RESUMO
BMP-1/tolloid-like proteinases (BTPs) are major players in tissue morphogenesis, growth and repair. They act by promoting the deposition of structural extracellular matrix proteins and by controlling the activity of matricellular proteins and TGF-ß superfamily growth factors. They have also been implicated in several pathological conditions such as fibrosis, cancer, metabolic disorders and bone diseases. Despite this broad range of pathophysiological functions, the putative existence of a specific endogenous inhibitor capable of controlling their activities could never be confirmed. Here, we show that procollagen C-proteinase enhancer-2 (PCPE-2), a protein previously reported to bind fibrillar collagens and to promote their BTP-dependent maturation, is primarily a potent and specific inhibitor of BTPs which can counteract their proteolytic activities through direct binding. PCPE-2 therefore differs from the cognate PCPE-1 protein and extends the possibilities to fine-tune BTP activities, both in physiological conditions and in therapeutic settings.
Assuntos
Glicoproteínas , Peptídeo Hidrolases , Humanos , Glicoproteínas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Morfogênese , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
Low birthweight and reduced height gain during infancy (stunting) may arise at least in part from adverse early life environments that trigger epigenetic reprogramming that may favor survival. We examined differential DNA methylation patterns using targeted methyl sequencing of regions regulating gene activity in groups of rural Gambian infants: (a) low and high birthweight (DNA from cord blood (n = 16 and n = 20, respectively), from placental trophoblast tissue (n = 21 and n = 20, respectively), and DNA from peripheral blood collected from infants at 12 months of age (n = 23 and n = 17, respectively)), and, (b) the top 10% showing rapid postnatal length gain (high, n = 20) and the bottom 10% showing slow postnatal length gain (low, n = 20) based on z score change between birth and 12 months of age (LAZ) (DNA from peripheral blood collected from infants at 12 months of age). Using BiSeq analysis to identify significant methylation marks, for birthweight, four differentially methylated regions (DMRs) were identified in trophoblast DNA, compared to 68 DMRs in cord blood DNA, and 54 DMRs in 12-month peripheral blood DNA. Twenty-five DMRs were observed to be associated with high and low length for age (LAZ) at 12 months. With the exception of five loci (associated with two different genes), there was no overlap between these groups of methylation marks. Of the 194 CpG methylation marks contained within DMRs, 106 were located to defined gene regulatory elements (promoters, CTCF-binding sites, transcription factor-binding sites, and enhancers), 58 to gene bodies (introns or exons), and 30 to intergenic DNA. Distinct methylation patterns associated with birthweight between comparison groups were observed in DNA collected at birth (at the end of intrauterine growth window) compared to those established by 12 months (near the infancy/childhood growth transition). The longitudinal differences in methylation patterns may arise from methylation adjustments, changes in cellular composition of blood or both that continue during the critical postnatal growth period, and in response to early nutritional and infectious environmental exposures with impacts on growth and longer-term health outcomes.
RESUMO
Microbial keratitis (MK) is a major cause of blindness worldwide. Despite adequate antimicrobial treatment, tissue damage can ensue. We compared the human corneal transcriptional profile in late stage MK to normal corneal tissue to identify pathways involved in pathogenesis. Total RNA from MK tissue and normal cadaver corneas was used to determine transcriptome profiles with Illumina HumanHT-12 v4 beadchips. We performed differential expression and network analysis of genes in bacterial keratitis (BK) and fungal keratitis (FK) compared with control (C) samples. Results were validated by RTqPCR for 45 genes in an independent series of 183 MK patients. For the microarray transcriptome analysis, 27 samples were used: 12 controls, 7 BK culture positive for Streptococcus pneumoniae (n = 6), Pseudomonas aeruginosa (n = 1), and 8 FK, culture positive for Fusarium sp. (n = 5), Aspergillus sp. (n = 2), or Lasiodiplodia sp. (n = 1). There were 185 unique differentially expressed genes in BK, 50 in FK, and 339 common to both [i.e., genes with fold-change (FC) < -4 or ≥4 and false discovery rate (FDR) adjusted P < 0.05]. MMP9 had the highest FC in BK (91 FC, adj p = 3.64 E-12) and FK (FC 64, adj. p = 6.10 E-11), along with other MMPs (MMP1, MMP7, MMP10, MMP12), pro-inflammatory cytokines (IL1B, TNF), and PRRs (TLR2, TLR4). HIF1A and its induced genes were upregulated uniquely in BK. Immune/defense response and extracellular matrix terms were the most enriched Gene Ontology terms in both BK and FK. In the network analysis, chemokines were prominent for FK, and actin filament reorganization for BK. Microarray and RTqPCR results were highly correlated for the same samples tested with both assays, and with the larger RTqPCR series. In conclusion, we found a great deal of overlap in the gene expression profile of late stage BK and FK, however genes unique to fungal infection highlighted a corneal epithelial wound healing response and for bacterial infection the prominence of HIF1A-induced genes. These sets of genes may provide new targets for future research into therapeutic agents.
Assuntos
Infecções Oculares Bacterianas/imunologia , Infecções Oculares Fúngicas/imunologia , Perfilação da Expressão Gênica/métodos , Ceratite/genética , Ceratite/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Aspergillus/patogenicidade , Análise por Conglomerados , Úlcera da Córnea/genética , Úlcera da Córnea/imunologia , Úlcera da Córnea/microbiologia , Citocinas/metabolismo , Feminino , Fusarium/patogenicidade , Expressão Gênica , Humanos , Ceratite/diagnóstico , Ceratite/microbiologia , Masculino , Metaloproteinase 9 da Matriz , Pessoa de Meia-Idade , Pseudomonas aeruginosa/patogenicidade , Streptococcus pneumoniae/patogenicidade , TranscriptomaRESUMO
RATIONALE: Improving the early detection and chemoprevention of lung cancer are key to improving outcomes. The pathobiology of early squamous lung cancer is poorly understood. We have shown that amplification of sex-determining region Y-box 2 (SOX2) is an early and consistent event in the pathogenesis of this disease, but its functional oncogenic potential remains uncertain. We tested the impact of deregulated SOX2 expression in a novel organotypic system that recreates the molecular and microenvironmental context in which squamous carcinogenesis occurs. OBJECTIVES: (1) To develop an in vitro model of bronchial dysplasia that recapitulates key molecular and phenotypic characteristics of the human disease; (2) to test the hypothesis that SOX2 deregulation is a key early event in the pathogenesis of bronchial dysplasia; and (3) to use the model for studies on pathogenesis and chemoprevention. METHODS: We engineered the inducible activation of oncogenes in immortalized bronchial epithelial cells. We used three-dimensional tissue culture to build an organotypic model of bronchial dysplasia. MEASUREMENTS AND MAIN RESULTS: We recapitulated human bronchial dysplasia in vitro. SOX2 deregulation drives dysplasia, and loss of tumor promoter 53 is a cooperating genetic event that potentiates the dysplastic phenotype. Deregulated SOX2 alters critical genes implicated in hallmarks of cancer progression. Targeted inhibition of AKT prevents the initiation of the dysplastic phenotype. CONCLUSIONS: In the appropriate genetic and microenvironmental context, acute deregulation of SOX2 drives bronchial dysplasia. This confirms its oncogenic potential in human cells and affords novel insights into the impact of SOX2 deregulation. This model can be used to test therapeutic agents aimed at chemoprevention.
Assuntos
Displasia Broncopulmonar/genética , Displasia Broncopulmonar/fisiopatologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/fisiopatologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatologia , Fatores de Transcrição SOXB1/genética , Técnicas de Cultura de Células , Humanos , Modelos BiológicosRESUMO
BACKGROUND: The Ashanti Dwarf Pig (ADP) of Ghana is an endangered pig breed with hardy and disease resistant traits. Characterisation of animal genetic resources provides relevant data for their conservation and sustainable use for food security and economic development. We investigated the origin and phylogenetic status of the local ADP of Ghana and their crosses with modern commercial breeds based on mtDNA, MC1R, Y-chromosome sequence polymorphisms, and genome-wide SNP genotyping. RESULTS: The study involved 164 local pigs sampled from the three agro-ecological zones of Ghana. Analyses of the mitochondrial D-loop region and Y-chromosome sequences revealed both European and Asian genetic signatures, with differences between the geographical zones. Black coat colour is the most predominant within the breed, with black MC1R alleles of both Asian and European origin. European alleles for spotting are present at a low frequency in the sample set, and may account for the occurrence of spotted piglets in some APD litters. PCA analysis of SNP data revealed a strong location and breed effect on clustering of local Ghanaian pigs. On a global level, Ghanaian local pigs cluster closely with European pigs of commercial origin, but we identified intervals via FST analyses that may elucidate loci for ADP specific traits. CONCLUSIONS: The presence of both European and Asian contributions, with differences between geographical zones probably reflects trading and colonial influences. Understanding the effects of admixture on important adaptive and economic traits of the ADP and other local breeds in Africa is critical for developing sustainable conservation programmes to prevent the decline of these genetic resources.
Assuntos
Suínos/genética , Animais , Técnicas de Genotipagem , Gana , Haplótipos/genética , Mitocôndrias/genética , Filogenia , Pigmentação/genética , Cromossomo Y/genéticaRESUMO
AIMS/HYPOTHESIS: Ageing is a major risk factor for development of metabolic diseases such as type 2 diabetes. Identification of the mechanisms underlying this association could help to elucidate the relationship between age-associated progressive loss of metabolic health and development of type 2 diabetes. We aimed to determine molecular signatures during ageing in the endocrine pancreas. METHODS: Global gene transcription was measured in pancreatic islets isolated from young and old rats by Ilumina BeadChip arrays. Promoter DNA methylation was measured by Sequenom MassArray in 46 genes that showed differential expression with age, and correlations with expression were established. Alterations in morphological and cellular processes with age were determined by immunohistochemical methods. RESULTS: Age-related changes in gene expression were found at 623 loci (>1.5-fold, false discovery rate [FDR] <5%), with a significant (FDR < 0.05) enrichment in genes previously implicated in islet-cell function (Enpp1, Abcc8), type 2 diabetes (Tspan8, Kcnq1), inflammatory processes (Cxcl9, Il33) and extracellular matrix organisation (Col3a1, Dpt). Age-associated transcriptional differences negatively correlated with promoter DNA methylation at several loci related to inflammation, glucose homeostasis, cell proliferation and cell-matrix interactions (Il33, Cxcl9, Gpr119, Fbp2, Col3a1, Dpt, Spp1). CONCLUSIONS/INTERPRETATION: Our findings suggest that a significant proportion of pancreatic islets develop a low-grade 'chronic' inflammatory status with ageing and this may trigger altered functional plasticity. Furthermore, we identified changes in expression of genes previously linked to type 2 diabetes and associated changes in DNA methylation that could explain their age-associated dysregulation. These findings provide new insights into key (epi)genetic signatures of the ageing process in islets.
Assuntos
Envelhecimento/fisiologia , Diabetes Mellitus Tipo 2/etiologia , Inflamação/genética , Ilhotas Pancreáticas/metabolismo , Envelhecimento/genética , Animais , Quimiocina CXCL9/genética , Colágeno Tipo III/genética , Metilação de DNA/genética , Diabetes Mellitus Tipo 2/metabolismo , Epigênese Genética/genética , Inflamação/metabolismo , Canal de Potássio KCNQ1/genética , Masculino , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Ratos , Receptores de Sulfonilureias/genética , Tetraspaninas/genéticaRESUMO
We describe a five generation family with dominantly inherited lymphedema, but no distichiasis, in which 3/3 affected offspring in the fifth generation have died of fetal hydrops and related birth defects. Mutational analysis disclosed a novel mutation in FOXC2 (R121C) in affected members. We searched for possible genetic influences on the greater severity of lymphedema (hydrops) in the fifth generation. Karyotypes disclosed an extra band in Xp in one affected fetus, but this was also found in the mother. Copy number variation (CNV) studies on four members of the pedigree (mother of the three severely affected fetuses/infants; one severely affected; a full, and a half, unaffected sibs) did not detect the source of the Xp band or a possible influence on the severe phenotype. However, use of SNP arrays did allow identification of the portion of the maternal proximal Xp shared by a hydrops-affected daughter and son which was not shared by an unaffected daughter from the same sibship.
Assuntos
Edema/diagnóstico , Edema/genética , Fatores de Transcrição Forkhead/genética , Linfedema/diagnóstico , Linfedema/genética , Mutação , Fenótipo , Variações do Número de Cópias de DNA , Feminino , Estudos de Associação Genética , Homozigoto , Humanos , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único , Índice de Gravidade de DoençaRESUMO
Size at birth, postnatal weight gain, and adult risk for type 2 diabetes may reflect environmental exposures during developmental plasticity and may be mediated by epigenetics. Both low birth weight (BW), as a marker of fetal growth restraint, and high birth weight (BW), especially after gestational diabetes mellitus (GDM), have been linked to increased risk of adult type 2 diabetes. We assessed DNA methylation patterns using a bead chip in cord blood samples from infants of mothers with GDM (group 1) and infants with prenatal growth restraint indicated by rapid postnatal catch-up growth (group 2), compared with infants with normal postnatal growth (group 3). Seventy-five CpG loci were differentially methylated in groups 1 and 2 compared with the controls (group 3), representing 72 genes, many relevant to growth and diabetes. In replication studies using similar methodology, many of these differentially methylated regions were associated with levels of maternal glucose exposure below that defined by GDM [the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study] or were identified as changes observed after randomized periconceptional nutritional supplementation in a Gambian cohort characterized by maternal deprivation. These studies provide support for the concept that similar epigenetic modifications may underpin different prenatal exposures and potentially increase long-term risk for diseases such as type 2 diabetes.
Assuntos
Peso ao Nascer/fisiologia , Metilação de DNA/genética , Diabetes Mellitus Tipo 2/etiologia , Diabetes Gestacional/etiologia , Desenvolvimento Fetal/fisiologia , Aumento de Peso/fisiologia , Adulto , Glicemia/metabolismo , Feminino , Humanos , Hiperglicemia/etiologia , Masculino , Gravidez , Risco , Adulto JovemRESUMO
BACKGROUND: Grade IV glioblastomas exist in two forms, primary (de novo) glioblastomas (pGBM) that arise without precursor lesions, and the less common secondary glioblastomas (sGBM) which develop from earlier lower grade lesions. Genetic heterogeneity between pGBM and sGBM has been documented as have differences in the methylation of individual genes. A hypermethylator phenotype in grade IV GBMs is now well documented however there has been little comparison between global methylation profiles of pGBM and sGBM samples or of methylation profiles between paired early and late sGBM samples. METHODS: We performed genome-wide methylation profiling of 20 matched pairs of early and late gliomas using the Infinium HumanMethylation450 BeadChips to assess methylation at >485,000 cytosine positions within the human genome. RESULTS: Clustering of our data demonstrated a frequent hypermethylator phenotype that associated with IDH1 mutation in sGBM tumors. In 80% of cases, the hypermethylator status was retained in both the early and late tumor of the same patient, indicating limited alterations to genome-wide methylation during progression and that the CIMP phenotype is an early event. Analysis of hypermethylated loci identified 218 genes frequently methylated across grade II, III and IV tumors indicating a possible role in sGBM tumorigenesis. Comparison of our sGBM data with TCGA pGBM data indicate that IDH1 mutated GBM samples have very similar hypermethylator phenotypes, however the methylation profiles of the majority of samples with WT IDH1 that do not demonstrate a hypermethylator phenotype cluster separately from sGBM samples, indicating underlying differences in methylation profiles. We also identified 180 genes that were methylated only in sGBM. Further analysis of these genes may lead to a better understanding of the pathology of sGBM vs pGBM. CONCLUSION: This is the first study to have documented genome-wide methylation changes within paired early/late astrocytic gliomas on such a large CpG probe set, revealing a number of genes that maybe relevant to secondary gliomagenesis.
Assuntos
Metilação de DNA , Glioblastoma/genética , Glioblastoma/patologia , Ilhas de CpG , Progressão da Doença , Genoma Humano , Humanos , Isocitrato Desidrogenase/genética , FenótipoRESUMO
BACKGROUND: Obesity, excess fat tissue in the body, can underlie a variety of medical complaints including heart disease, stroke and cancer. The pig is an excellent model organism for the study of various human disorders, including obesity, as well as being the foremost agricultural species. In order to identify genetic variants associated with fatness, we used a selective genomic approach sampling DNA from animals at the extreme ends of the fat and lean spectrum using estimated breeding values derived from a total population size of over 70,000 animals. DNA from 3 breeds (Sire Line Large White, Duroc and a white Pietrain composite line (Titan)) was used to interrogate the Illumina Porcine SNP60 Genotyping Beadchip in order to identify significant associations in terms of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs). RESULTS: By sampling animals at each end of the fat/lean EBV (estimate breeding value) spectrum the whole population could be assessed using less than 300 animals, without losing statistical power. Indeed, several significant SNPs (at the 5% genome wide significance level) were discovered, 4 of these linked to genes with ontologies that had previously been correlated with fatness (NTS, FABP6, SST and NR3C2). Quantitative analysis of the data identified putative CNV regions containing genes whose ontology suggested fatness related functions (MCHR1, PPARα, SLC5A1 and SLC5A4). CONCLUSIONS: Selective genotyping of EBVs at either end of the phenotypic spectrum proved to be a cost effective means of identifying SNPs and CNVs associated with fatness and with estimated major effects in a large population of animals.
Assuntos
Tecido Adiposo , Variações do Número de Cópias de DNA/genética , Estudo de Associação Genômica Ampla , Obesidade/genética , Animais , Cruzamento , Genótipo , Humanos , Obesidade/patologia , Polimorfismo de Nucleotídeo Único , Suínos/genéticaRESUMO
Reduced trophoblast invasion and vascular conversion in decidua are thought to be the primary defect of common pregnancy disorders including preeclampsia and fetal growth restriction. Genetic studies suggest these conditions are linked to combinations of polymorphic killer cell Ig-like receptor (KIR) genes expressed by maternal decidual NK cells (dNK) and HLA-C genes expressed by fetal trophoblast. Inhibitory KIR2DL1 and activating KIR2DS1 both bind HLA-C2, but confer increased risk or protection from pregnancy disorders, respectively. The mechanisms underlying these genetic associations with opposing outcomes are unknown. We show that KIR2DS1 is highly expressed in dNK, stimulating strong activation of KIR2DS1+ dNK. We used microarrays to identify additional responses triggered by binding of KIR2DS1 or KIR2DL1 to HLA-C2 and found different responses in dNK coexpressing KIR2DS1 with KIR2DL1 compared with dNK only expressing KIR2DL1. Activation of KIR2DS1+ dNK by HLA-C2 stimulated production of soluble products including GM-CSF, detected by intracellular FACS and ELISA. We demonstrated that GM-CSF enhanced migration of primary trophoblast and JEG-3 trophoblast cells in vitro. These findings provide a molecular mechanism explaining how recognition of HLA class I molecules on fetal trophoblast by an activating KIR on maternal dNK may be beneficial for placentation.
Assuntos
Decídua/citologia , Células Matadoras Naturais/metabolismo , Placentação , Receptores KIR/fisiologia , Movimento Celular , Células Cultivadas , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Humanos , Fenótipo , Gravidez , Receptores KIR2DL1/metabolismo , Transcrição Gênica , Transcriptoma , Útero/citologiaRESUMO
Oncostatin M receptor (OSMR) is commonly over-expressed in advanced cervical squamous cell carcinoma (SCC), producing a significantly worse clinical outcome. Cervical SCC cells that over-express OSMR show enhanced responsiveness to the major ligand OSM, which induces multiple pro-malignant effects, including increased cell migration and invasiveness. Here, we show that tissue transglutaminase (TGM2) is an important mediator of the ligand-dependent phenotypic effects of OSMR over-expression in SCC cells. TGM2 expression correlated with disease progression and with OSMR levels in clinical samples of cervical and oral SCC. TGM2 depletion in cervical SCC cells abrogated OSM-induced migration on fibronectin-coated surfaces and invasiveness through extracellular matrix, while ectopic expression of TGM2 increased cell motility and invasiveness. Confocal microscopy and co-immunoprecipitation assays showed that TGM2 interacted with integrin-α5ß1 in the presence of fibronectin in cervical SCC cells, with OSM treatment strengthening the interaction. Importantly, integrin-α5ß1 and fibronectin were also over-expressed in cervical and oral SCC, where levels correlated with those of OSMR and TGM2. This combined tissue and in vitro study demonstrates for the first time that stimulation of over-expressed OSMR in cervical SCC cells activates TGM2/integrin-α5ß1 interactions and induces pro-malignant changes. We conclude that an OSMR/TGM2/integrin-α5ß1/fibronectin pathway is of biological significance in cervical SCC and a candidate for therapeutic targeting.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores de Oncostatina M/metabolismo , Transdução de Sinais/fisiologia , Transglutaminases/metabolismo , Neoplasias do Colo do Útero/metabolismo , Western Blotting , Carcinoma de Células Escamosas/patologia , Feminino , Fibronectinas/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunoprecipitação , Integrina alfa5beta1/metabolismo , Microscopia Confocal , Invasividade Neoplásica/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteína 2 Glutamina gama-Glutamiltransferase , Reação em Cadeia da Polimerase em Tempo Real , Análise Serial de Tecidos , Regulação para Cima , Neoplasias do Colo do Útero/patologiaRESUMO
In addition to the genetic constitution inherited by an organism, the developmental trajectory and resulting mature phenotype are also determined by mechanisms acting during critical windows in early life that influence and establish stable patterns of gene expression. This is the crux of the developmental origins of health and disease hypothesis that suggests undernutrition during gestation and infancy predisposes to ill health in later life. The hypothesis that periconceptional maternal micronutrient supplementation might affect fetal genome-wide methylation within gene promoters was explored in cord blood samples from offspring of Gambian women enrolled into a unique randomized, double blind controlled trial. Significant changes in the epigenome in cord blood DNA samples were further explored in a subset of offspring at 9 months. Gender-specific changes related to periconceptional nutritional supplementation were identified in cord blood DNA samples, some of which showed persistent changes in infant blood DNA samples. Significant effects of periconceptional micronutrient supplementation were also observed in postnatal samples which were not evident in cord blood. In this Gambian population, the increased death rate of individuals born in nutritionally poor seasons has been related to infection and it is of interest that we identified differential methylation at genes associated with defence against infection and immune response. Although the sample size was relatively small, these pilot data suggest that periconceptional nutrition in humans is an important determinant of newborn whole genome methylation patterns but may also influence postnatal developmental patterns of gene promoter methylation linking early with disease risk.
Assuntos
Suplementos Nutricionais , Epigênese Genética , Fertilização/genética , Micronutrientes/administração & dosagem , Adolescente , Adulto , Ilhas de CpG , Metilação de DNA , Método Duplo-Cego , Feminino , Sangue Fetal/metabolismo , Gâmbia , Impressão Genômica , Humanos , Lactente , Recém-Nascido , Masculino , Desnutrição/complicações , Desnutrição/dietoterapia , Desnutrição/genética , Fenômenos Fisiológicos da Nutrição Materna/genética , Pessoa de Meia-Idade , Gravidez , Complicações na Gravidez/dietoterapia , Complicações na Gravidez/genética , Efeitos Tardios da Exposição Pré-Natal/genética , Regiões Promotoras Genéticas , Caracteres Sexuais , Adulto JovemRESUMO
Oncostatin M receptor (OSMR) shows frequent copy number gain and overexpression in advanced cervical squamous cell carcinoma (SCC). We used cell-based in vitro assays, RNA interference, and integrative gene expression profiling to investigate the functional significance of this observation. CaSki and SW756 were selected as representative cervical SCC cells that overexpressed OSMR, and ME180 and MS751 as cells that did not. The STAT-dependent pro-angiogenic factors VEGF-A and ID1 were rapidly induced by OSM in CaSki/SW756 but not in ME180/MS751. However, rapid induction did occur in MS751 following forced OSMR overexpression, while depleting OSMR in CaSki abrogated VEGF-A expression. Conditioned medium from both CaSki and SW756 stimulated endothelial tube formation in vitro, effects that were inhibited by depleting OSMR in the SCC cells. For both CaSki and SW756, migration in a wound healing assay and invasion through Matrigel were stimulated by OSM and consistently inhibited by OSMR depletion. The phenotype was rescued by transfection with OSMR containing a silent mutation that provided specific siRNA resistance. Overall, there was a positive correlation between OSMR levels and invasiveness. We used gene expression profiling to identify genes induced by OSM in CaSki/SW756 but not in ME180/MS751. The most prominent gene ontology category groups for the differentially expressed genes were cell motility/invasion, angiogenesis, signal transduction, and apoptosis. We also profiled 23 cervical SCC samples, identifying genes that were differentially expressed in cases with OSMR overexpression versus those without. Integration of the datasets identified 15 genes that showed consistent differential expression in association with OSMR levels in vitro and in vivo. We conclude that OSMR overexpression in cervical SCC cells provides increased sensitivity to OSM, which induces pro-malignant changes. OSMR is a potential prognostic and therapeutic target in cervical SCC. The genes that mediate OSM:OSMR effects will be valuable indicators of the effectiveness of antibody blockade in pre-clinical systems.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neovascularização Patológica/metabolismo , Subunidade beta de Receptor de Oncostatina M/biossíntese , Neoplasias do Colo do Útero/metabolismo , Antineoplásicos/farmacologia , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oncostatina M/farmacologia , Subunidade beta de Receptor de Oncostatina M/genética , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/irrigação sanguínea , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The molecular basis of CNS myelin regeneration (remyelination) is poorly understood. We generated a comprehensive transcriptional profile of the separate stages of spontaneous remyelination that follow focal demyelination in the rat CNS and found that transcripts that encode the retinoid acid receptor RXR-γ were differentially expressed during remyelination. Cells of the oligodendrocyte lineage expressed RXR-γ in rat tissues that were undergoing remyelination and in active and remyelinated multiple sclerosis lesions. Knockdown of RXR-γ by RNA interference or RXR-specific antagonists severely inhibited oligodendrocyte differentiation in culture. In mice that lacked RXR-γ, adult oligodendrocyte precursor cells efficiently repopulated lesions after demyelination, but showed delayed differentiation into mature oligodendrocytes. Administration of the RXR agonist 9-cis-retinoic acid to demyelinated cerebellar slice cultures and to aged rats after demyelination caused an increase in remyelinated axons. Our results indicate that RXR-γ is a positive regulator of endogenous oligodendrocyte precursor cell differentiation and remyelination and might be a pharmacological target for regenerative therapy in the CNS.
Assuntos
Sistema Nervoso Central/metabolismo , Bainha de Mielina/metabolismo , Regeneração Nervosa/fisiologia , Receptores do Ácido Retinoico/fisiologia , Idoso , Alitretinoína , Animais , Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Diferenciação Celular/fisiologia , Linhagem da Célula/genética , Células Cultivadas , Sistema Nervoso Central/patologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/genética , Regeneração Nervosa/genética , Neurotoxinas , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Oligodendroglia/fisiologia , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/fisiologia , Tretinoína/farmacologia , Receptor gama de Ácido RetinoicoRESUMO
Trachoma is a poorly understood immunofibrogenic disease process, initiated by Chlamydia trachomatis. Differences in conjunctival gene expression profiles between Ethiopians with trachomatous trichiasis (with [TTI] or without [TT] inflammation) and controls (C) were investigated to identify relevant host responses. Tarsal conjunctival swab samples were collected for RNA isolation and C. trachomatis PCR. Transcriptome-wide microarray experiments were conducted on 42 samples (TTI, n = 13; TT, n = 15; C, n =14). Specific results were confirmed by using multiplex quantitative reverse transcription-PCR for 16 mRNA targets in an independent collection of case-control samples: 386 case-control pairs (TTI, n = 244; TT, n = 142; C, n = 386). The gene expression profiles of cases were consistent with squamous metaplasia (keratins, SPRR), proinflammatory cytokine production (IL1ß, CXCL5, and S100A7), and tissue remodeling (MMP7, MMP9, MMP12, and HAS3). There was no difference in the level of IFNγ between cases and controls. However, cases had increased INDO, NOS2A, and IL13RA2 and reduced IL13. C. trachomatis was detected in 1/772. Cases show evidence of ongoing inflammation and tissue remodeling, which were more marked where clinical inflammation was also present. Significantly, these processes appear to be active in the absence of current C. trachomatis infection. There was limited evidence of a T(H)1 response (INDO and NOS2A) and no association between a T(H)2 response and cases. The epithelium appears to be actively involved in late cicatricial stages of trachoma through the production of proinflammatory factors (IL1ß, CXCL5, and S100A7). Longitudinal studies are needed to investigate which etiological factors and pathways are associated with progressive scarring and whether simply controlling chlamydial infection will halt progression in people with established cicatricial disease.
Assuntos
Cicatriz/patologia , Túnica Conjuntiva/metabolismo , Tracoma/complicações , Triquíase/metabolismo , Adulto , Estudos de Casos e Controles , Etiópia/epidemiologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Tracoma/epidemiologia , Tracoma/patologia , Triquíase/epidemiologia , Triquíase/patologiaRESUMO
BACKGROUND: X monosomic mice (39,XO) have a remarkably mild phenotype when compared to women with Turner syndrome (45,XO). The generally accepted hypothesis to explain this discrepancy is that the number of genes on the mouse X chromosome which escape X inactivation, and thus are expressed at higher levels in females, is very small. However this hypothesis has never been tested and only a small number of genes have been assayed for their X-inactivation status in the mouse. We performed a global expression analysis in four somatic tissues (brain, liver, kidney and muscle) of adult 40,XX and 39,XO mice using the Illumina Mouse WG-6 v1_1 Expression BeadChip and an extensive validation by quantitative real time PCR, in order to identify which genes are expressed from both X chromosomes. RESULTS: We identified several genes on the X chromosome which are overexpressed in XX females, including those previously reported as escaping X inactivation, as well as new candidates. However, the results obtained by microarray and qPCR were not fully concordant, illustrating the difficulty in ascertaining modest fold changes, such as those expected for genes escaping X inactivation. Remarkably, considerable variation was observed between tissues, suggesting that inactivation patterns may be tissue-dependent. Our analysis also exposed several autosomal genes involved in mitochondrial metabolism and in protein translation which are differentially expressed between XX and XO mice, revealing secondary transcriptional changes to the alteration in X chromosome dosage. CONCLUSIONS: Our results support the prediction that the mouse inactive X chromosome is largely silent, while providing a list of the genes potentially escaping X inactivation in rodents. Although the lower expression of X-linked genes in XO mice may not be relevant in the particular tissues/systems which are affected in human X chromosome monosomy, genes deregulated in XO mice are good candidates for further study in an involvement in Turner Syndrome phenotype.