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1.
Curr Opin Cell Biol ; 11(6): 683-8, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10600704

RESUMO

Skeletal muscle development involves a multistep pathway in which mesodermal precursor cells are selected, in response to inductive cues, to form myoblasts that later withdraw from the cell cycle and differentiate. The transcriptional circuitry controlling muscle differentiation is intimately linked to the cell cycle machinery, such that muscle differentiation genes do not become transcribed until myoblasts have exited the cell cycle. Members of the MyoD and MEF2 families of transcription factors associate combinatorially to control myoblast specification, differentiation and proliferation. Recent studies have revealed multiple signaling systems that stimulate and inhibit myogenesis by altering MEF2 phosphorylation and its association with other transcriptional cofactors.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Músculo Esquelético/embriologia , Proteína MyoD/fisiologia , Transdução de Sinais , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Divisão Celular , Proteínas de Ligação a DNA/metabolismo , Sistema de Sinalização das MAP Quinases , Fatores de Transcrição MEF2 , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Proteína MyoD/metabolismo , Fatores de Regulação Miogênica , Fosforilação , Receptores Notch , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador beta/fisiologia
2.
J Clin Invest ; 105(10): 1395-406, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10811847

RESUMO

Hypertrophic growth is an adaptive response of the heart to diverse pathological stimuli and is characterized by cardiomyocyte enlargement, sarcomere assembly, and activation of a fetal program of cardiac gene expression. A variety of Ca(2+)-dependent signal transduction pathways have been implicated in cardiac hypertrophy, but whether these pathways are independent or interdependent and whether there is specificity among them are unclear. Previously, we showed that activation of the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin or its target transcription factor NFAT3 was sufficient to evoke myocardial hypertrophy in vivo. Here, we show that activated Ca(2+)/calmodulin-dependent protein kinases-I and -IV (CaMKI and CaMKIV) also induce hypertrophic responses in cardiomyocytes in vitro and that CaMKIV overexpressing mice develop cardiac hypertrophy with increased left ventricular end-diastolic diameter and decreased fractional shortening. Crossing this transgenic line with mice expressing a constitutively activated form of NFAT3 revealed synergy between these signaling pathways. We further show that CaMKIV activates the transcription factor MEF2 through a posttranslational mechanism in the hypertrophic heart in vivo. Activated calcineurin is a less efficient activator of MEF2-dependent transcription, suggesting that the calcineurin/NFAT and CaMK/MEF2 pathways act in parallel. These findings identify MEF2 as a downstream target for CaMK signaling in the hypertrophic heart and suggest that the CaMK and calcineurin pathways preferentially target different transcription factors to induce cardiac hypertrophy.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Animais , Fator Natriurético Atrial/genética , Calcineurina/metabolismo , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Cardiomegalia/genética , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Fatores de Transcrição MEF2 , Camundongos , Camundongos Transgênicos , Miocárdio/metabolismo , Fatores de Regulação Miogênica , Cadeias Pesadas de Miosina/genética , Fatores de Transcrição NFATC , Regiões Promotoras Genéticas , Ratos , Transdução de Sinais
3.
Mol Cell Biol ; 16(2): 626-33, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8552091

RESUMO

Using degenerate PCR cloning we have identified a novel basic helix-loop-helix (bHLH) transcription factor, BETA3, from a hamster insulin tumor (HIT) cell cDNA library. Sequence analysis revealed that this factor belongs to the class B bHLH family and has the highest degree of homology with another bHLH transcription factor recently isolated in our laboratory, BETA2 (neuroD) (J. E. Lee, S. M. Hollenberg, L. Snider, D. L. Turner, N. Lipnick, and H. Weintraub, Science 268:836-844, 1995; F. J. Naya, C. M. M. Stellrecht, and M.-J. Tsai, Genes Dev. 8:1009-1019, 1995). BETA2 is a brain- and pancreatic-islet-specific bHLH transcription factor and is largely responsible for the tissue-specific expression of the insulin gene. BETA3 was found to be tissue restricted, with the highest levels of expression in HIT, lung, kidney, and brain cells. Surprisingly, despite the homology between BETA2 and BETA3 and its intact basic region, BETA3 is unable to bind the insulin E box in bandshift analysis as a homodimer or as a heterodimer with the class A bHLH factors E12, E47, or BETA1. Instead, BETA3 inhibited both the E47 homodimer and the E47/BETA2 heterodimer binding to the insulin E box. In addition, BETA3 greatly repressed the BETA2/E47 induction of the insulin enhancer in HIT cells as well as the MyoD/E47 induction of a muscle-specific E box in the myoblast cell line C2C12. In contrast, expression of BETA3 had no significant effect on the GAL4-VP16 transcriptional activity. Immunoprecipitation analysis demonstrates that the mechanism of repression is via direct protein-protein interaction, presumably by heterodimerization between BETA3 and class A bHLH factors.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Sequências Hélice-Alça-Hélice , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Cricetinae , DNA Complementar/genética , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Dados de Sequência Molecular , Proteína MyoD/metabolismo , Ligação Proteica , Conformação Proteica , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Transcrição TCF , Distribuição Tecidual , Transativadores/metabolismo , Proteína 1 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas
4.
J Neurosci ; 20(10): 3714-24, 2000 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-10804213

RESUMO

The transcription factors neuronal helix-loop-helix protein (NEX)/mammalian atonal homolog 2 (Math-2), BETA2/neuronal determination factor (NeuroD), and NeuroD-related factor (NDRF)/NeuroD2 comprise a family of Drosophila atonal-related basic helix-loop-helix (bHLH) proteins with highly overlapping expression in the developing forebrain. The ability of BETA2/NeuroD and NDRF to convert ectodermal cells into neurons after mRNA injection into Xenopus oocytes suggested a role in specifying neuronal cell fate. However, neuronal bHLH genes are largely transcribed in CNS neurons, which are fully committed. Here we analyze a defect in mice lacking BETA2/NeuroD, and in NEX*BETA2/NeuroD double mutants, demonstrating that bHLH proteins are required in vivo for terminal neuronal differentiation. Most strikingly, presumptive granule cells of the dentate gyrus are generated but fail to mature, lack normal sodium currents, and show little dendritic arborization. Long-term hippocampal slice cultures demonstrate secondary alterations of entorhinal and commissural/associational projections. The primary developmental arrest appears to be restricted to granule cells in which an autoregulatory system involving all three neuronal bHLH genes has failed.


Assuntos
Giro Denteado/citologia , Sequências Hélice-Alça-Hélice/genética , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Proteínas Virais , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Apoptose/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Moléculas de Adesão Celular Neuronais/análise , Diferenciação Celular/fisiologia , Giro Denteado/crescimento & desenvolvimento , Proteínas da Matriz Extracelular/análise , Regulação da Expressão Gênica no Desenvolvimento , Marcação In Situ das Extremidades Cortadas , Integrases/metabolismo , Antígeno Ki-67/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/química , Neurônios/enzimologia , Técnicas de Patch-Clamp , Proteína Reelina , Serina Endopeptidases , Ativação Transcricional/fisiologia
5.
Diabetes ; 46(6): 1069-74, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9166681

RESUMO

Recent genome searches suggest a putative linkage of many loci to susceptibility to type I diabetes. The chromosome 2q31-35 region is reported to be linked to susceptibility to type I diabetes and is thought to contain several diabetes susceptibility loci. These candidate genes include the HOXD gene cluster, BETA2, CTLA4, CD28, IGFBP2, and IGFBP5. Association studies in populations and families are required to confirm and/or identify the actual susceptibility loci. We hereby report several previously unknown DNA polymorphisms for HOXD8, BETA2, and IGFBP5, which we have used along with previously known polymorphisms of HOXD8 and CTLA4 to test whether these candidate loci are the susceptibility genes on chromosome 2q31-35. Using a case-control design with a subsequent family-association approach to confirm associations, we find no evidence that these candidate genes are associated with susceptibility to type I diabetes.


Assuntos
Cromossomos Humanos Par 2/genética , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 1/genética , Proteínas de Homeodomínio/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Transativadores/genética , Fatores de Transcrição/genética , Alelos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Antígenos CD28/genética , Estudos de Casos e Controles , Mapeamento Cromossômico , Primers do DNA/química , Diabetes Mellitus Tipo 1/imunologia , Família , Frequência do Gene , Ligação Genética , Antígenos HLA-DR/imunologia , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Repetições de Microssatélites/genética , Família Multigênica , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples
6.
Cell Differ ; 22(2): 115-23, 1988 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-3342449

RESUMO

Retina cognin, a cell membrane glycoprotein which mediates cell-cell recognition and adhesion in vitro, is initially present throughout the retina and becomes confined to the ganglion cell layer at 14-15 days of embryogenesis. Within this layer it is found on membranes of virtually all ganglion and displaced amacrine cells, but not on membranes of retinal glial cells (Müller fibers) which traverse this layer. The distribution of cognin as determined by immunocytochemistry is described and compared with that of choline acetyltransferase. The significance of cognin as a possible address marker during development of neural retina is discussed.


Assuntos
Colina O-Acetiltransferase/análise , Proteínas de Membrana/análise , Retina/análise , Retina/embriologia , Células Ganglionares da Retina/análise , Animais , Antígenos de Diferenciação/imunologia , Embrião de Galinha , Células Ganglionares da Retina/enzimologia
7.
Genes Dev ; 9(8): 1009-19, 1995 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-7774807

RESUMO

The insulin gene is one of the best paradigms of tissue-specific gene expression. It is developmentally regulated and is expressed exclusively in the pancreatic beta-cell. This restricted expression is directed by a tissue-specific enhancer, within the promoter, which contains an E-box sequence. The insulin E-box binds an islet-specific protein complex, termed 3a1. E-boxes bind proteins belonging to the basic helix-loop-helix (bHLH) family of transcription factors. The bHLH proteins function as potent transcriptional activators of tissue-specific genes by forming heterodimers between ubiquitous and cell-restricted family members. In addition, the cell-restricted bHLH members play an important role in specifying cell fate. To isolate the tissue-specific bHLH factor controlling insulin gene expression and study its role in islet cell differentiation, a modified yeast two-hybrid system was utilized to clone a novel bHLH factor, BETA2 (beta-cell E-box trans-activator 2), from a hamster insulin tumor (HIT) cell cDNA library. Northern analysis demonstrates that high-level expression of the BETA2 gene is restricted to pancreatic alpha- and beta-cell lines. As expected of tissue-specific bHLH members, BETA2 binds to the insulin E-box sequence with high affinity as a heterodimer with the ubiquitous bHLH factor E47. More importantly, antibody supershift experiments clearly show that BETA2 is a component of the native insulin E-box-binding complex. Transient transfection assays demonstrate that the BETA2/E47 heterodimer synergistically interacts with a neighboring beta-cell-specific complex to activate an insulin enhancer. In contrast, other bHLH factors such as MyoD and E47, which can bind to the insulin E-box with high affinity, fail to do so. Thus, a unique, cooperative interaction is the basis by which the insulin E-box enhancer discriminates between various bHLH factors to achieve tissue-specific activation of the insulin gene.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Sequências Hélice-Alça-Hélice , Insulina/biossíntese , Ilhotas Pancreáticas/metabolismo , Transativadores/genética , Fatores de Transcrição , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Insulina/genética , Ilhotas Pancreáticas/citologia , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Sequências Reguladoras de Ácido Nucleico , Seleção Genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Transcrição TCF , Proteína 1 Semelhante ao Fator 7 de Transcrição , Transcrição Gênica , Leveduras/genética
8.
Genes Dev ; 12(6): 820-30, 1998 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-9512516

RESUMO

The major epithelial cell types lining the intestine comprise a perpetually self-renewing population of cells that differentiate continuously from a stem cell in the intestinal crypts. Secretin-producing enteroendocrine cells represent a nondividing subpopulation of intestinal epithelial cells, suggesting that expression of the hormone is coordinated with cell cycle arrest during the differentiation of this cell lineage. Here we report that the basic helix-loop-helix protein BETA2 associates functionally with the coactivator, p300 to activate transcription of the secretin gene as well as the gene encoding the cyclin-dependent kinase inhibitor p21. Overexpression of BETA2 in cell lines induces both cell cycle arrest and apoptosis suggesting that BETA2 may regulate proliferation of secretin cells. Consistent with this role, we observed both reentry of normally quiescent cells into the cell cycle and disrupted cell number regulation in the small intestine of BETA2 null mice. Thus, BETA2 may function to coordinate transcriptional activation of the secretin gene, cell cycle arrest, and cell number regulation, providing one of the first examples of a transcription factor that controls terminal differentiation of cells in the intestinal epithelium.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Glândulas Endócrinas/citologia , Glândulas Endócrinas/metabolismo , Intestinos/citologia , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sítios de Ligação , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Genes/genética , Células HeLa , Sequências Hélice-Alça-Hélice , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Secretina/genética , Transativadores/genética , Transativadores/fisiologia , Transcrição Gênica/genética , Transcrição Gênica/fisiologia , Células Tumorais Cultivadas
9.
Development ; 126(10): 2045-52, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10207130

RESUMO

The four members of the MEF2 family of MADS-box transcription factors, MEF2-A, MEF2-B, MEF2-C and MEF2-D, are expressed in overlapping patterns in developing muscle and neural cell lineages during embryogenesis. However, during late fetal development and postnatally, MEF2 transcripts are also expressed in a wide range of cell types. Because MEF2 expression is controlled by translational and post-translational mechanisms, it has been unclear whether the presence of MEF2 transcripts in the embryo reflects transcriptionally active MEF2 proteins. To define the temporospatial expression pattern of transcriptionally active MEF2 proteins during mouse embryogenesis, we generated transgenic mice harboring a lacZ reporter gene controlled by three tandem copies of the MEF2 site and flanking sequences from the desmin enhancer, which is active in cardiac, skeletal and smooth muscle cells. Expression of this MEF2-dependent transgene paralleled expression of MEF2 mRNAs in developing myogenic lineages and regions of the adult brain. However, it was not expressed in other cell types that express MEF2 transcripts. Tandem copies of the MEF2 site from the c-jun promoter directed expression in a similar pattern to the desmin MEF2 site, suggesting that transgene expression reflects the presence of transcriptionally active MEF2 proteins, rather than other factors specific for DNA sequences flanking the MEF2 site. These results demonstrate the presence of transcriptionally active MEF2 proteins in the early muscle and neural cell lineages during embryogenesis and argue against the existence of lineage-restricted MEF2 cofactors that discriminate between MEF2 sites with different immediate flanking sequences. The discordance between MEF2 mRNA expression and MEF2 transcriptional activity in nonmuscle cell types of embryos and adults also supports the notion that post-transcriptional mechanisms regulate the expression of MEF2 proteins.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/genética , Transcrição Gênica , Animais , Sítios de Ligação , Desmina/genética , Desenvolvimento Embrionário e Fetal , Feminino , Expressão Gênica , Óperon Lac , Fatores de Transcrição MEF2 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Miocárdio/metabolismo , Fatores de Regulação Miogênica , Precursores de Proteínas , Proteínas Proto-Oncogênicas c-jun/genética , Somitos , Transgenes
10.
Nature ; 400(6744): 581-5, 1999 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-10448862

RESUMO

Localized synthesis of insulin-like growth factors (IGFs) has been broadly implicated in skeletal muscle growth, hypertrophy and regeneration. Virally delivered IGF-1 genes induce local skeletal muscle hypertrophy and attenuate age-related skeletal muscle atrophy, restoring and improving muscle mass and strength in mice. Here we show that the molecular pathways underlying the hypertrophic action of IGF-1 in skeletal muscle are similar to those responsible for cardiac hypertrophy. Transfected IGF-1 gene expression in postmitotic skeletal myocytes activates calcineurin-mediated calcium signalling by inducing calcineurin transcripts and nuclear localization of calcineurin protein. Expression of activated calcineurin mimics the effects of IGF-1, whereas expression of a dominant-negative calcineurin mutant or addition of cyclosporin, a calcineurin inhibitor, represses myocyte differentiation and hypertrophy. Either IGF-1 or activated calcineurin induces expression of the transcription factor GATA-2, which accumulates in a subset of myocyte nuclei, where it associates with calcineurin and a specific dephosphorylated isoform of the transcription factor NF-ATc1. Thus, IGF-1 induces calcineurin-mediated signalling and activation of GATA-2, a marker of skeletal muscle hypertrophy, which cooperates with selected NF-ATc isoforms to activate gene expression programs.


Assuntos
Calcineurina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fator de Crescimento Insulin-Like I/fisiologia , Músculo Esquelético/patologia , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Animais , Inibidores de Calcineurina , Cardiomegalia/metabolismo , Linhagem Celular , Ciclosporina/farmacologia , Fator de Transcrição GATA2 , Regulação da Expressão Gênica , Hipertrofia , Fator de Crescimento Insulin-Like I/genética , Camundongos , Camundongos Transgênicos , Miocárdio/metabolismo , Fatores de Transcrição NFATC , Transdução de Sinais
11.
Proc Natl Acad Sci U S A ; 94(8): 3560-4, 1997 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-9108015

RESUMO

The gene encoding the hormone secretin is expressed only in enteroendocrine S cells and insulin-producing pancreatic beta cells during development. A 120-bp enhancer directs cell-specific expression of the rat secretin gene in secretin-expressing cells. The enhancer includes an E-box sequence, CAGCTG, which is important for transcriptional activity. To further characterize the role of the E box, a consensus binding site for basic helix-loop-helix (bHLH) proteins, we have examined factors that interact with this element in the secretin gene. The results suggest that transcription is activated by a recently isolated tissue-specific bHLH protein, BETA2, heterodimerized to the ubiquitously expressed bHLH proteins, Pan 1 and Pan 2, the rodent homologues of E47 and E12. The importance of BETA2 for transcriptional activation of secretin is further illustrated by antisense experiments inhibiting BETA2 expression in secretin-producing cell lines, which resulted in the inhibition of most E box-dependent transcription. Expression of BETA2 in a nonendocrine cell line conferred the ability to express secretin-reporter genes that are transcribed at minimal levels in the absence of BETA2. Secretin-producing enteroendocrine cells in the murine small intestine showed specific immunostaining with BETA2 antibodies, corroborating observations in cell lines. Thus BETA2 is to our knowledge the first transcription factor identified that specifically activates cell type-specific expression of an intestinal hormone gene.


Assuntos
Colo/metabolismo , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Secretina/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sequências Hélice-Alça-Hélice/genética , Humanos , Mamíferos , Proteínas do Tecido Nervoso/biossíntese , Ratos , Secretina/metabolismo
12.
J Biol Chem ; 275(7): 4545-8, 2000 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-10671477

RESUMO

Adult skeletal muscle fibers can be categorized into fast and slow twitch subtypes based on specialized contractile and metabolic properties and on distinctive patterns of muscle gene expression. Muscle fiber-type characteristics are dependent on the frequency of motor nerve stimulation and are thought to be controlled by calcium-dependent signaling. The calcium, calmodulin-dependent protein phosphatase, calcineurin, stimulates slow fiber-specific gene promoters in cultured skeletal muscle cells, and the calcineurin inhibitor, cyclosporin A, inhibits slow fiber gene expression in vivo, suggesting a key role of calcineurin in activation of the slow muscle fiber phenotype. Calcineurin has also been shown to induce hypertrophy of cardiac muscle and to mediate the hypertrophic effects of insulin-like growth factor-1 on skeletal myocytes in vitro. To determine whether activated calcineurin was sufficient to induce slow fiber gene expression and hypertrophy in adult skeletal muscle in vivo, we created transgenic mice that expressed activated calcineurin under control of the muscle creatine kinase enhancer. These mice exhibited an increase in slow muscle fibers, but no evidence for skeletal muscle hypertrophy. These results demonstrate that calcineurin activation is sufficient to induce the slow fiber gene regulatory program in vivo and suggest that additional signals are required for skeletal muscle hypertrophy.


Assuntos
Calcineurina/fisiologia , Regulação da Expressão Gênica/fisiologia , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Animais , Sequência de Bases , Creatina Quinase/genética , Primers do DNA , Elementos Facilitadores Genéticos , Camundongos , Camundongos Transgênicos , Fibras Musculares de Contração Lenta/enzimologia , Músculo Esquelético/enzimologia
13.
Genes Dev ; 11(18): 2323-34, 1997 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-9308961

RESUMO

Candidate transcription factors involved in pancreatic endocrine development have been isolated using insulin gene regulation as a paradigm. The cell-type restricted basic helix-loop-helix (bHLH) gene, BETA2/NeuroD, expressed in pancreatic endocrine cells, the intestine, and the brain, activates insulin gene transcription and can induce neurons to differentiate. To understand the importance of BETA2 in pancreatic endocrine cell differentiation, mice lacking a functional BETA2 gene were generated by gene targeting experiments. Mice carrying a targeted disruption of the BETA2 gene developed severe diabetes and died perinatally. Homozygous BETA2 null mice had a striking reduction in the number of insulin-producing beta cells and failed to develop mature islets. Islet morphogenesis appeared to be arrested between E14.5 and E17.5, a period characterized by major expansion of the beta cell population. The presence of severe diabetes in these mice suggests that proper islet structure plays an important role in blood glucose homeostasis. In addition, secretin- and cholecystokinin-producing enteroendocrine cells failed to develop in the absence of BETA2. The absence of these two pancreatic secretagogs may explain the abnormal cellular polarity and inability to secrete zymogen granules in pancreatic acinar exocrine cells. The nervous system appeared to develop normally, despite abundant expression of BETA2 in differentiating neurons. Thus, BETA2 is critical for the normal development of several specialized cell types arising from the gut endoderm.


Assuntos
Proteínas de Ligação a DNA/genética , Diabetes Mellitus Experimental/genética , Pâncreas/patologia , Transativadores/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Colecistocinina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Marcação de Genes , Sequências Hélice-Alça-Hélice/genética , Camundongos , Camundongos Mutantes , Morfogênese/genética , Pâncreas/metabolismo , Secretina/metabolismo , Transativadores/metabolismo
14.
Proc Natl Acad Sci U S A ; 97(2): 865-70, 2000 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-10639171

RESUMO

BETA2/NeuroD is a homologue of the Drosophila atonal gene that is widely expressed during development in the mammalian brain and pancreas. Although studies in Xenopus suggest that BETA2/NeuroD is involved in cellular differentiation, its function in the mammalian nervous system is unclear. Here we show that mutant mice homozygous for a deletion at the BETA2/NeuroD locus fail to develop a granule cell layer within the dentate gyrus, one of the principal structures of the hippocampal formation. To understand the basis of this abnormality, we analyzed dentate gyrus development by using immunocytochemical markers in BETA2/NeuroD-deficient mice. The early cell populations in the dentate gyrus, including Cajal-Retzius cells and radial glia, are present and appear normally organized. The migration of dentate precursor cells and newly born granule cells from the neuroepithelium to the dentate gyrus remains intact. However, there is a dramatic defect in the proliferation of precursor cells once they reach the dentate and a significant delay in the differentiation of granule cells. This leads to malformation of the dentate granule cell layer and excess cell death. BETA2/NeuroD null mice also exhibit spontaneous limbic seizures associated with electrophysiological evidence of seizure activity in the hippocampus and cortex. These findings thus establish a critical role of BETA2/NeuroD in the development of a specific class of neurons. Furthermore, failure to express BETA2/NeuroD leads to a stereotyped pattern of pathological excitability of the adult central nervous system.


Assuntos
Proteínas de Ligação a DNA/genética , Giro Denteado/patologia , Epilepsia/genética , Transativadores/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular , Divisão Celular , Giro Denteado/embriologia , Giro Denteado/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Hipocampo/citologia , Hipocampo/embriologia , Hipocampo/metabolismo , Hibridização In Situ , Sistema Límbico/metabolismo , Sistema Límbico/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Mutantes , Mutação , Neurônios/citologia , Neurônios/metabolismo , Fenótipo
15.
EMBO J ; 20(22): 6414-23, 2001 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11707412

RESUMO

Gene expression in skeletal muscles of adult vertebrates is altered profoundly by changing patterns of contractile work. Here we observed that the functional activity of MEF2 transcription factors is stimulated by sustained periods of endurance exercise or motor nerve pacing, as assessed by expression in trans genic mice of a MEF2-dependent reporter gene (desMEF2-lacZ). This response is accompanied by transformation of specialized myofiber subtypes, and is blocked either by cyclosporin A, a specific chemical inhibitor of calcineurin, or by forced expression of the endogenous calcineurin inhibitory protein, myocyte-enriched calcineurin interacting protein 1. Calcineurin removes phosphate groups from MEF2, and augments the potency of the transcriptional activation domain of MEF2 fused to a heterologous DNA binding domain. Across a broad range, the enzymatic activity of calcineurin correlates directly with expression of endogenous genes that are transcriptionally activated by muscle contractions. These results delineate a molecular pathway in which calcineurin and MEF2 participate in the adaptive mechanisms by which skeletal myofibers acquire specialized contractile and metabolic properties as a function of changing patterns of muscle contraction.


Assuntos
Calcineurina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ciclosporina/farmacologia , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Genes Reporter , Immunoblotting , Cinética , Fatores de Transcrição MEF2 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Contração Muscular , Músculo Esquelético/metabolismo , Fatores de Regulação Miogênica , Mioglobina/biossíntese , Condicionamento Físico Animal , Esforço Físico , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , beta-Galactosidase/metabolismo
16.
EMBO J ; 19(9): 1963-73, 2000 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-10790363

RESUMO

Different patterns of motor nerve activity drive distinctive programs of gene transcription in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Recently, we proposed that the influence of motor nerve activity on skeletal muscle fiber type is transduced to the relevant genes by calcineurin, which controls the functional activity of NFAT (nuclear family of activated T cell) proteins. Here we demonstrate that calcineurin-dependent gene regulation in skeletal myocytes is mediated also by MEF2 transcription factors, and is integrated with additional calcium-regulated signaling inputs, specifically calmodulin-dependent protein kinase activity. In skeletal muscles of transgenic mice, both NFAT and MEF2 binding sites are necessary for properly regulated function of a slow fiber-specific enhancer, and either forced expression of activated calcineurin or motor nerve stimulation up-regulates a MEF2-dependent reporter gene. These results provide new insights into the molecular mechanisms by which specialized characteristics of skeletal myofiber subtypes are established and maintained.


Assuntos
Calcineurina/metabolismo , Sinalização do Cálcio , Cálcio/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Calcineurina/genética , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Estimulação Elétrica , Elementos Facilitadores Genéticos/genética , Fatores de Transcrição MEF2 , Camundongos , Camundongos Transgênicos , Neurônios Motores/fisiologia , Fibras Musculares de Contração Rápida/citologia , Fibras Musculares de Contração Rápida/enzimologia , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares de Contração Lenta/citologia , Fibras Musculares de Contração Lenta/enzimologia , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Músculo Esquelético/inervação , Fatores de Regulação Miogênica , Fatores de Transcrição NFATC , Especificidade de Órgãos , Fosforilação , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Ativação Transcricional
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