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1.
PLoS One ; 12(2): e0171510, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28158249

RESUMO

Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia in vivo. To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice (TgK18GT121; K18 mice). We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size.


Assuntos
Proliferação de Células , Queratina-18/metabolismo , Proteína do Retinoblastoma/fisiologia , Linfócitos T/citologia , Timo/citologia , Animais , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos , Proteína do Retinoblastoma/genética , Células Estromais/metabolismo , Linfócitos T/imunologia , Timo/imunologia , Timo/metabolismo , Transgenes
2.
PLoS One ; 8(12): e80459, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24312475

RESUMO

Carcinomas arise in a complex microenvironment consisting of multiple distinct epithelial lineages surrounded by a variety of stromal cell types. Understanding cancer etiologies requires evaluating the relationship among cell types during disease initiation and through progression. Genetically engineered mouse (GEM) models facilitate the prospective examination of early oncogenic events, which is not possible in humans. Since most solid tumors harbor aberrations in the RB network, we developed an inducible GEM approach for the establishment and assessment of carcinoma initiation in a diverse range of epithelial tissues and subtypes upon inactivation of RB-mediated tumor suppression (RB-TS). The system allows independent assessment of epithelial subtypes that express either cytokeratins (K) 18 or 19. By Cre-dependent expression of a protein that dominantly inactivates RB and functionally redundant proteins p107 and p130, neoplasia could be initiated in either K18 or K19 expressing cells of numerous tissues. By design, because only a single pathway aberration was engineered, carcinomas developed stochastically only after long latency. Hence, this system, which allows for directed cell type-specific carcinoma initiation, facilitates further definition of events that can progress neoplasms to aggressive cancers via engineered, carcinogen-induced and/or spontaneous evolution.


Assuntos
Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias/metabolismo , Proteína do Retinoblastoma/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Queratina-18/biossíntese , Queratina-18/genética , Queratina-19/biossíntese , Queratina-19/genética , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/patologia , Especificidade de Órgãos , Proteína do Retinoblastoma/genética
3.
Proc Natl Acad Sci U S A ; 110(44): 17933-8, 2013 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-24114272

RESUMO

Glioblastoma (GBM), the most common brain malignancy, remains fatal with no effective treatment. Analyses of common aberrations in GBM suggest major regulatory pathways associated with disease etiology. However, 90% of GBMs are diagnosed at an advanced stage (primary GBMs), providing no access to early disease stages for assessing disease progression events. As such, both understanding of disease mechanisms and the development of biomarkers and therapeutics for effective disease management are limited. Here, we describe an adult-inducible astrocyte-specific system in genetically engineered mice that queries causation in disease evolution of regulatory networks perturbed in human GBM. Events yielding disease, both engineered and spontaneous, indicate ordered grade-specific perturbations that yield high-grade astrocytomas (anaplastic astrocytomas and GBMs). Impaired retinoblastoma protein RB tumor suppression yields grade II histopathology. Additional activation of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) network drives progression to grade III disease, and further inactivation of phosphatase and tensin homolog (PTEN) yields GBM. Spontaneous missense mutation of tumor suppressor Trp53 arises subsequent to KRAS activation, but before grade III progression. The stochastic appearance of mutations identical to those observed in humans, particularly the same spectrum of p53 amino acid changes, supports the validity of engineered lesions and the ensuing interpretations of etiology. Absence of isocitrate dehydrogenase 1 (IDH1) mutation, asymptomatic low grade disease, and rapid emergence of GBM combined with a mesenchymal transcriptome signature reflect characteristics of primary GBM and provide insight into causal relationships.


Assuntos
Astrocitoma/etiologia , Evolução Biológica , Modelos Animais de Doenças , Engenharia Genética/métodos , Glioblastoma/etiologia , Animais , Sequência de Bases , Progressão da Doença , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/genética , Camundongos , Camundongos Transgênicos , Análise em Microsséries , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/genética
4.
PLoS One ; 7(9): e42446, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22984402

RESUMO

Humans with Hermansky-Pudlak Syndrome (HPS) or ocular albinism (OA1) display abnormal aspects of organelle biogenesis. The multigenic disorder HPS displays broad defects in biogenesis of lysosome-related organelles including melanosomes, platelet dense granules, and lysosomes. A phenotype of ocular pigmentation in OA1 is a smaller number of macromelanosomes, in contrast to HPS, where in many cases the melanosomes are smaller than normal. In these studies we define the role of the Mreg(dsu) gene, which suppresses the coat color dilution of Myo5a, melanophilin, and Rab27a mutant mice in maintaining melanosome size and distribution. We show that the product of the Mreg(dsu) locus, melanoregulin (MREG), interacts both with members of the HPS BLOC-2 complex and with Oa1 in regulating melanosome size. Loss of MREG function facilitates increase in the size of micromelanosomes in the choroid of the HPS BLOC-2 mutants ruby, ruby2, and cocoa, while a transgenic mouse overexpressing melanoregulin corrects the size of retinal pigment epithelium (RPE) macromelanosomes in Oa1(ko/ko) mice. Collectively, these results suggest that MREG levels regulate pigment incorporation into melanosomes. Immunohistochemical analysis localizes melanoregulin not to melanosomes, but to small vesicles in the cytoplasm of the RPE, consistent with a role for this protein in regulating membrane interactions during melanosome biogenesis. These results provide the first link between the BLOC pathway and Oa1 in melanosome biogenesis, thus supporting the hypothesis that intracellular G-protein coupled receptors may be involved in the biogenesis of other organelles. Furthermore these studies provide the foundation for therapeutic approaches to correct the pigment defects in the RPE of HPS and OA1.


Assuntos
Albinismo Ocular/genética , Proteínas de Transporte/metabolismo , Loci Gênicos/genética , Organelas/metabolismo , Albinismo Ocular/patologia , Animais , Proteínas de Transporte/genética , Linhagem Celular , Corioide/metabolismo , Corioide/patologia , Corioide/ultraestrutura , Dosagem de Genes/genética , Síndrome de Hermanski-Pudlak/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Melanossomas/metabolismo , Melanossomas/patologia , Melanossomas/ultraestrutura , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Mutação/genética , Tamanho das Organelas , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/patologia , Epitélio Pigmentado Ocular/ultraestrutura , Transporte Proteico , Proteínas de Transporte Vesicular
5.
Genetics ; 183(2): 581-94, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19635938

RESUMO

The microphthalmia-associated transcription factor (Mitf) has emerged as an important model for gene regulation in eukaryotic organisms. In vertebrates, it regulates the development of several cell types including melanocytes and has also been shown to play an important role in melanoma. In vitro, the activity of MITF is regulated by multiple signaling pathways, including the KITL/KIT/B-Raf pathway, which results in phosphorylation of MITF on serine residues 73 and 409. However, the precise role of signaling to MITF in vivo remains largely unknown. Here, we use a BAC transgene rescue approach to introduce specific mutations in MITF to study the importance of specific phospho-acceptor sites and protein domains. We show that mice that carry a BAC transgene where single-amino-acid substitutions have been made in the Mitf gene rescue the phenotype of the loss-of-function mutations in Mitf. This may indicate that signaling from KIT to MITF affects other phospho-acceptor sites in MITF or that alternative sites can be phosphorylated when Ser73 and Ser409 have been mutated. Our results have implications for understanding signaling to transcription factors. Furthermore, as MITF and signaling mechanisms have been shown to play an important role in melanomas, our findings may lead to novel insights into this resilient disease.


Assuntos
Cromossomos Artificiais Bacterianos/genética , Olho/metabolismo , Cor de Cabelo/genética , Fator de Transcrição Associado à Microftalmia/genética , Transgenes/genética , Processamento Alternativo , Animais , Sítios de Ligação/genética , Éxons/genética , Olho/crescimento & desenvolvimento , Feminino , Deleção de Genes , Masculino , Melanócitos/metabolismo , Camundongos , Camundongos Transgênicos , Fator de Transcrição Associado à Microftalmia/metabolismo , Mutação , Miocárdio/metabolismo , Fenótipo , Fosforilação , Serina/genética , Serina/metabolismo , Pele/crescimento & desenvolvimento , Pele/metabolismo
6.
Proc Natl Acad Sci U S A ; 101(48): 16831-6, 2004 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-15550542

RESUMO

MYO5A is a major actin-based vesicle transport motor that binds to one of its cargos, the melanosome, by means of a RAB27A/MLPH receptor. When one of the members of this receptor-motor complex is mutated, the melanosomes clump in the perinuclear region of the melanocyte and are transferred unevenly to the developing hair, leading to a dilution of coat color. Mutation of a fourth gene, dilute suppressor (dsu), suppresses this coat color dilution. MYO5A is required for the peripheral accumulation of melanosomes in melanocytes, but its role in melanosome transfer to neighboring keratinocytes and the hair is unknown. Here, we show that MYO5A is nonessential for melanosome transfer, although pigment incorporation into the hair in MYO5A-deficient mice is uneven, probably due to the clumping of melanosomes that occurs in the perinuclear region of mutant melanocytes. We also show that dsu is caused by a loss-of-function mutation in a unique vertebrate-specific protein that appears to function in an MYO5A-independent pathway to alter pigment incorporation into the hair. Therefore, dsu identifies a unique protein involved in pigmentation of the mammalian hair.


Assuntos
Cor de Cabelo/genética , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Animais , Western Blotting , Cromossomos Bacterianos , Teste de Complementação Genética , Camundongos , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética
7.
Nat Genet ; 33(2): 145-53, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12548288

RESUMO

Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous disease involving abnormalities of melanosomes, platelet dense granules and lysosomes. Here we have used positional candidate and transgenic rescue approaches to identify the genes mutated in ruby-eye 2 and ruby-eye mice (ru2 and ru, respectively), two 'mimic' mouse models of HPS. We also show that these genes are orthologs of the genes mutated in individuals with HPS types 5 and 6, respectively, and that their protein products directly interact. Both genes are previously unknown and are found only in higher eukaryotes, and together represent a new class of genes that have evolved in higher organisms to govern the synthesis of highly specialized lysosome-related organelles.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Proteínas de Drosophila , Síndrome de Hermanski-Pudlak/genética , Proteínas de Insetos/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Mutação/genética , Proteínas/genética , Complexo 3 de Proteínas Adaptadoras , Subunidades beta do Complexo de Proteínas Adaptadoras , Adulto , Sequência de Aminoácidos , Animais , Células COS , Pré-Escolar , Chlorocebus aethiops , Cromossomos Artificiais Bacterianos/genética , Cromossomos Artificiais de Bacteriófago P1/genética , Modelos Animais de Doenças , Feminino , Síndrome de Hermanski-Pudlak/metabolismo , Síndrome de Hermanski-Pudlak/patologia , Humanos , Masculino , Melanossomas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Oligopeptídeos , Peptídeos/imunologia , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Proteínas Proto-Oncogênicas c-myc/imunologia , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Técnicas do Sistema de Duplo-Híbrido
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