-vascular fraction (SVF) under conditions that promote white adipose tissue (WAT) browning in mice. Gain- and loss-of-function experiments reveal that miR-327 targets FGF10 to prevent beige adipocyte differentiation. Pharmacological and physiological ß-adrenergic stimulation upregulates FGF10 levels and promotes preadipocyte differentiation into beige adipocytes. In vivo local delivery of miR-327 to WATs significantly compromises the beige phenotype and thermogenesis. Contrarily, systemic inhibition of miR-327 in mice induces browning and increases whole-body metabolic rate under thermoneutral conditions. Our data provide mechanistic insight into an autocrine regulatory signaling loop that regulates beige adipocyte formation and suggests that the miR-327-FGF10-FGFR2 signaling axis may be a therapeutic targets for treatment of obesity and metabolic diseases.
Assuntos
Tecido Adiposo Bege/fisiologia , Tecido Adiposo Branco/fisiologia , Comunicação Autócrina/genética , Fator 10 de Crescimento de Fibroblastos/genética , MicroRNAs/metabolismo , Adipócitos/fisiologia , Tecido Adiposo Bege/citologia , Tecido Adiposo Bege/efeitos dos fármacos , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Regulação para Baixo , Metabolismo Energético/fisiologia , Feminino , Fator 10 de Crescimento de Fibroblastos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , MicroRNAs/genética , Modelos Animais , Obesidade/tratamento farmacológico , Obesidade/metabolismo , RNA Interferente Pequeno/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The impact of discontinuation of anti-VEGF cancer therapy in promoting cancer metastasis is unknown. Here we show discontinuation of anti-VEGF treatment creates a time-window of profound structural changes of liver sinusoidal vasculatures, exhibiting hyper-permeability and enlarged open-pore sizes of the fenestrated endothelium and loss of VE-cadherin. The drug cessation caused highly leaky hepatic vasculatures permit tumour cell intravasation and extravasation. Discontinuation of an anti-VEGF antibody-based drug and sunitinib markedly promotes liver metastasis. Mechanistically, host hepatocyte, but not tumour cell-derived vascular endothelial growth factor (VEGF), is responsible for cancer metastasis. Deletion of hepatocyte VEGF markedly ablates the 'off-drug'-induced metastasis. These findings provide mechanistic insights on anti-VEGF cessation-induced metastasis and raise a new challenge for uninterrupted and sustained antiangiogenic therapy for treatment of human cancers.