RESUMO
Ferroptosis is a non-apoptotic form of regulated cell death that is triggered by the discoordination of regulatory redox mechanisms culminating in massive peroxidation of polyunsaturated phospholipids. Ferroptosis inducers have shown considerable effectiveness in killing tumour cells in vitro, yet there has been no obvious success in experimental animal models, with the notable exception of immunodeficient mice1,2. This suggests that the effect of ferroptosis on immune cells remains poorly understood. Pathologically activated neutrophils (PMNs), termed myeloid-derived suppressor cells (PMN-MDSCs), are major negative regulators of anti-tumour immunity3-5. Here we found that PMN-MDSCs in the tumour microenvironment spontaneously die by ferroptosis. Although decreasing the presence of PMN-MDSCs, ferroptosis induces the release of oxygenated lipids and limits the activity of human and mouse T cells. In immunocompetent mice, genetic and pharmacological inhibition of ferroptosis abrogates suppressive activity of PMN-MDSCs, reduces tumour progression and synergizes with immune checkpoint blockade to suppress the tumour growth. By contrast, induction of ferroptosis in immunocompetent mice promotes tumour growth. Thus, ferroptosis is a unique and targetable immunosuppressive mechanism of PMN-MDSCs in the tumour microenvironment that can be pharmacologically modulated to limit tumour progression.
Assuntos
Neoplasias , Humanos , Camundongos , Animais , Microambiente TumoralRESUMO
Chromatin modifiers play critical roles in epidermal development, but the functions of histone deacetylases in this context are poorly understood. The class I HDAC, HDAC3, is of particular interest because it plays divergent roles in different tissues by partnering with tissue-specific transcription factors. We found that HDAC3 is expressed broadly in embryonic epidermis and is required for its orderly stepwise stratification. HDAC3 protein stability in vivo relies on NCoR and SMRT, which function redundantly in epidermal development. However, point mutations in the NCoR and SMRT deacetylase-activating domains, which are required for HDAC3's enzymatic function, permit normal stratification, indicating that HDAC3's roles in this context are largely independent of its histone deacetylase activity. HDAC3-bound sites are significantly enriched for predicted binding motifs for critical epidermal transcription factors including AP1, GRHL, and KLF family members. Our results suggest that among these, HDAC3 operates in conjunction with KLF4 to repress inappropriate expression of Tgm1, Krt16, and Aqp3 In parallel, HDAC3 suppresses expression of inflammatory cytokines through a Rela-dependent mechanism. These data identify HDAC3 as a hub coordinating multiple aspects of epidermal barrier acquisition.
Assuntos
Diferenciação Celular/genética , Células Epidérmicas/citologia , Epiderme/embriologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Animais , Embrião de Mamíferos , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais/genética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.
Assuntos
Adenocarcinoma/terapia , Fatores Imunológicos/imunologia , Imunoterapia , Subpopulações de Linfócitos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Pancreáticas/terapia , Microambiente Tumoral/imunologia , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Linfócitos T CD8-Positivos/imunologia , Epigenômica , Feminino , Perfilação da Expressão Gênica , Humanos , Fatores Imunológicos/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Cultura Primária de Células , Neoplasias PancreáticasRESUMO
Mutations in the retinoblastoma (RB) tumour suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma1-3. Although RB was the first tumour suppressor to be identified, the molecular and cellular basis that underlies selection for persistent RB loss in cancer remains unclear4-6. Methods that reactivate the RB pathway using inhibitors of cyclin-dependent kinases CDK4 and CDK6 are effective in some cancer types and are currently under evaluation for the treatment of lung adenocarcinoma7-9. Whether RB pathway reactivation will have therapeutic effects and whether targeting CDK4 and CDK6 is sufficient to reactivate RB pathway activity in lung cancer remains unknown. Here we model RB loss during lung adenocarcinoma progression and pathway reactivation in established oncogenic KRAS-driven tumours in mice. We show that RB loss enables cancer cells to bypass two distinct barriers during tumour progression. First, RB loss abrogates the requirement for amplification of the MAPK signal during malignant progression. We identify CDK2-dependent phosphorylation of RB as an effector of MAPK signalling and critical mediator of resistance to inhibition of CDK4 and CDK6. Second, RB inactivation deregulates the expression of cell-state-determining factors, facilitates lineage infidelity and accelerates the acquisition of metastatic competency. By contrast, reactivation of RB reprograms advanced tumours towards a less metastatic cell state, but is nevertheless unable to halt cancer cell proliferation and tumour growth due to adaptive rewiring of MAPK pathway signalling, which restores a CDK-dependent suppression of RB. Our study demonstrates the power of reversible gene perturbation approaches to identify molecular mechanisms of tumour progression, causal relationships between genes and the tumour suppressive programs that they control and critical determinants of successful cancer therapy.
Assuntos
Linhagem da Célula , Progressão da Doença , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metástase Neoplásica , Retinoblastoma/metabolismo , Células 3T3 , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Linhagem da Célula/genética , Quinase 2 Dependente de Ciclina/deficiência , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Metástase Neoplásica/genética , Retinoblastoma/genéticaRESUMO
LC3b (Map1lc3b) plays an essential role in canonical autophagy and is one of several components of the autophagy machinery that mediates non-canonical autophagic functions. Phagosomes are often associated with lipidated LC3b to promote phagosome maturation in a process called LC3-associated phagocytosis (LAP). Specialized phagocytes, such as mammary epithelial cells, retinal pigment epithelial (RPE) cells, and sertoli cells, utilize LAP for optimal degradation of phagocytosed material, including debris. In the visual system, LAP is critical to maintain retinal function, lipid homeostasis, and neuroprotection. In a mouse model of retinal lipid steatosis-mice lacking LC3b (LC3b-/-), we observed increased lipid deposition, metabolic dysregulation, and enhanced inflammation. Herein, we present a non-biased approach to determine if loss of LAP mediated processes modulate the expression of various genes related to metabolic homeostasis, lipid handling, and inflammation. A comparison of the RPE transcriptome of WT and LC3b-/- mice revealed 1533 DEGs, with ~73% upregulated and 27% downregulated. Enriched gene ontology (GO) terms included inflammatory response (upregulated DEGs), fatty acid metabolism, and vascular transport (downregulated DEGs). Gene set enrichment analysis (GSEA) identified 34 pathways; 28 were upregulated (dominated by inflammation/related pathways) and 6 were downregulated (dominated by metabolic pathways). Analysis of additional gene families identified significant differences for genes in the solute carrier family, RPE signature genes, and genes with a potential role in age-related macular degeneration. These data indicate that loss of LC3b induces robust changes in the RPE transcriptome contributing to lipid dysregulation and metabolic imbalance, RPE atrophy, inflammation, and disease pathophysiology.
Assuntos
Proteínas Associadas aos Microtúbulos , Transcriptoma , Animais , Masculino , Camundongos , Autofagia/genética , Inflamação/genética , Inflamação/metabolismo , Lipídeos , Proteínas Associadas aos Microtúbulos/metabolismo , Fagocitose/genética , Epitélio Pigmentado da Retina/metabolismoRESUMO
Notch1 is required to generate the earliest embryonic hematopoietic stem cells (HSCs); however since Notch-deficient embryos die early in gestation, additional functions for Notch in embryonic HSC biology have not been described. We used two complementary genetic models to address this important biological question. Unlike Notch1-deficient mice, mice lacking the conserved Notch1 transcriptional activation domain (TAD) show attenuated Notch1 function in vivo and survive until late gestation, succumbing to multiple cardiac abnormalities. Notch1 TAD-deficient HSCs emerge and successfully migrate to the fetal liver but are decreased in frequency by embryonic day 14.5. In addition, TAD-deficient fetal liver HSCs fail to compete with wild-type HSCs in bone marrow transplant experiments. This phenotype is independently recapitulated by conditional knockout of Rbpj, a core Notch pathway component. In vitro analysis of Notch1 TAD-deficient cells shows that the Notch1 TAD is important to properly assemble the Notch1/Rbpj/Maml trimolecular transcription complex. Together, these studies reveal an essential role for the Notch1 TAD in fetal development and identify important cell-autonomous functions for Notch1 signaling in fetal HSC homeostasis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/fisiologia , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Células-Tronco Fetais , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Camundongos , Mutação , Estrutura Terciária de Proteína/genética , Receptor Notch1/genética , Análise de SobrevidaRESUMO
As development proceeds, inductive cues are interpreted by competent tissues in a spatially and temporally restricted manner. While key inductive signaling pathways within competent cells are well-described at a molecular level, the mechanisms by which tissues lose responsiveness to inductive signals are not well understood. Localized activation of Wnt signaling before zygotic gene activation in Xenopus laevis leads to dorsal development, but competence to induce dorsal genes in response to Wnts is lost by the late blastula stage. We hypothesize that loss of competence is mediated by changes in histone modifications leading to a loss of chromatin accessibility at the promoters of Wnt target genes. We use ATAC-seq to evaluate genome-wide changes in chromatin accessibility across several developmental stages. Based on overlap with p300 binding, we identify thousands of putative cis-regulatory elements at the gastrula stage, including sites that lose accessibility by the end of gastrulation and are enriched for pluripotency factor binding motifs. Dorsal Wnt target gene promoters are not accessible after the loss of competence in the early gastrula while genes involved in mesoderm and neural crest development maintain accessibility at their promoters. Inhibition of histone deacetylases increases acetylation at the promoters of dorsal Wnt target genes and extends competence for dorsal gene induction by Wnt signaling. Histone deacetylase inhibition, however, is not sufficient to extend competence for mesoderm or neural crest induction. These data suggest that chromatin state regulates the loss of competence to inductive signals in a context-dependent manner.
Assuntos
Cromatina/metabolismo , Indução Embrionária/genética , Histonas/metabolismo , Acetilação , Animais , Blástula/metabolismo , Cromatina/genética , Gástrula/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/genética , Mesoderma/metabolismo , Crista Neural/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismoRESUMO
Retinoic acid (RA) induces spermatogonial differentiation, but the mechanism by which it operates remains largely unknown. We developed a germ cell culture assay system to study genes involved in spermatogonial differentiation triggered by RA. Stimulated by RA 8 (Stra8), a RA-inducible gene, is indispensable for meiosis initiation, and its deletion results in a complete block of spermatogenesis at the pre-leptotene/zygotene stage. To interrogate the role of Stra8 in RA mediated differentiation of spermatogonia, we derived germ cell cultures from the neonatal testis of both wild type and Stra8 knock-out mice. We provide the first evidence that Stra8 plays a crucial role in modulating the responsiveness of undifferentiated spermatogonia to RA and facilitates transition to a differentiated state. Stra8-mediated differentiation is achieved through the downregulation of a large portfolio of genes and pathways, most notably including genes involved in the spermatogonial stem cell self-renewal process. We also report here for the first time the role of transcription elongation regulator-1 like (Tcerg1l) as a downstream effector of RA-induced spermatogonial differentiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Embrião de Mamíferos/embriologia , Camundongos/genética , Espermatogônias , Fatores de Elongação da Transcrição/genética , Tretinoína/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Masculino , Camundongos/embriologia , Fatores de Elongação da Transcrição/metabolismoRESUMO
BACKGROUND: Fibrosis and stricture are major comorbidities in patients with eosinophilic esophagitis (EoE). Lysyl oxidase (LOX), a collagen cross-linking enzyme, has not been investigated in the context of EoE. OBJECTIVE: We investigated regulation of epithelial LOX expression as a novel biomarker and functional effector of fibrostenotic disease conditions associated with EoE. METHODS: LOX expression was analyzed by using RNA-sequencing, PCR assays, and immunostaining in patients with EoE; cytokine-stimulated esophageal 3-dimensional organoids; and fibroblast-epithelial cell coculture, the latter coupled with fluorescence-activated cell sorting. RESULTS: Gene ontology and pathway analyses linked TNF-α and LOX expression in patients with EoE, which was validated in independent sets of patients with fibrostenotic conditions. TNF-α-mediated epithelial LOX upregulation was recapitulated in 3-dimensional organoids and coculture experiments. We find that fibroblast-derived TNF-α stimulates epithelial LOX expression through activation of nuclear factor κB and TGF-ß-mediated signaling. In patients receiver operating characteristic analyses suggested that LOX upregulation indicates disease complications and fibrostenotic conditions in patients with EoE. CONCLUSIONS: There is a novel positive feedback mechanism in epithelial LOX induction through fibroblast-derived TNF-α secretion. Esophageal epithelial LOX might have a role in the development of fibrosis with substantial translational implications.
Assuntos
Biomarcadores/metabolismo , Esofagite Eosinofílica/genética , Células Epiteliais/fisiologia , Esôfago/patologia , Fibroblastos/fisiologia , Proteína-Lisina 6-Oxidase/genética , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Idoso , Células Cultivadas , Criança , Pré-Escolar , Técnicas de Cocultura , Constrição Patológica , Esofagite Eosinofílica/diagnóstico , Feminino , Fibrose , Ontologia Genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Proteína-Lisina 6-Oxidase/metabolismo , Regulação para Cima , Adulto JovemRESUMO
The origin and nature of age-associated B cells (ABCs) in mice are poorly understood. In this article, we show that their emergence required MHC class II and CD40/CD40L interactions. Young donor B cells were adoptively transferred into congenic recipients and allowed to remain for 1 mo in the absence of external Ag. B cells expressing the T-bet transcription factor, a marker for ABCs, were generated after multiple cell divisions from C57BL/6 donors but not from MHC class II- or CD40-deficient donors. Furthermore, old CD154 (CD40L)-deficient mice did not accrue ABCs, confirming that they arise primarily through T-dependent interactions. To determine what Igs ABCs express, we sequenced VH and Vκ rearranged genes from unimmunized 22-mo-old C57BL/6 mice and showed that they had a heterogeneous repertoire, which was comparable to that seen in old follicular and marginal zone B cell subsets. However, in contrast to the follicular and marginal zone cells, ABCs displayed significant somatic hypermutation. The mutation frequency was lower than found in germinal center cells after deliberate immunization, suggesting that ABCs have undergone mild stimulation from endogenous Ags over time. These observations show that quiescent ABCs are Ag-experienced cells that accumulate during T cell-dependent responses to diverse Ags during the life of an individual.
Assuntos
Envelhecimento/imunologia , Subpopulações de Linfócitos B/imunologia , Anticorpos de Domínio Único/genética , Hipermutação Somática de Imunoglobulina , Animais , Subpopulações de Linfócitos B/metabolismo , Antígenos CD40/deficiência , Antígenos CD40/imunologia , Rearranjo Gênico , Genes MHC da Classe II , Centro Germinativo/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de DNARESUMO
BACKGROUND: Canonical Wnt pathway signaling is necessary for maintaining the proliferative capacity of mammalian intestinal crypt base columnar stem cells (CBCs). Furthermore, dysregulation of the Wnt pathway is a major contributor to disease, including oncogenic transformation of the intestinal epithelium. Given the critical importance of this pathway, numerous tools have been used as proxy measures for Wnt pathway activity, yet the relationship between Wnt target gene expression and reporter allele activity within individual cells at the crypt base remains unclear. RESULTS: Here, we describe a novel Axin2-CreERT2-tdTomato allele that efficiently marks both Wnt(High) CBCs and radioresistant reserve intestinal stem cells. We analyze the molecular and functional identity of Axin2-CreERT2-tdTomato-marked cells using single cell gene expression profiling and tissue regeneration assays and find that Axin2 reporter activity does not necessarily correlate with expression of Wnt target genes and, furthermore, that Wnt target genes themselves vary in their expression patterns at the crypt base. CONCLUSIONS: Wnt target genes and reporter alleles can vary greatly in their cell-type specificity, demonstrating that these proxies cannot be used interchangeably. Furthermore, Axin2-CreERT2-tdTomato is a robust marker of both active and reserve intestinal stem cells and is thus useful for understanding the intestinal stem cell compartment. Developmental Dynamics 245:822-833, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Citometria de Fluxo , Imunofluorescência , Imunoquímica , Mucosa Intestinal/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/fisiologia , Via de Sinalização Wnt/genéticaRESUMO
Dysregulation of iron homeostasis may be a pathogenic factor in age-related macular degeneration (AMD). Meanwhile, the formation of complement-containing deposits under the retinal pigment epithelial (RPE) cell layer is a pathognomonic feature of AMD. In this study, we investigated the molecular mechanisms by which complement component 3 (C3), a central protein in the complement cascade, is up-regulated by iron in RPE cells. Modulation of TGF-ß signaling, involving ERK1/2, SMAD3, and CCAAT/enhancer-binding protein-δ, is responsible for iron-induced C3 expression. The differential effects of spatially distinct SMAD3 phosphorylation sites at the linker region and at the C terminus determined the up-regulation of C3. Pharmacologic inhibition of either ERK1/2 or SMAD3 phosphorylation decreased iron-induced C3 expression levels. Knockdown of SMAD3 blocked the iron-induced up-regulation and nuclear accumulation of CCAAT/enhancer-binding protein-δ, a transcription factor that has been shown previously to bind the basic leucine zipper 1 domain in the C3 promoter. We show herein that mutation of this domain reduced iron-induced C3 promoter activity. In vivo studies support our in vitro finding of iron-induced C3 up-regulation. Mice with a mosaic pattern of RPE-specific iron overload demonstrated co-localization of iron-induced ferritin and C3d deposits. Humans with aceruloplasminemia causing RPE iron overload had increased RPE C3d deposition. The molecular events in the iron-C3 pathway represent therapeutic targets for AMD or other diseases exacerbated by iron-induced local complement dysregulation.
Assuntos
Complemento C3/metabolismo , Degeneração Macular/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Ferro/química , Sobrecarga de Ferro , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Transdução de Sinais , Proteína Smad3/metabolismo , Regulação para CimaRESUMO
Gastric cancers are the most frequent gastric malignancy and usually arise in the sequence of Helicobacter pylori-associated chronic gastritis. CpG methylation is a central mechanism of epigenetic gene regulation affecting cancer-related genes, and occurs early in gastric carcinogenesis. DNA samples from non-metaplastic gastric mucosa with variable levels of gastritis (non-metaplastic mucosa), intestinal metaplasia, or gastric cancer were screened with methylation arrays for CpG methylation of cancer-related genes and 30 gene targets were further characterized by high-definition bisulfite next-generation sequencing. In addition, data from The Cancer Genome Atlas were analyzed for correlation of methylation with gene expression. Overall, 13 genes had significantly increased CpG methylation in gastric cancer vs non-metaplastic mucosa (BRINP1, CDH11, CHFR, EPHA5, EPHA7, FGF2, FLI1, GALR1, HS3ST2, PDGFRA, SEZ6L, SGCE, and SNRPN). Further, most of these genes had corresponding reduced expression levels in gastric cancer compared with intestinal metaplasia, including novel hypermethylated genes in gastric cancer (FLI1, GALR1, SGCE, and SNRPN), suggesting that they may regulate neoplastic transformation from non-malignant intestinal metaplasia to cancer. Our data suggest a tumor-suppressor role for FLI1 in gastric cancer, consistent with recently reported data in breast cancer. For the genes with strongest methylation/expression correlation, namely FLI1, the expression was lowest in microsatellite-unstable tumors compared with other gastric cancer molecular subtypes. Importantly, reduced expression of hypermethylated BRINP1 and SGCE was significantly associated with favorable survival in gastric cancer. In summary, we report novel methylation gene targets that may have functional roles in discrete stages of gastric carcinogenesis and may serve as biomarkers for diagnosis and prognosis of gastric cancer.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Transformação Celular Neoplásica/genética , Ilhas de CpG , Metilação de DNA , Epigênese Genética , Mucosa Gástrica/química , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA/métodos , Neoplasias Gástricas/genética , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Transformação Celular Neoplásica/patologia , Biologia Computacional , Bases de Dados Genéticas , Progressão da Doença , Gastrectomia , Mucosa Gástrica/patologia , Mucosa Gástrica/cirurgia , Gastrite/genética , Gastrite/patologia , Predisposição Genética para Doença , Humanos , Metaplasia , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgiaRESUMO
The neural crest (NC) is a multipotent population of migratory cells unique to the vertebrate embryo, contributing to the development of multiple organ systems. Transcription factors pax3 and zic1 are among the earliest genes activated in NC progenitors, and they are both necessary and sufficient to promote NC fate. In order to further characterize the function of these transcription factors during NC development we have used hormone inducible fusion proteins in a Xenopus animal cap assay, and DNA microarray to identify downstream targets of Pax3 and Zic1. Here we present the results of this screen and the initial validation of these targets using quantitative RT-PCR, in situ hybridization and morpholinos-mediated knockdown. Among the targets identified we found several well-characterized NC-specific genes, including snail2, foxd3, gbx2, twist, sox8 and sox9, which validate our approach. We also obtained several factors with no known function in Xenopus NC, which represent novel regulators of NC fate. The comprehensive characterization of Pax3 and Zic1 targets function in the NC gene regulatory network, are essential to understanding the mechanisms regulating the emergence of this important cell population.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Crista Neural/embriologia , Fatores de Transcrição Box Pareados/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes/fisiologia , Hibridização In Situ , Análise em Microsséries , Morfolinos/genética , Crista Neural/metabolismo , Fator de Transcrição PAX3 , Reação em Cadeia da Polimerase em Tempo Real , Xenopus laevis/genéticaRESUMO
Anaplastic lymphoma kinase (ALK), physiologically expressed only by nervous system cells, displays a remarkable capacity to transform CD4(+) T lymphocytes and other types of nonneural cells. In this study, we report that activity of nucleophosmin (NPM)/ALK chimeric protein, the dominant form of ALK expressed in T cell lymphomas (TCLs), closely resembles cell activation induced by IL-2, the key cytokine supporting growth and survival of normal CD4(+) T lymphocytes. Direct comparison of gene expression by ALK(+) TCL cells treated with an ALK inhibitor and IL-2-dependent ALK(-) TCL cells stimulated with the cytokine revealed a very similar, albeit inverse, gene-regulation pattern. Depending on the analysis method, up to 67% of the affected genes were modulated in common by NPM/ALK and IL-2. Based on the gene expression patterns, Jak/STAT- and IL-2-signaling pathways topped the list of pathways identified as affected by both IL-2 and NPM/ALK. The expression dependence on NPM/ALK and IL-2 of the five selected genes-CD25 (IL-2Rα), Egr-1, Fosl-1, SOCS3, and Irf-4-was confirmed at the protein level. In both ALK(+) TCL and IL-2-stimulated ALK(-) TCL cells, CD25, SOCS3, and Irf-4 genes were activated predominantly by the STAT5 and STAT3 transcription factors, whereas transcription of Egr-1 and Fosl-1 was induced by the MEK-ERK pathway. Finally, we found that Egr-1, a protein not associated previously with either IL-2 or ALK, contributes to the cell proliferation. These findings indicate that NPM/ALK transforms the target CD4(+) T lymphocytes, at least in part, by using the pre-existing, IL-2-dependent signaling pathways.
Assuntos
Linfócitos T CD4-Positivos/patologia , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Interleucina-2/fisiologia , Linfoma de Células T/enzimologia , Proteínas de Fusão Oncogênica/fisiologia , Proteínas Tirosina Quinases/fisiologia , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/metabolismo , Carbazóis/farmacologia , Linhagem Celular Tumoral , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/genética , Ativação Enzimática , Humanos , Linfoma de Células T/genética , Sistema de Sinalização das MAP Quinases , Mimetismo Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Compostos de Fenilureia/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/antagonistas & inibidores , Fator de Transcrição STAT3/fisiologia , Fator de Transcrição STAT5/fisiologia , Transdução de Sinais/genéticaRESUMO
Cystogenesis and tubulogenesis are basic building blocks for many epithelial organs, including the kidney. Most researchers have used two-dimensional (2D) cell culture to investigate signaling pathways downstream of hepatocyte growth factor (HGF). We hypothesize that three-dimensional (3D) collagen-grown Madin-Darby canine kidney (MDCK) cells, which form cysts and then tubulate in response to HGF, are a much more in vivo-like system for the identification of novel tubulogenes. With the use of a canine microarray containing over 20,000 genes, 2,417 genes were identified as potential tubulogenes that were differentially regulated, exclusively in 3D-grown MDCK cells. Among these, 840 were dependent on MAPK signaling. Importantly, this work shows that many putative tubulogenes, previously identified via microarray analysis of 2D cultures, including by us, do not change in 3D culture and vice versa. The use of a 3D-culture system allowed for the identification of novel MAPK-dependent and -independent genes that regulate early renal tubulogenesis in vitro, e.g., matrix metalloproteinase 1 (MMP1). Knockdown of MMP1 led to defects in cystogenesis and tubulogenesis in 3D-grown MDCK cells, most likely due to problems establishing normal polarity. We suggest that data obtained from 2D cultures, even those using MDCK cells treated with HGF, should not be automatically extrapolated to factors important for cystogenesis and tubulogenesis. Instead, 3D culture, which more closely replicates the biological environment and is therefore a more accurate model for identifying tubulogenes, is preferred. Results from the present analysis will be used to build a more accurate model of the signaling pathways that control cystogenesis and tubulogenesis.
Assuntos
Perfilação da Expressão Gênica/métodos , Túbulos Renais/enzimologia , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Cultura de Tecidos , Animais , Polaridade Celular , Cães , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Fator de Crescimento de Hepatócito/metabolismo , Túbulos Renais/crescimento & desenvolvimento , Túbulos Renais/patologia , Células Madin Darby de Rim Canino , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Organogênese , Interferência de RNA , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos TestesRESUMO
Tribbles homolog 2 (Trib2) was identified as a downregulated transcript in leukemic cells undergoing growth arrest. To investigate the effects of Trib2 in hematopoietic progenitors, mice were reconstituted with hematopoietic stem cells retrovirally expressing Trib2. Trib2-transduced bone marrow cells exhibited a growth advantage ex vivo and readily established factor-dependent cell lines. In vivo, Trib2-reconstituted mice uniformly developed fatal transplantable acute myelogenous leukemia (AML). In mechanistic studies, we found that Trib2 associated with and inhibited C/EBPalpha. Furthermore, Trib2 expression was elevated in a subset of human AML patient samples. Together, our data identify Trib2 as an oncogene that induces AML through a mechanism involving inactivation of C/EBPalpha.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Transplante de Medula Óssea , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mieloide Aguda/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Oncogenes , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Taxa de Sobrevida , Quimeras de TransplanteRESUMO
MicroRNAs (miRs) play a key role in the control of gene expression in a wide array of tissue systems, where their functions include the regulation of self-renewal, cellular differentiation, proliferation, and apoptosis. However, the functional importance of individual miRs in controlling spermatogonial stem cell (SSC) homeostasis has not been investigated. Using high-throughput sequencing, we profiled the expression of miRs in the Thy1(+) testis cell population, which is highly enriched for SSCs, and the Thy1(-) cell population, composed primarily of testis somatic cells. In addition, we profiled the global expression of miRs in cultured germ cells, also enriched for SSCs. Our results demonstrate that miR-21, along with miR-34c, -182, -183, and -146a, are preferentially expressed in the Thy1(+) SSC-enriched population, compared with Thy1(-) somatic cells. Importantly, we demonstrate that transient inhibition of miR-21 in SSC-enriched germ cell cultures increased the number of germ cells undergoing apoptosis and significantly reduced the number of donor-derived colonies of spermatogenesis formed from transplanted treated cells in recipient mouse testes, indicating that miR-21 is important in maintaining the SSC population. Moreover, we show that in SSC-enriched germ cell cultures, miR-21 is regulated by the transcription factor ETV5, known to be critical for SSC self-renewal.
Assuntos
Proliferação de Células , MicroRNAs/genética , Espermatogônias/citologia , Células-Tronco/metabolismo , Animais , Apoptose/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Biblioteca Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Espermatogênese/genética , Espermatogônias/metabolismo , Transplante de Células-Tronco/métodos , Testículo/citologia , Testículo/metabolismo , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: Facial asymmetry is a common comorbid condition in patients with jaw deformation malocclusion. Heritability of malocclusion is advancing rapidly, but very little is known regarding genetic contributions to asymmetry. This study identifies differences in expression of key asymmetry-producing genes that are down-regulated in patients with facial asymmetry. METHODS: Masseter muscle samples were collected during bilateral sagittal split osteotomy orthognathic surgery to correct skeletal-based malocclusion. Patients were classified as class II or III and open or deep bite malocclusion with or without facial asymmetry. Muscle samples were analyzed for gene expression differences on Affymetrix HT2.0 microarray global expression chips. RESULTS: Overall gene expression was different for asymmetric patients compared with other malocclusion classifications by principal component analysis (P < 0.05). We identified differences in the nodal signaling pathway, which promotes development of mesoderm and endoderm and left-right patterning during embryogenesis. Nodal and Lefty expression was 1.39- to 1.84-fold greater (P < 3.41 × 10), whereas integral membrane Nodal modulators Nomo1,2,3 were -5.63 to -5.81 (P < 3.05 × 10) less in asymmetry subjects. Fold differences among intracellular pathway members were negative in the range of -7.02 to -2.47 (P < 0.003). Finally Pitx2, an upstream effector of Nodal known to influence the size of type II skeletal muscle fibers was also significantly decreased in facial asymmetry (P < 0.05). CONCLUSIONS: When facial asymmetry is part of skeletal malocclusion, there are decreases in nodal signaling pathway genes in masseter muscle. This data suggest that the nodal signaling pathway is down-regulated to help promote development of asymmetry. Pitx2 expression differences also contributed to both skeletal and muscle development in this condition.
Assuntos
Regulação para Baixo/genética , Assimetria Facial/genética , Proteína Nodal/genética , Transdução de Sinais/genética , Adulto , Assimetria Facial/patologia , Feminino , Proteínas de Homeodomínio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fatores de Determinação Direita-Esquerda/genética , Masculino , Má Oclusão/genética , Má Oclusão/patologia , Músculo Masseter/metabolismo , Músculo Masseter/patologia , Proteínas de Membrana/genética , Fatores de Transcrição/genética , Proteína Homeobox PITX2RESUMO
The world-wide prevalence of myopia (nearsightedness) is increasing, but its pathogenesis is incompletely understood. Among many putative mechanisms, laboratory and clinical findings have implicated circadian biology in the etiology of myopia. Consistent with a circadian hypothesis, we recently reported a marked variability in diurnal patterns of gene expression in two crucial tissues controlling post-natal refractive development - the retina and choroid-at the onset of form-deprivation myopia in chick, a widely studied and validated model. To extend these observations, we assayed gene expression by RNA-Seq in retina and choroid during the progression of established unilateral form-deprivation myopia of chick. We assayed gene expression every 4 hours during a single day from myopic and contralateral control eyes. Retinal and choroidal gene expression in myopic vs. control eyes during myopia progression differed strikingly at discrete times during the day. Very few differentially expressed genes occurred at more than one time in either tissue during progressing myopia. Similarly, Gene Set Enrichment Analysis pathways varied markedly by time during the day. Some of the differentially expressed genes in progressing myopia coincided with candidate genes for human myopia, but only partially corresponded with genes previously identified at myopia onset. Considering other laboratory findings and human genetics and epidemiology, these results further link circadian biology to the pathogenesis of myopia; but they also point to important mechanistic differences between the onset of myopia and the progression of established myopia. Future laboratory and clinical investigations should systematically incorporate circadian mechanisms in studying the etiology of myopia and in seeking more effective treatments to normalize eye growth in children.