RESUMO
Plasmalemma vesicle-associated protein (Plvap) is an endothelial protein with roles in endothelial diaphragm formation and maintenance of basal vascular permeability. At the same time, Plvap has roles in immunity by facilitating leukocyte diapedesis at inflammatory sites and controlling peripheral lymph node morphogenesis and the entry of soluble Ags into lymph node conduits. Based on its postulated role in diapedesis, we have investigated the role of Plvap in hematopoiesis and show that deletion of Plvap results in a dramatic decrease of IgM+IgDlo B cells in both the spleen and the peritoneal cavity. Tissue-specific deletion of Plvap demonstrates that the defect is B cell extrinsic, because B cell and pan-hematopoietic Plvap deletion has no effect on IgM+IgDlo B cell numbers. Endothelial-specific deletion of Plvap in the embryo or at adult stage recapitulates the full Plvap knockout phenotype, whereas endothelial-specific reconstitution of Plvap under the Chd5 promoter rescues the IgM+IgDlo B cell phenotype. Taken together, these results show that Plvap expression in endothelial cells is important in the maintenance of IgM+ B cells in the spleen and peritoneal cavity.
Assuntos
Linfócitos B/metabolismo , Proteínas de Transporte/metabolismo , Homeostase , Proteínas de Membrana/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Baço/citologia , Animais , Linfócitos B/patologia , Permeabilidade Capilar , Proteínas de Transporte/genética , DNA Helicases/genética , Células Endoteliais/química , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Imunoglobulina D/genética , Imunoglobulina D/metabolismo , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Cavidade Peritoneal/citologia , Fenótipo , Baço/imunologia , Migração Transendotelial e Transepitelial/imunologiaRESUMO
PV1 is an endothelial-specific protein with structural roles in the formation of diaphragms in endothelial cells of normal vessels. PV1 is also highly expressed on endothelial cells of many solid tumours. On the basis of in vitro data, PV1 is thought to actively participate in angiogenesis. To test whether or not PV1 has a function in tumour angiogenesis and in tumour growth in vivo, we have treated pancreatic tumour-bearing mice by single-dose intratumoural delivery of lentiviruses encoding for two different shRNAs targeting murine PV1. We find that PV1 down-regulation by shRNAs inhibits the growth of established tumours derived from two different human pancreatic adenocarcinoma cell lines (AsPC-1 and BxPC-3). The effect observed is because of down-regulation of PV1 in the tumour endothelial cells of host origin, PV1 being specifically expressed in tumour vascular endothelial cells and not in cancer or other stromal cells. There are no differences in vascular density of tumours treated or not with PV1 shRNA, and gain and loss of function of PV1 in endothelial cells does not modify either their proliferation or migration, suggesting that tumour angiogenesis is not impaired. Together, our data argue that down-regulation of PV1 in tumour endothelial cells results in the inhibition of tumour growth via a mechanism different from inhibiting angiogenesis.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Proteínas de Transporte/genética , Proteínas de Membrana/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Adenocarcinoma/irrigação sanguínea , Animais , Sequência de Bases , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Regulação para Baixo , Ensaios de Seleção de Medicamentos Antitumorais , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Lentivirus/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Camundongos Nus , Dados de Sequência Molecular , Neovascularização Patológica/genética , Neoplasias Pancreáticas/irrigação sanguínea , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
BACKGROUND & AIMS: Rb1 encodes a cell-cycle regulator that is functionally disrupted in most human cancers. Pancreatic ductal adenocarcinomas (PDACs) have a high frequency of mutations in KRAS and INK4A/CDKN2A that might allow cells to bypass the regulatory actions of retinoblastoma (RB). To determine the role of loss of RB function in PDAC progression, we investigated the effects of Rb disruption during pancreatic malignant transformation initiated by oncogenic Kras. METHODS: We generated mice with pancreas-specific disruption of Rb, in the absence or presence of oncogenic Kras, to examine the role of RB in pancreatic carcinogenesis. RESULTS: In the presence of oncogenic Kras, loss of Rb from the pancreatic epithelium accelerated formation of pancreatic intraepithelial neoplasia (PanIN), increased the frequency of cystic neoplasms, and promoted rapid progression toward PDAC. Early stage cancers were characterized by acute pancreatic inflammation, associated with up-regulation of proinflammatory cytokines within the pancreas. Despite the presence of markers associated with oncogene-induced senescence, low-grade PanIN were highly proliferative and expressed high levels of p53. Pancreatic cancer cell lines derived from these mice expressed high levels of cytokines, and transcriptional activity of p53 was impaired. CONCLUSIONS: Rb encodes a tumor suppressor that attenuates progression of oncogenic Kras-induced carcinogenesis in the pancreas by mediating the senescence response and promoting activity of the tumor suppressor p53.
Assuntos
Carcinoma in Situ/fisiopatologia , Senescência Celular/fisiologia , Deleção de Genes , Neoplasias Pancreáticas/fisiopatologia , Lesões Pré-Cancerosas/fisiopatologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/fisiologia , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Animais , Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/fisiopatologia , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/patologia , Citocinas/fisiologia , Modelos Animais de Doenças , Progressão da Doença , Camundongos , Camundongos Mutantes , Neoplasias Pancreáticas/patologia , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Enhancers have been functionally described for >35 years, but the molecular principles underlying the integration of regulatory inputs to alternate gene enhancers used during mammalian organogenesis remain incompletely understood. Using a combination of in vivo enhancer mapping and proteomics approaches, we have established that two distant and distinct early enhancers, each requiring different transcription complexes, are required for full activation of the gene encoding the pituitary lineage determining factor, Pit1. A transcription factor belonging to the "giant, multiple-homeodomain and zinc finger family," Atbf1, serves as a novel pituitary regulator for one of the two required enhancers as shown by genetic and in vitro analysis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição Pit-1/metabolismo , Animais , Sequência de Bases , Linhagem da Célula , Células-Tronco Embrionárias/metabolismo , Epistasia Genética , Genoma/genética , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação/genética , Hipófise/metabolismo , Ligação Proteica , Proteômica , Fatores de Tempo , Fator de Transcrição Pit-1/genéticaRESUMO
Negative checkpoint regulators (NCRs) temper the T cell immune response to self-antigens and limit the development of autoimmunity. Unlike all other NCRs that are expressed on activated T lymphocytes, V-type immunoglobulin domain-containing suppressor of T cell activation (VISTA) is expressed on naïve T cells. We report an unexpected heterogeneity within the naïve T cell compartment in mice, where loss of VISTA disrupted the major quiescent naïve T cell subset and enhanced self-reactivity. Agonistic VISTA engagement increased T cell tolerance by promoting antigen-induced peripheral T cell deletion. Although a critical player in naïve T cell homeostasis, the ability of VISTA to restrain naïve T cell responses was lost under inflammatory conditions. VISTA is therefore a distinctive NCR of naïve T cells that is critical for steady-state maintenance of quiescence and peripheral tolerance.
Assuntos
Antígenos B7/fisiologia , Proteínas de Membrana/fisiologia , Tolerância Periférica/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais , Antígenos B7/genética , Ativação Linfocitária , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tolerância Periférica/genética , Receptores de Antígenos de Linfócitos T/fisiologiaRESUMO
Chronic pancreatitis increases by 16-fold the risk of developing pancreatic ductal adenocarcinoma (PDAC), one of the deadliest human cancers. It also appears to accelerate cancer progression in genetically engineered mouse models. We now report that in a mouse model where oncogenic Kras is activated in all pancreatic cell types, two brief episodes of acute pancreatitis caused rapid PanIN progression and accelerated pancreatic cancer development. Thus, a brief inflammatory insult to the pancreas, when occurring in the context of oncogenic Kras(G12D), can initiate a cascade of events that dramatically enhances the risk for pancreatic malignant transformation.
Assuntos
Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/patologia , Pancreatite/complicações , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Animais , Modelos Animais de Doenças , Progressão da Doença , Camundongos , Camundongos Endogâmicos , Neoplasias Pancreáticas/metabolismo , Pancreatite/metabolismo , Pancreatite/patologia , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.
Assuntos
Proteínas Sanguíneas/metabolismo , Capilares/fisiologia , Capilares/ultraestrutura , Permeabilidade Capilar , Proteínas de Transporte/metabolismo , Endotélio Vascular/fisiologia , Endotélio Vascular/ultraestrutura , Proteínas de Membrana/metabolismo , Animais , Proteínas de Transporte/genética , Cavéolas/fisiologia , Membrana Celular/metabolismo , Endotélio Vascular/citologia , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Enteropatias Perdedoras de Proteínas/fisiopatologiaRESUMO
Targeting of oncogenic Kras to the pancreatic Nestin-expressing embryonic progenitor cells and subsequently to the adult acinar compartment and Nestin-expressing cells is sufficient for the development of low grade pancreatic intraepithelial neoplasia (PanIN) between 2 and 4 months. The mice die around 6 month-old of unrelated causes, and it is therefore not possible to assess whether the lesions will progress to carcinoma. We now report that two brief episodes of caerulein-induced acute pancreatitis in 2 month-old mice causes rapid PanIN progression and pancreatic ductal adenocarcinoma (PDAC) development by 4 months of age. These events occur with similar frequency as observed in animals where the oncogene is targeted during embryogenesis to all pancreatic cell types. Thus, these data show that oncogenic Kras-driven PanIN originating in a non-ductal compartment can rapidly progress to PDAC when subjected to a brief inflammatory insult.
Assuntos
Linhagem da Célula , Progressão da Doença , Proteínas de Filamentos Intermediários/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite/patologia , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Ceruletídeo , Marcação de Genes , Humanos , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , Nestina , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/metabolismo , Pancreatite/metabolismo , Lesões Pré-Cancerosas/metabolismo , Fator de Transcrição STAT3/metabolismo , Células-Tronco/metabolismo , Transgenes/genéticaRESUMO
OBJECTIVES: The present study was conducted to evaluate the expression and function of AP-2α isoforms in pancreatic ductal adenocarcinoma. METHODS: The expression of AP-2α was evaluated at the RNA level by reverse transcription-polymerase chain reaction and at the protein level by Western blotting and immunofluorescence. Its function as a transcription factor was evaluated in transient transfection experiments: DNA binding properties by electromobility shift assay and transactivation capabilities by luciferase assay. RESULTS: Multiple alternative splicing events of AP-2α messenger occurred in all human pancreatic cancer cell lines, including a novel isoform, termed variant 6, which was not present in HeLa cells. At the protein level, except for 1 cell line, all pancreatic cancer cell lines expressed high nuclear levels of AP-2α. We also showed that AP-2α expressed by the pancreatic cancer cell lines could bind its cognate recognition site and activate transcription. However, variant 6, although not able to activate transcription, did not act in a dominant negative manner when cotransfected with the full-length protein. CONCLUSIONS: Multiple isoforms of AP-2α are highly expressed in pancreatic cancer cell lines including a new isoform, AP-2α variant 6, which seems to be pancreatic cancer specific and is deprived of transcriptional activity.
Assuntos
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Expressão Gênica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fator de Transcrição AP-2/genética , Fator de Transcrição AP-2/metabolismo , Processamento Alternativo , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA/genética , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Variação Genética , Humanos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Deleção de Sequência , Fator de Transcrição AP-2/química , Ativação TranscricionalRESUMO
Primary cilia have been proposed to participate in the modulation of growth factor signaling pathways. In this study, we determined that ciliogenesis is suppressed in both pancreatic cancer cells and pancreatic intraepithelial neoplasia (PanIN) lesions in human pancreatic ductal adenocarcinoma (PDAC). Primary cilia were absent in these cells even when not actively proliferating. Cilia were also absent from mouse PanIN cells in three different mouse models of PDAC driven by an endogenous oncogenic Kras allele. Inhibition of Kras effector pathways restored ciliogenesis in a mouse pancreatic cancer cell line, raising the possibility that ciliogenesis may be actively repressed by oncogenic Kras. By contrast, normal duct, islet, and centroacinar cells retained primary cilia in both human and mouse pancreata. Thus, arrested ciliogenesis is a cardinal feature of PDAC and its precursor PanIN lesions, does not require ongoing proliferation, and could potentially be targeted pharmacologically.
Assuntos
Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Lesões Pré-Cancerosas/patologia , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/ultraestrutura , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Centrossomo/patologia , Cílios/patologia , Células Epiteliais/patologia , Genes ras , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/ultraestrutura , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/ultraestrutura , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
To determine the cell compartment in which initial oncogenic mutations occur in pancreatic ductal adenocarcinoma (PDAC), we generated a mouse model in which endogenous expression of mutated Kras (Kras(G12D)) was initially directed to a population of pancreatic exocrine progenitors characterized by the expression of Nestin. Targeting of oncogenic Kras to such a restricted cell compartment was sufficient for the formation of pancreatic intraepithelial neoplasias (PanINs), putative precursors to PDAC. PanINs appeared with the same grade and frequency as observed when Kras(G12D) was targeted to the whole pancreas by a Pdx1-driven Cre recombinase strategy. Thus, the Nestin cell lineage is highly responsive to Kras oncogenic activation and may represent the elusive progenitor population in which PDAC arises.