RESUMO
We determined the role of p38 mitogen-activated protein kinase (MAPK) in the increased airway smooth muscle (ASM) contractile responses following ozone and modulation by corticosteroids. Mice were exposed to air or ozone (3 ppm for 3 h) and isometric contractile responses of bronchial rings to acetylcholine (ACh) were measured using a myograph in the presence of p38 MAPK inhibitor, SB239063 (10â»6 M) or dexamethasone (10â»6 M). Because MAPK phosphatase (MKP)-1 is a negative regulator of p38 MAPK, we also studied these effects in MKP-1(-/-) mice. Bronchial rings from ozone-exposed wild-type and MKP-1(-/-) mice showed increased contractile responses, with a leftward shift of the dose-response curve in MKP-1(-/-) mice. SB239063 inhibited bronchial contraction equally in air- and ozone-exposed C57/BL6 and MKP-1(-/-) mice. Dexamethasone inhibited ACh-induced bronchial contraction in both air- and ozone-exposed C57/BL6 mice, but not in air- or ozone-exposed MKP-1(-/-) mice. ACh-stimulated p38 MAPK and heat shock protein (HSP)27 phosphorylation, as measured by Western blotting, and this effect was suppressed by SB239063 in C57/BL6 and MKP-1(-/-) mice, but not by dexamethasone in either air- or ozone-exposed MKP-1(-/-) mice. p38 MAPK plays a role in maximal ACh-induced isometric contractile responses and increased contractility induced by ozone. Dexamethasone inhibits ACh-induced ASM contraction through phosphorylation of p38 MAPK and HSP27.
Assuntos
Corticosteroides/farmacologia , Brônquios/patologia , Ozônio/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Acetilcolina/farmacologia , Animais , Brônquios/efeitos dos fármacos , Dexametasona/farmacologia , Fosfatase 1 de Especificidade Dupla/genética , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Imidazóis/farmacologia , Contração Isométrica , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso/metabolismo , Fosforilação , Pirimidinas/farmacologiaRESUMO
OBJECTIVES: The expression of the human cardiac troponin I (hTnIc) gene is developmentally regulated and tissue-specific. In analysing the putative binding elements within the proximal promoter, a CACC-box sequence overlapping a consensus Sp1 element has been identified. The aim of this study was to characterise the factors binding to this element and to determine their importance in the transcriptional activity of the promoter. METHODS: A combination of supershift and competition electrophoretic mobility shift assays (EMSA) were used to identify the binding of factors to the overlapping CACC-box/Sp1 consensus element. The functional importance of this element was tested by transient transfection into primary neonatal rat cardiac myocytes in culture. RESULTS: At least four factors were able to interact with this region including the zinc finger proteins Sp1, Sp3 and two potentially novel factors. Whereas both Sp1 and Sp3 bound to the consensus Sp1 element, and to a lesser extent the CACC-box, two of the complexes required the intact CACC-box for binding. Site-directed mutagenesis of this region showed that the CACC-box is essential for hTnIc promoter-reporter activity. Further characterisation using EMSA indicated that the factors binding the hTnIc CACC-box are unlikely to be zinc finger proteins as they are insensitive to the addition of divalent cation chelating agents. They were also unable to bind to other known CACC-box elements. These factors are present in both human and rat cardiac muscle but absent from a number of cell lines including several derived from skeletal muscle. CONCLUSION: The human cardiac troponin I gene promoter requires an upstream CACC-box element for full activity. This element binds at least two complexes which represent novel, tissue-restricted DNA-binding activity present in the heart which we have named HCB1 and HCB2 for heart CACC-box binding factors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Troponina I/genética , Animais , Técnicas de Cultura de Células , Linhagem Celular , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Plasmídeos , Ratos , Ratos Sprague-Dawley , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3 , Fatores de Transcrição/genética , Transfecção , Troponina I/metabolismo , Dedos de Zinco/genéticaRESUMO
We have used antibodies specific for troponin I proteins to examine human cardiac development and have detected a transiently expressed developmental isoform. This isoform is distinct from adult cardiac troponin I (TnIc) but is indistinguishable, on the basis of electrophoretic mobility and antibody reactivity, from the isoform found in slow skeletal muscle (TnIs). Furthermore, we show that mRNA for TnIs is present in fetal, but not adult, heart. Analysis of a developmental series of fetal samples indicates that there is a transition in expression from TnIs to TnIc which occurs between 20 weeks fetal and 9 months postnatal development.
Assuntos
Coração/embriologia , Miocárdio/metabolismo , Troponina/metabolismo , Sequência de Bases , Northern Blotting , Western Blotting , Humanos , Dados de Sequência Molecular , Músculos/embriologia , Músculos/metabolismo , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Troponina IRESUMO
BACKGROUND: The presence of lipids in alveolar macrophages (AMs) may impair their phagocytic response, and determine airway inflammation and obstruction. OBJECTIVE: To determine the factors such as severity of asthma, chronic cough, airway inflammation and obesity that may influence the presence of lipids in lung macrophages. METHODS: Bronchoalveolar lavage fluid (BALF) was obtained from 38 asthmatics (21 severe and 17 mild/moderate), 16 subjects with chronic cough and 11 healthy control subjects. The presence of lipids in macrophages was detected using an Oil-red-O stain and an index of lipid-laden macrophages (LLMI) was obtained. RESULTS: LLMI scores were higher in healthy subjects (median 48 [IQR 10-61]) and the severe asthma group (37 [11.5-61]) compared to mild/moderate asthmatics (7 [0.5-37]; p < 0.05 each). Subjects reporting a history of gastro-oesophageal reflux disease (GORD) had higher LLMI values (41.5 [11.3-138] versus 13 [0-39.3], p = 0.02). There was no significant correlation between LLMI and chronic cough, BAL cell differential counts, FEV1, FEV1/FVC or body mass index (BMI). CONCLUSIONS: The reduced LLMI in mild/moderate asthma may be related to lower incidence of GORD. However, this was not related to the degree of airflow obstruction, obesity or airway inflammation.
Assuntos
Asma/patologia , Líquido da Lavagem Broncoalveolar/química , Tosse/patologia , Lipídeos/análise , Macrófagos Alveolares/química , Adulto , Asma/metabolismo , Índice de Massa Corporal , Líquido da Lavagem Broncoalveolar/citologia , Broncoscopia , Estudos de Casos e Controles , Contagem de Células , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
Troponin I (TnI) is a constituent protein of the troponin complex located on the thin filament of striated muscle that provides a calcium-sensitive switch for striated muscle contraction. Unlike other contractile proteins, the cardiac isoform of troponin I (TnIc) is expressed only in cardiac muscle and therefore offers a model for cardiac-specific expression. It is also subject to developmental regulation with increased expression occurring at the time of birth. Here we describe the isolation and characterization of the human TnIc gene (HGMW-approved symbol TNNI3) and its promoter. The gene comprises eight exons contained within 6.2 kb of genomic DNA. The proximal promoter and 1.1-kb 5'-flanking region were sequenced, and several putative cis-acting elements that are conserved between the human and the mouse TnIc genes were identified. In addition, multiple copies of a 37-bp chromosome 19-specific mini-satellite sequence were identified within this region. Following transfection, 2300 bp of 5' sequence is active in both cardiac myocytes and skeletal muscle cells but is inactive in fibroblasts, indicating that it can drive expression but is insufficient to confer cardiac specificity.
Assuntos
Genes , Miocárdio/metabolismo , Troponina/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 19/genética , Clonagem Molecular , Coturnix , Éxons/genética , Expressão Gênica , Humanos , Íntrons/genética , Camundongos , Repetições de Microssatélites , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Alinhamento de Sequência , Homologia de Sequência , Transcrição Gênica , Transfecção , Troponina IRESUMO
The human cardiac troponin I (TnIc) gene exhibits both cardiac-specific and developmentally regulated expression. The structure and expression of this gene as well as the identification of putative regulatory elements have been described previously. This study shows that a minimal promoter containing 98 bp of sequence is sufficient to drive transcription in neonatal rat cardiac myocytes. This region contains several putative cis -regulatory elements including an Initiator element surrounding the start site of transcription, an A/T-rich (TATA/MEF-2) element, two GATA elements and a cytosine-rich region containing overlapping CACC box and Sp1 elements. Using electrophoretic mobility shift assays (EMSAs) this study demonstrates the binding of MEF-2, Oct-1, and recombinant TBP to the A/T-rich element and of GATA-4 to both GATA elements. The CACC/Sp element binds the zinc finger transcription factors Sp1 and Sp3 in addition to an unidentified complex present in neonatal rat cardiac myocytes. Mutation of each of these sites has a deleterious effect on promoter activity as assayed by transient transfection into cardiac myocytes. The data suggest that transcriptional activity of the human TnIc gene can be driven by a compact promoter region and that within this region GATA, MEF-2 Sp1 and CACC box-binding factors are required for optimal activity. Furthermore, a comparison with data obtained for identical elements in the promoters of rodent TnIc genes identifies differences between species which may be of consequence for species-specific promoter function.
Assuntos
Regulação da Expressão Gênica , Miocárdio/metabolismo , Regiões Promotoras Genéticas , Troponina I/genética , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Citosina , Humanos , Camundongos , Dados de Sequência Molecular , Miocárdio/citologia , Ratos , Ratos Sprague-Dawley , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The vertebrate heart forms as two concentric epithelial cylinders of myocardium and endocardium separated by an extended basement membrane matrix commonly referred to as cardiac jelly. Subsequent maturation involves a complex series of events including asymmetric changes in cell shape and division which contribute to bending and the formation of the bulboventricular loop, the formation of specialised tissues including endocardial cushion tissue of the atrioventricular (AV) and outflow tract regions, the development of conductive tissue and myocyte maturation leading to the overall pattern of expression characteristic of mature heart muscle. These processes depend on a precise spatial and temporal control of gene expression both of genes encoding regulatory molecules and those encoding structural components of the heart. In this chapter we address three aspects of cardiac development, namely, the determination of cell fate during formation of endocardial cushion tissue in the embryonic heart, transitions in troponin gene expression during fetal myocyte maturation, and the use of cloning techniques based on the polymerase chain reaction for identifying transcription factors present in the heart.
Assuntos
Expressão Gênica/fisiologia , Coração/embriologia , Troponina/genética , Animais , Sequência de Bases , Embrião de Galinha , DNA/química , Genes Homeobox/fisiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Fatores de Transcrição/fisiologiaRESUMO
Based on chromosomal mapping data, we recently revealed an unexpected linkage of troponin genes in the human genome: the six genes encoding striated muscle troponin I and troponin T isoforms are located at three chromosomal sites, each of which carries a troponin I-troponin T gene pair. Here we have investigated the organization of these genes at the DNA level in isolated P1 and PAC genomic clones and demonstrate close physical linkage in two cases through the isolation of individual clones containing a complete troponin I-troponin T gene pair. As an initial step toward fully characterizing this pattern of linkage, we have determined the organization and complete sequence of the locus encoding cardiac troponin I and slow skeletal troponin T and thereby also provide the first determination of the structure and sequence of a slow skeletal troponin T gene. Our data show that the genes are organized head to tail and are separated by only 2.6 kb of intervening sequence. In contrast to other troponin genes, and despite their close proximity, the cardiac troponin I and slow skeletal troponin T genes show independent tissue-specific expression. Such close physical linkage has implications for the evolution of the troponin gene families, for their regulation, and for the analysis of mutations implicated in cardiomyopathy.