RESUMO
The Hedgehog (Hh) signaling pathway has been implicated in the most common childhood brain tumor, medulloblastoma (MB). Given the toxicity of post-surgical treatments for MB, continued need exists for new, targeted therapies. Based upon our finding that Neuropilin (Nrp) transmembrane proteins are required for Hh signal transduction, we investigated the role of Nrp in MB cells. Cultured cells derived from a mouse Ptch (+/-) ;LacZ MB (Med1-MB), effectively modeled the Hh pathway-related subcategory of human MBs in vitro. Med1-MB cells maintained constitutively active Hh target gene transcription, and consistently formed tumors within one month after injection into mouse cerebella. The proliferation rate of Med1-MBs in culture was dependent upon Nrp2, while reducing Nrp1 function had little effect. Knockdown of Nrp2 prior to cell implantation significantly increased mouse survival, compared to transfection with a non-targeting siRNA. Knocking down Nrp2 specifically in MB cells avoided any direct effect on tumor vascularization. Nrp2 should be further investigated as a potential target for adjuvant therapy in patients with MB.
Assuntos
Transformação Celular Neoplásica/patologia , Neoplasias Cerebelares/patologia , Modelos Animais de Doenças , Proteínas Hedgehog/metabolismo , Meduloblastoma/patologia , Neuropilina-1/metabolismo , Neuropilina-2/metabolismo , Receptores de Superfície Celular/fisiologia , Animais , Western Blotting , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Neoplasias Cerebelares/metabolismo , Humanos , Masculino , Meduloblastoma/metabolismo , Camundongos , Camundongos Knockout , Camundongos Nus , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/genética , Neuropilina-2/antagonistas & inibidores , Neuropilina-2/genética , Receptores Patched , Receptor Patched-1 , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Central nervous system tumors carry grave clinical prognoses due to limited effectiveness of surgical resection, radiation, and chemotherapy. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine knot (knottin) peptide that binds with high affinity to αvß3, αvß5, and α5ß1 integrin receptors. This integrin-binding knottin peptide, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB. Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared with the normal surrounding brain tissue, as measured by optical imaging. The imaging signal intensity correlated with tumor volume. Due to its unique ability to bind to α5ß1 integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared with other engineered integrin-binding knottin peptides and with c(RGDfK), a well-studied integrin-binding peptidomimetic. Next, EETI 2.5F was fused to an antibody fragment crystallizable (Fc) domain (EETI 2.5F-Fc) to determine if a larger integrin-binding protein could also target intracranial brain tumors. EETI 2.5F-Fc, conjugated to a fluorescent dye, illuminated MB following i.v. injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity. These results highlight the potential of using EETI 2.5F and EETI 2.5-Fc as targeted molecular probes for brain tumor imaging.
Assuntos
Neoplasias Cerebelares/diagnóstico , Miniproteínas Nó de Cistina/metabolismo , Diagnóstico por Imagem/métodos , Meduloblastoma/diagnóstico , Animais , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Miniproteínas Nó de Cistina/química , Miniproteínas Nó de Cistina/genética , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Integrina alfa5beta1/metabolismo , Masculino , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Microscopia de Fluorescência , Imagem Molecular/métodos , Receptores Patched , Ligação Proteica , Engenharia de Proteínas , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Sensibilidade e EspecificidadeRESUMO
Pyramidal neurons in the deep layers of the cerebral cortex can be classified into two major classes: callosal projection neurons and long-range subcortical neurons. We and others have shown that a gene expressed specifically by subcortical projection neurons, Fezf2, is required for the formation of axonal projections to the spinal cord, tectum, and pons. Here, we report that Fezf2 regulates a decision between subcortical vs. callosal projection neuron fates. Fezf2(-/-) neurons adopt the fate of callosal projection neurons as assessed by their axonal projections, electrophysiological properties, and acquisition of Satb2 expression. Ctip2 is a major downstream effector of Fezf2 in regulating the extension of axons toward subcortical targets and can rescue the axonal phenotype of Fezf2 mutants. When ectopically expressed, either Fezf2 or Ctip2 can alter the axonal targeting of corticocortical projection neurons and cause them to project to subcortical targets, although Fezf2 can promote a subcortical projection neuron fate in the absence of Ctip2 expression.
Assuntos
Axônios/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Piramidais/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Mutantes , Proteínas do Tecido Nervoso/genética , Fenótipo , Células Piramidais/citologia , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
In addition to the apical-basal polarity pathway operating in epithelial cells, a planar cell polarity (PCP) pathway establishes polarity within the plane of epithelial tissues and is conserved from Drosophila to mammals. In Drosophila, a 'core' group of PCP genes including frizzled (fz), flamingo/starry night, dishevelled (dsh), Van Gogh/strabismus and prickle, function to regulate wing hair, bristle and ommatidial polarity. In vertebrates, the PCP pathway regulates convergent extension movements and neural tube closure, as well as the orientation of stereociliary bundles of sensory hair cells in the inner ear. Here we show that a mutation in the mouse protein tyrosine kinase 7 (PTK7) gene, which encodes an evolutionarily conserved transmembrane protein with tyrosine kinase homology, disrupts neural tube closure and stereociliary bundle orientation, and shows genetic interactions with a mutation in the mouse Van Gogh homologue vangl2. We also show that PTK7 is dynamically localized during hair cell polarization, and that the Xenopus homologue of PTK7 is required for neural convergent extension and neural tube closure. These results identify PTK7 as a novel regulator of PCP in vertebrates.
Assuntos
Polaridade Celular , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Sistema Nervoso Central/citologia , Sistema Nervoso Central/embriologia , Cóclea/citologia , Cóclea/embriologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Células Epiteliais/citologia , Camundongos , Mutação/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Proteínas de Xenopus/genéticaRESUMO
Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) belong to the largest mammalian superfamily of proteins with seven-transmembrane domains. LGRs can be divided into three subgroups based on their unique domain arrangement. Although two subgroups have been found to be receptors for glycoprotein hormones and relaxin-related ligands, respectively, the third LGR subgroup, consisting of LGR4-6, are orphan receptors with unknown physiological roles. To elucidate the functions of this subgroup of LGRs, LGR4 null mice were generated using a secretory trap approach to delete the majority of the LGR4 gene after the insertion of a beta-galactosidase reporter gene immediately after exon 1. Tissues expressing LGR4 were analyzed based on histochemical staining of the transgene driven by the endogenous LGR4 promoter. LGR4 was widely expressed in kidney, adrenal gland, stomach, intestine, heart, bone/cartilage, and other tissues. The expression of LGR4 in these tissues was further confirmed by immunohistochemical studies in wild-type animals. Analysis of the viability of 250 newborn animals suggested a skewed inheritance pattern, indicating that only 40% of the expected LGR4 null mice were born. For the LGR4 null mice viable at birth, most of them died within 2 d. Furthermore, the LGR4 null mice showed intrauterine growth retardation as reflected by a 14% decrease in body weight at birth, together with 30% and 40% decreases in kidney and liver weights, respectively. The present findings demonstrate the widespread expression of LGR4, and an essential role of LGR4 for embryonic growth, as well as kidney and liver development. The observed pre- and postnatal lethality of LGR4 null mice illustrates the importance of the LGR4 signaling system for the survival and growth of animals during the perinatal stage.
Assuntos
Retardo do Crescimento Fetal/etiologia , Leucina/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologia , Sequência de Aminoácidos , Animais , Células Epiteliais/imunologia , Feminino , Expressão Gênica/genética , Genes Letais , Genes Reporter/genética , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Rim/citologia , Rim/imunologia , Rim/metabolismo , Leucina/análise , Fígado/imunologia , Fígado/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Insercional/genética , Gravidez , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Receptor IGF Tipo 1/genética , Receptores Acoplados a Proteínas G/análise , Sequências Repetitivas de Aminoácidos , Distribuição Tecidual , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
Endothelial cell production of anticoagulant heparan sulfate (HS(act)) is controlled by the Hs3st1 gene, which encodes the rate-limiting enzyme heparan sulfate 3-O-sulfotransferase-1 (3-OST-1). In vitro, HS(act) dramatically enhances the neutralization of coagulation proteases by antithrombin. The in vivo role of HS(act) was evaluated by generating Hs3st1(-/-) knockout mice. Hs3st1(-/-) animals were devoid of 3-OST-1 enzyme activity in plasma and tissue extracts. Nulls showed dramatic reductions in tissue levels of HS(act) but maintained wild-type levels of tissue fibrin accumulation under both normoxic and hypoxic conditions. Given that vascular HS(act) predominantly occurs in the subendothelial matrix, mice were subjected to a carotid artery injury assay in which ferric chloride administration induces de-endothelialization and occlusive thrombosis. Hs3st1(-/-) and Hs3st1(+/+) mice yielded indistinguishable occlusion times and comparable levels of thrombin.antithrombin complexes. Thus, Hs3st1(-/-) mice did not show an obvious procoagulant phenotype. Instead, Hs3st1(-/-) mice exhibited genetic background-specific lethality and intrauterine growth retardation, without evidence of a gross coagulopathy. Our results demonstrate that the 3-OST-1 enzyme produces the majority of tissue HS(act). Surprisingly, this bulk of HS(act) is not essential for normal hemostasis in mice. Instead, 3-OST-1-deficient mice exhibited unanticipated phenotypes suggesting that HS(act) or additional 3-OST-1-derived structures may serve alternate biologic roles.
Assuntos
Anticoagulantes/farmacologia , Heparitina Sulfato/farmacologia , Animais , Antitrombinas/metabolismo , Artérias Carótidas/patologia , Cruzamentos Genéticos , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Éxons , Feminino , Genótipo , Hemostasia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Retroviridae/genética , Sulfotransferases/metabolismo , Trombina/metabolismo , Distribuição TecidualRESUMO
The WT1 tumor-suppressor gene is expressed by many forms of acute myeloid leukemia. Inhibition of this expression can lead to the differentiation and reduced growth of leukemia cells and cell lines, suggesting that WT1 participates in regulating the proliferation of leukemic cells. However, the role of WT1 in normal hematopoiesis is not well understood. To investigate this question, we have used murine cells in which the WT1 gene has been inactivated by homologous recombination. We have found that cells lacking WT1 show deficits in hematopoietic stem cell function. Embryonic stem cells lacking WT1, although contributing efficiently to other organ systems, make only a minimal contribution to the hematopoietic system in chimeras, indicating that hematopoietic stem cells lacking WT1 compete poorly with healthy stem cells. In addition, fetal liver cells lacking WT1 have an approximately 75% reduction in erythroid blast-forming unit (BFU-E), erythroid colony-forming unit (CFU-E), and colony-forming unit-granulocyte macrophage-erythroid-megakaryocyte (CFU-GEMM). However, transplantation of fetal liver hematopoietic cells lacking WT1 will repopulate the hematopoietic system of an irradiated adult recipient in the absence of competition. We conclude that the absence of WT1 in hematopoietic cells leads to functional defects in growth potential that may be of consequence to leukemic cells that have alterations in the expression of WT1.