RESUMO
Adenine nucleotide translocases (ANTs) transport ADP and ATP through mitochondrial inner membrane, thus playing an essential role for energy metabolism of eukaryotic cells. Mice have three ANT paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. While knockout mice have been characterized with Ant1 and Ant4 genes, which resulted in exercise intolerance and male infertility, respectively, the role of the ubiquitously expressed Ant2 gene in animal development has not been fully demonstrated. Here, we generated Ant2 hypomorphic mice by targeted disruption of the gene, in which Ant2 expression is largely depleted. The mice showed apparently normal embryonic development except pale phenotype along with a reduced birth rate. However, postnatal growth was severely retarded with macrocytic anemia, B lymphocytopenia, lactic acidosis and bloated stomach, and died within 4 weeks. Ant2 depletion caused anemia in a cell-autonomous manner by maturation arrest of erythroid precursors with increased reactive oxygen species and premature deaths. B-lymphocyte development was similarly affected by Ant2 depletion, and splenocytes showed a reduction in maximal respiration capacity and cellular ATP levels as well as an increase in cell death accompanying mitochondrial permeability transition pore opening. In contrast, myeloid, megakaryocyte and T-lymphocyte lineages remained apparently intact. Erythroid and B-cell development may be particularly vulnerable to Ant2 depletion-mediated mitochondrial dysfunction and oxidative stress.
Assuntos
Translocador 2 do Nucleotídeo Adenina/deficiência , Linfócitos B/citologia , Eritrócitos/citologia , Eritropoese/fisiologia , Linfopoese/fisiologia , Translocases Mitocondriais de ADP e ATP/metabolismo , Translocador 2 do Nucleotídeo Adenina/genética , Translocador 2 do Nucleotídeo Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Anemia/genética , Anemia/metabolismo , Animais , Linfócitos B/metabolismo , Eritrócitos/metabolismo , Eritropoese/genética , Feminino , Técnicas de Inativação de Genes , Transtornos do Crescimento/genética , Transtornos do Crescimento/metabolismo , Humanos , Linfopoese/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismoRESUMO
Pnn (PNN) is a nuclear and cell adhesion-related protein. Previous work has suggested that Pnn/DRS/memA is a potential tumor suppressor involved in the regulation of cell adhesion and cell migration. Using the ecdysone-inducible mammalian expression system, a stable inducible GFP-tagged human Pnn gene (PNNGFP) expressing 293 cell line was created (EcR293-PNNGFP). Cells induced to express PNNGFP not only exhibited increased cell-cell adhesion but also exhibited changes in cell growth and cell cycle progression. cDNA array analyses, together with real time PCR, revealed that the effects of exogenously expressed Pnn on cellular behavior may be linked to the regulation of the expression of specific subset genes. This subset includes cell cycle-related genes such as p21(cip1/waf1), CDK4, CPR2; cell migration and invasion regulatory genes such as RhoA, CDK5, TIMP-1, MMP-7, and EMMPRIN; and MIC-1. Concordant with previous observations of Pnn-induced phenotype changes, genes coding for epithelial associated processes and cell division controls were elevated, while those coding for increased cell motility and cellular reorganizations were downregulated. We utilized p21 promoter-luciferase reporter constructs and demonstrated that a marked stimulation of p21 promoter activity in 293 cells correlated with increased Pnn expression. Taken together, these data indicate that Pnn may participate in the regulation of gene expression, thereby, positively promoting cell-cell adhesion, and negatively affecting cell migration and cell proliferation.
Assuntos
Moléculas de Adesão Celular/fisiologia , Ciclinas/biossíntese , Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , Adesão Celular/genética , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Linhagem Celular , Movimento Celular/genética , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , DNA Complementar/genética , Ecdisona/farmacologia , Perfilação da Expressão Gênica , Genes Reporter , Genes Sintéticos , Humanos , Rim/citologia , Luciferases/biossíntese , Luciferases/genética , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/fisiologia , Transcrição Gênica/genéticaRESUMO
Foregut development produces a characteristic sequence of gastrointestinal and respiratory organs, but the signaling pathways that ensure this developmental order remain largely unknown. Here, mutations of activin receptors ActRIIA and ActRIIB are shown to disrupt the development of posterior foregut-derived organs, including the stomach, pancreas, and spleen. Foregut expression of genes including Shh and Isl1 is shifted in mutant mice. The endocrine pancreas is particularly sensitive to the type and extent of receptor inactivation. ActRIIA(+/-)B(+/-) animals lack axial defects, but have hypoplastic pancreatic islets, hypoinsulinemia, and impaired glucose tolerance. Thus, activin receptor-mediated signaling regulates axial patterning, cell differentiation, and function of foregut-derived organs.
Assuntos
Padronização Corporal/fisiologia , Proteínas do Tecido Nervoso , Pâncreas/embriologia , Pâncreas/fisiologia , Receptores de Fatores de Crescimento/metabolismo , Transativadores , Receptores de Activinas Tipo II , Animais , Diferenciação Celular , Sistema Digestório/embriologia , Fenômenos Fisiológicos do Sistema Digestório , Teste de Tolerância a Glucose , Proteínas Hedgehog , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hiperplasia , Insulina/sangue , Proteínas com Homeodomínio LIM , Masculino , Camundongos , Camundongos Mutantes , Pâncreas/patologia , Proteínas/genética , Proteínas/metabolismo , Receptores de Fatores de Crescimento/genética , Baço/anormalidades , Baço/fisiologia , Estômago/anormalidades , Estômago/embriologia , Fatores de TranscriçãoRESUMO
The activin receptor-like kinase 1 (ALK1) is a type I receptor for transforming growth factor-beta (TGF-beta) family proteins. Expression of ALK1 in blood vessels and mutations of the ALK1 gene in human type II hereditary hemorrhagic telangiectasia patients suggest that ALK1 may have an important role during vascular development. To define the function of ALK1 during development, we inactivated the ALK1 gene in mice by gene targeting. The ALK1 homozygous embryos die at midgestation, exhibiting severe vascular abnormalities characterized by excessive fusion of capillary plexes into cavernous vessels and hyperdilation of large vessels. These vascular defects are associated with enhanced expression of angiogenic factors and proteases and are characterized by deficient differentiation and recruitment of vascular smooth muscle cells. The blood vessel defects in ALK1-deficient mice are reminiscent of mice lacking TGF-beta1, TGF-beta type II receptor (TbetaR-II), or endoglin, suggesting that ALK1 may mediate TGF-beta1 signal in endothelial cells. Consistent with this hypothesis, we demonstrate that ALK1 in endothelial cells binds to TGF-beta1 and TbetaR-II. Furthermore, the ALK1 signaling pathway can inhibit TGF-beta1-dependent transcriptional activation mediated by the known TGF-beta1 type I receptor, ALK5. Taken together, our results suggest that the balance between the ALK1 and ALK5 signaling pathways in endothelial cells plays a crucial role in determining vascular endothelial properties during angiogenesis.
Assuntos
Receptores de Ativinas Tipo I , Neovascularização Fisiológica , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Receptores de Ativinas , Animais , Capilares/fisiologia , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/citologia , Mutação , Fosfoproteínas/metabolismo , Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Smad , Proteína Smad5 , Transativadores/metabolismo , Transfecção , Regulação para CimaRESUMO
The type II activin receptors, ActRIIA and ActRIIB, have been shown to play critical roles in axial patterning and organ development in mice. To investigate whether their function is required for mesoderm formation and gastrulation as implicated in Xenopus studies, we generated mice carrying both receptor mutations by interbreeding the ActRIIA and ActRIIB knockout mutants. We found that embryos homozygous for both receptor mutations were growth arrested at the egg cylinder stage and did not form mesoderm. Further analyses revealed that ActRIIA(-/-)ActRIIB(+/-) and about 15% of the ActRIIA(-/-) embryos failed to form an elongated primitive streak, resulting in severe disruption of mesoderm formation in the embryo proper. Interestingly, we observed similar gastrulation defects in ActRIIA(-/-)nodal(+/-) double mutants, which, if they developed beyond the gastrulation stage, displayed rostral head defects and cyclopia. These results provide genetic evidence that type II activin receptors are required for egg cylinder growth, primitive streak formation, and rostral head development in mice.
Assuntos
Padronização Corporal/fisiologia , Desenvolvimento Embrionário e Fetal/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Receptores de Activinas Tipo II , Animais , Sequência de Bases , Padronização Corporal/genética , Cruzamentos Genéticos , Primers do DNA/genética , Desenvolvimento Embrionário e Fetal/genética , Feminino , Gástrula/citologia , Cabeça/embriologia , Hibridização In Situ , Mesoderma/citologia , Camundongos , Camundongos Knockout , Gravidez , Receptores de Fatores de Crescimento/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/fisiologiaRESUMO
The transcriptional coactivator and integrator p300 and its closely related family member CBP mediate multiple, signal-dependent transcriptional events. We have generated mice lacking a functional p300 gene. Animals nullizygous for p300 died between days 9 and 11.5 of gestation, exhibiting defects in neurulation, cell proliferation, and heart development. Cells derived from p300-deficient embryos displayed specific transcriptional defects and proliferated poorly. Surprisingly, p300 heterozygotes also manifested considerable embryonic lethality. Moreover, double heterozygosity for p300 and cbp was invariably associated with embryonic death. Thus, mouse development is exquisitely sensitive to the overall gene dosage of p300 and cbp. Our results provide genetic evidence that a coactivator endowed with histone acetyltransferase activity is essential for mammalian cell proliferation and development.
Assuntos
Desenvolvimento Embrionário e Fetal/genética , Dosagem de Genes , Transativadores/fisiologia , Animais , Encéfalo/anormalidades , Proteínas Estimuladoras de Ligação a CCAAT , Divisão Celular , Células Cultivadas , Cruzamentos Genéticos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Coração/embriologia , Heterozigoto , Histona Acetiltransferases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Defeitos do Tubo Neural/genética , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Coativador 3 de Receptor Nuclear , Receptores do Ácido Retinoico/genética , Transativadores/genética , Transcrição GênicaRESUMO
Vertebrate animals exhibit segmented axial skeletons and lateral asymmetry of the visceral organs. The segment identity of individual vertebrae is believed to be determined by a combination of functionally active Hox genes that have defined expression boundaries along the anteroposterior axis (known as the axial Hox code). Disturbance of the Hox code by ectopic expression or mutation of Hox genes often leads to homeotic transformation of the vertebrae. Largely unknown, however, are the signaling molecules that provide the positional cues for the precise establishment and maintenance of the Hox code. In this study we show that disruption of the type IIB activin receptor (ActRIIB) by gene targeting results in altered expression of multiple Hox genes and abnormal patterning of the vertebrae, similar to but severer than retinoic acid (RA)-induced anterior transformation. We further show that RA and ActRIIB mutation have synergistic effects on vertebral patterning. Activin, Vg-1 and, type II activin receptors have been implicated in regulation of lateral asymmetry during chick and Xenopus development. We show here that the ActRIIB-/- mice die after birth with complicated cardiac defects including randomized heart position, malposition of the great arteries, and ventricular and atrial septal defects. In addition, the heart anomalies are associated with right pulmonary isomerism and splenic abnormalities, recapitulating the clinical symptoms of the human asplenia syndrome. These findings provide genetic evidence that the ActRIIB-mediated signaling pathway plays a critical role in patterning both anteroposterior and left-right axes in vertebrate animals.
Assuntos
Padronização Corporal , Desenvolvimento Embrionário e Fetal/genética , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Receptores de Ativinas , Animais , Osso e Ossos/embriologia , Genes Homeobox , Mutação em Linhagem Germinativa , Cardiopatias Congênitas/genética , Camundongos , Receptores de Fatores de Crescimento/genéticaRESUMO
It has been a controversial issue as to how many DNA cytosine methyltransferase mammalian cells have and whether de novo methylation and maintenance methylation activities are encoded by a single gene or two different genes. To address these questions, we have generated a null mutation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-somite stage. Surprisingly, the null mutant embryonic stem cells are viable and contain low but stable levels of methyl cytosine and methyltransferase activity, suggesting the existence of a second DNA methyltransferase in mammalian cells. Further studies indicate that de novo methylation activity is not impaired by the mutation as integrated provirus DNA in MoMuLV-infected homozygous embryonic stem cells become methylated at a similar rate as in wild-type cells. Differentiation of mutant cells results in further reduction of methyl cytosine levels, consistent with the de novo methylation activity being down regulated in differentiated cells. These results provide the first evidence that an independently encoded DNA methyltransferase is present in mammalian cells which is capable of de novo methylating cellular and viral DNA in vivo.
Assuntos
DNA-Citosina Metilases/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Metilação de DNA , DNA Viral/metabolismo , DNA-Citosina Metilases/genética , Desenvolvimento Embrionário , Feminino , Deleção de Genes , Camundongos , Vírus da Leucemia Murina de Moloney/genética , Gravidez , Provírus/genéticaRESUMO
We have isolated overlapping cDNAs encoding the N-terminal non-triple-helical region of mouse alpha 1(XVIII) collagen and shown that three different variants of alpha 1(XVIII) collagen exist. Each of the three variants shows characteristic tissue-specific expression patterns. Immunohistochemical studies show positive staining for alpha 1(XVIII) collagen along the basement membrane zones of vessels in the intestinal villi, the choroid plexus, skin, liver, and kidney. Thus, we conclude that alpha 1(XVIII) collagen may interact (directly or indirectly) with components in basement membrane zones or on the basal surface of endothelial/epithelial cells.
Assuntos
Processamento Alternativo , Membrana Basal/química , Colágeno/genética , Variação Genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Vasos Sanguíneos/química , Northern Blotting , Clonagem Molecular , Colágeno/biossíntese , Colágeno/isolamento & purificação , DNA Complementar/genética , Embrião de Mamíferos/química , Embrião de Mamíferos/citologia , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Distribuição TecidualRESUMO
We have isolated overlapping mouse cDNAs encoding a collagenous polypeptide that we have designated alpha 1(XVIII) collagen. Nucleotide sequence analysis shows that alpha 1(XVIII) collagen contains 10 triple-helical domains separated and flanked by non-triple-helical regions. Within the non-triple-helical regions, there are several Ser-Gly-containing sequences that conform to consensus sequences for glycosaminoglycan attachment sites in proteoglycan core proteins. Northern blots show that alpha 1(XVIII) transcripts are present in multiple organs, with the highest levels in liver, lung, and kidney. We have also isolated overlapping cDNAs encoding human alpha 1(XV) collagen, and their sequence extends a published partial alpha 1(XV) sequence to the 3' end. Comparison of the alpha 1(XV) and alpha 1(XVIII) sequences reveals a striking similarity in the lengths of the six most carboxyl-terminal triple-helical domains. In addition, within the carboxyl non-triple-helical domain NC1 of the two chains, a region of 177 amino acid residues shows about 60% identity at the amino acid level. We suggest, therefore, that alpha 1(XV) and alpha 1(XVIII) collagens are structurally related. Their structure is different from that of other known collagen types. We conclude that they belong to a subfamily of extracellular matrix proteins and we suggest the designation multiplexins (for protein with multiple triple-helix domains and interruptions) for members of this subfamily.
Assuntos
Colágeno/biossíntese , Colágeno/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Colágeno/genética , DNA Complementar/química , DNA Complementar/isolamento & purificação , Biblioteca Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Família Multigênica , Estrutura Secundária de Proteína , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
Types XV and XVIII collagen belong to a unique and novel subclass of the collagen superfamily for which we have proposed the name the MULTIPLEXIN family. Members of this class contain polypeptides with multiple triple-helical domains separated and flanked by non-triple-helical regions. In this paper, we report the isolation of human cDNAs and genomic DNAs encoding the alpha 1(XVIII) collagen chain. Utilizing a genomic clone as probe, we have mapped the COL18A1 gene to chromosome 21q22.3 by fluorescence in situ hybridization. In addition, using an interspecific backcross panel, we have shown that the murine Col18a1 locus is on chromosome 10, close to the loci for Col6a1 and Col6a2.
Assuntos
Cromossomos Humanos Par 10 , Cromossomos Humanos Par 21 , Colágeno/genética , DNA Complementar/genética , DNA/genética , Família Multigênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Colágeno/classificação , Cruzamentos Genéticos , Feminino , Humanos , Hibridização Genética , Hibridização in Situ Fluorescente , Masculino , Camundongos/genética , Dados de Sequência Molecular , Muridae/genética , Especificidade de Órgãos , Alinhamento de Sequência , Homologia de Sequência , Especificidade da EspécieRESUMO
Polyclonal antibodies were raised in rabbits against a fusion peptide representing a portion of the amino-terminal non-triple-helical domain of mouse type XII collagen. The antibodies reacted with bands of 220 and 350 kDa on Western blots of mouse tissue extracts. Immunohistochemical analyses of mouse embryos demonstrated that type XII collagen is expressed mainly in dense connective tissues of tendons, ligaments, dermis, cornea, blood vessel walls, meninges, and developing membranous bones. Comparison of skin extracts and medium of cultured mouse skin fibroblasts by Western blotting showed that while tissue contain short 220 kDa type XII collagen polypeptides as well as the long form, cultured cells produce mainly the long form with 350 kDa polypeptides.
Assuntos
Colágeno/biossíntese , Animais , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Células Cultivadas/metabolismo , Colágeno/química , Córnea/embriologia , Córnea/metabolismo , Fibroblastos/metabolismo , Imuno-Histoquímica , Ligamentos/embriologia , Ligamentos/metabolismo , Camundongos , Camundongos Endogâmicos , Proteínas Recombinantes de Fusão/química , Tendões/embriologia , Tendões/metabolismoRESUMO
Type XII collagen is a member of the FACIT (fibril-associated collagens with interrupted triple helices) group of extracellular matrix proteins. Like the other members of this group, collagen types IX and XIV, type XII has alternating triple-helical and non-triple-helical domains. Because of its structure, its association with collagen fibrils, and its distribution in dense connective tissues, type XII is thought possibly to act as a cross-bridge between fibrils and resist shear forces caused by tension. A portion of the ffuse gene was isolated by screening a genomic library with a chicken alpha 1 (XII) cDNA probe, followed by subcloning and sequence analysis. Comparison of exon sequences with the sequence of a mouse cDNA clone allowed the mouse gene to be identified as the alpha 1 (XII) collagen gene. In the mouse, Col12a1 is located on chromosome 9, as determined by linkage analysis using DNA from interspecific backcrosses with Mus spretus. Screening of a human genomic library also allowed the isolation of a human alpha 1(XII)-like gene (CoL12A1). This gene was mapped to chromosome 6 by blot hybridization to DNA from human/hamster hybrid cell lines. This information should prove useful in determining the role of type XII collagen genes as candidate genes in inheritable connective tissue diseases.