RESUMO
Familial exudative vitreoretinopathy (FEVR) is a hereditary ocular disorder characterized by a failure of peripheral retinal vascularization. Loci associated with FEVR map to 11q13-q23 (EVR1; OMIM 133780, ref. 1), Xp11.4 (EVR2; OMIM 305390, ref. 2) and 11p13-12 (EVR3; OMIM 605750, ref. 3). Here we have confirmed linkage to the 11q13-23 locus for autosomal dominant FEVR in one large multigenerational family and refined the disease locus to a genomic region spanning 1.55 Mb. Mutations in FZD4, encoding the putative Wnt receptor frizzled-4, segregated completely with affected individuals in the family and were detected in affected individuals from an additional unrelated family, but not in normal controls. FZD genes encode Wnt receptors, which are implicated in development and carcinogenesis. Injection of wildtype and mutated FZD4 into Xenopus laevis embryos revealed that wildtype, but not mutant, frizzled-4 activated calcium/calmodulin-dependent protein kinase II (CAMKII) and protein kinase C (PKC), components of the Wnt/Ca(2+) signaling pathway. In one of the mutants, altered subcellular trafficking led to defective signaling. These findings support a function for frizzled-4 in retinal angiogenesis and establish the first association between a Wnt receptor and human disease.
Assuntos
Neovascularização Patológica/genética , Proteínas/genética , Doenças Retinianas/genética , Vasos Retinianos/patologia , Sequência de Aminoácidos , Pré-Escolar , Feminino , Receptores Frizzled , Marcadores Genéticos , Haplótipos , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Linhagem , Polimorfismo Genético , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Retina/patologia , Doenças Retinianas/patologia , Alinhamento de Sequência , Transdução de SinaisRESUMO
Dapper was isolated in a screen for proteins interacting with Dishevelled, a key factor in Wnt signaling. Dapper and Dishevelled colocalize intracellularly and form a complex with Axin, GSK-3, CKI, and beta-catenin. Overexpression of Dapper increases Axin and GSK-3 in this complex, resulting in decreased soluble beta-catenin and decreased activation of beta-catenin-responsive genes. Dapper also inhibits activation by Dishevelled of c-Jun N-terminal kinase (JNK), a component of beta-catenin-independent Frizzled signaling. Inhibition of Dapper activates both beta-catenin-responsive genes and an AP1-responsive promoter, demonstrating that Dapper is a general Dishevelled antagonist. Depletion of maternal Dapper RNA from Xenopus embryos results in loss of notochord and head structures, demonstrating that Dapper is required for normal vertebrate development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Notocorda/embriologia , Proteínas Nucleares , Fosfoproteínas/metabolismo , Proteínas Repressoras , Transativadores , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteínas de Peixe-Zebra , Animais , Proteína Axina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Transporte/química , Caseína Quinases , Sequência Conservada , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas Desgrenhadas , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase , Células HeLa , Humanos , Ligação de Hidrogênio , Técnicas In Vitro , MAP Quinase Quinase 4 , Dados de Sequência Molecular , Notocorda/metabolismo , Fenótipo , Ligação Proteica/fisiologia , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Homologia de Sequência de Aminoácidos , Vertebrados , Proteínas Wnt , Proteínas de Xenopus/química , Xenopus laevis , beta CateninaRESUMO
Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Embrião não Mamífero/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase C/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Desgrenhadas , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/enzimologia , Hibridização In Situ , Toxina Pertussis/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
Numerous preclinical studies suggest that gonadal steroids, particularly estrogen, may be neuroprotective against insult or disease progression. This paper reviews the mechanisms contributing to estrogen-mediated neuroprotection. Rapid signaling pathways, such as MAPK, PI3K, Akt, and PKC, are required for estrogen's ability to provide neuroprotection. These rapid signaling pathways converge on genomic pathways to modulate transcription of E2-responsive genes via ERE-dependent and ERE-independent mechanisms. It is clear that both rapid signaling and transcription are important for estrogen's neuroprotective effects. A mechanistic understanding of estrogen-mediated neuroprotection is crucial for the development of therapeutic interventions that enhance quality of life without deleterious side effects.
Assuntos
Estradiol/fisiologia , Regulação da Expressão Gênica , Hormônios Esteroides Gonadais/fisiologia , Transdução de Sinais , Animais , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/metabolismo , Estradiol/farmacologia , Terapia de Reposição de Estrogênios , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios Esteroides Gonadais/farmacologia , Proteínas de Membrana/metabolismo , Modelos Biológicos , Doenças do Sistema Nervoso/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Elementos Reguladores de Transcrição , Caracteres Sexuais , Transdução de Sinais/efeitos dos fármacosRESUMO
Estrogen reduces brain injury after experimental cerebral ischemia in part through a genomic mechanism of action. Using DNA microarrays, we analyzed the genomic response of the brain to estradiol, and we identified a transcript, cocaine- and amphetamine-regulated transcript (CART), that is highly induced in the cerebral cortex by estradiol under ischemic conditions. Using in vitro and in vivo models of neural injury, we confirmed and characterized CART mRNA and protein up-regulation by estradiol in surviving neurons, and we demonstrated that i.v. administration of a rat CART peptide is protective against ischemic brain injury in vivo. We further demonstrated binding of cAMP response element (CRE)-binding protein to a CART promoter CRE site in ischemic brain and rapid activation by CART of ERK in primary cultured cortical neurons. The findings suggest that CART is an important player in estrogen-mediated neuroprotection and a potential therapeutic agent for stroke and other neurodegenerative diseases.
Assuntos
Estradiol/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/metabolismo , Animais , Isquemia Encefálica/induzido quimicamente , Células Cultivadas , Córtex Cerebral/patologia , Estradiol/farmacologia , Feminino , Infarto da Artéria Cerebral Média/induzido quimicamente , Camundongos , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Regulação para Cima/efeitos dos fármacosRESUMO
The Wnt/beta-catenin signaling pathway plays multiple roles during embryonic development, only a few of which have been extensively characterized. Although domains of Wnt expression have been identified throughout embryogenesis, anatomical and molecular characterization of responding cells has been mostly unexplored. We have generated a transgenic zebrafish line that expresses a destabilized green fluorescent protein (GFP) variant under the control of a beta-catenin responsive promoter. Early zygotic expression of this transgene (TOPdGFP) mirrors known domains of Wnt signaling in the embryo. Loss of Lef1 activity results in decreased reporter expression and posterior defects, while loss of Tcf3 (Headless, Hdl) activity does not alter reporter expression, even though it results in loss of forebrain structures. In addition, ectopic Wnt1 expression can activate the reporter. In older embryos, we identify a number of transgene-expressing cell populations as novel sites of beta-catenin signaling. We conclude that our TOP-dGFP reporter line faithfully illustrates domains of beta-catenin activity and enables the identification of responsive cell populations.