RESUMO
The asthma susceptibility gene, a disintegrin and metalloprotease-33 (ADAM33), is selectively expressed in mesenchymal cells, and the activity of soluble ADAM33 has been linked to angiogenesis and airway remodeling. Transforming growth factor (TGF)-ß is a profibrogenic growth factor, the expression of which is increased in asthma, and recent studies show that it enhances shedding of soluble ADAM33. In this study, we hypothesized that TGF-ß also affects ADAM33 expression in bronchial fibroblasts in asthma. Primary fibroblasts were grown from bronchial biopsies from donors with and those without asthma, and treated with TGF-ß(2) to induce myofibroblast differentiation. ADAM33 expression was assessed using quantitative RT-PCR and Western blotting. To examine the mechanisms whereby TGF-ß(2) affected ADAM33 expression, quantitative methylation-sensitive PCR, chromatin immunoprecipitation, and nuclear accessibility assays were conducted on the ADAM33 promoter. We found that TGF-ß(2) caused a time- and concentration-dependent reduction in ADAM33 mRNA expression in normal and asthmatic fibroblasts, affecting levels of splice variants similarly. TGF-ß(2) also induced ADAM33 protein turnover and appearance of a cell-associated C-terminal fragment. TGF-ß(2) down-regulated ADAM33 mRNA expression by causing chromatin condensation around the ADAM33 promoter with deacetylation of histone H3, demethylation of H3 on lysine-4, and hypermethylation of H3 on lysine-9. However, the methylation status of the ADAM33 promoter did not change. Together, these data suggest that TGF-ß(2) suppresses expression of ADAM33 mRNA in normal or asthmatic fibroblasts. This occurs by altering chromatin structure, rather than by gene silencing through DNA methylation as in epithelial cells. This may provide a mechanism for fine regulation of levels of ADAM33 expression in fibroblasts, and may self-limit TGF-ß(2)-induced ectodomain shedding of ADAM33.
Assuntos
Proteínas ADAM/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Proteínas ADAM/genética , Acetilação , Adulto , Diferenciação Celular , Imunoprecipitação da Cromatina , Feminino , Humanos , Masculino , Metilação , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Splicing de RNA , RNA Mensageiro/genéticaRESUMO
The ADAM (A Disintegrin and Metalloprotease) family of Zn++-dependent metalloproteases are multidomain proteins involved in diverse cellular activities. Polymorphic variation in ADAM33 is strongly associated with asthma and bronchial hyperresponsiveness. Identification of those isoforms of ADAM33 that are expressed in airways is fundamental to dissecting the role of ADAM33 in asthma. Analysis of primary human airways fibroblasts has shown the presence of a number of alternatively spliced forms of ADAM33, including one encoding a putative secreted variant, and many transcripts lacking the metalloproteinase domain. The relative abundance of these transcripts has been quantified using reverse transcription real-time polymerase chain reaction, in both nuclear and cytoplasmic fractions of RNA. These results demonstrate that a number of splice variants of ADAM33 are transported into the cytoplasm. Ninety percent of ADAM33 mRNA is retained in the nucleus and the subtle differences in the composition of nuclear and cytoplasmic RNA suggest important events in the splicing and selection of ADAM33 transcripts. Western blot analysis confirmed that several protein isoforms of ADAM33 are expressed in primary airways fibroblasts. These findings demonstrate that ADAM33 exists in multiple isoforms, suggesting that it is a complex molecule that plays multiple roles within mesenchymal cells.
Assuntos
Asma/genética , Brônquios/metabolismo , Fibroblastos/metabolismo , Metaloendopeptidases/genética , RNA Mensageiro/metabolismo , Proteínas ADAM , Adulto , Processamento Alternativo/genética , Asma/metabolismo , Asma/fisiopatologia , Brônquios/citologia , Brônquios/fisiopatologia , Núcleo Celular/genética , Células Cultivadas , Citoplasma/genética , Éxons/genética , Fibroblastos/citologia , Regulação da Expressão Gênica/genética , Humanos , Metaloendopeptidases/biossíntese , Polimorfismo Genético/genética , Isoformas de Proteínas/genética , Estrutura Terciária de Proteína/genética , Transcrição Gênica/genéticaRESUMO
Asthma is increasing in prevalence worldwide as a result of factors associated with a Western lifestyle. The prevalence and chronic nature of the disease represent significant economic burdens. Despite advances in understanding the inflammatory and immunologic components of asthma, there is relatively little understanding of the cellular and molecular mechanisms underlying the structural changes seen in the asthmatic lung (airway remodeling). These changes include hypertrophy of bronchial smooth muscle, transformation of fibroblasts to myofibroblasts, and deposition of subepithelial collagen. Airway remodeling is linked to bronchial hyperresponsiveness to diverse triggers and a steeper trajectory of long-term decrease in lung function in asthmatic patients. Until recently, these remodeling changes have been considered to be secondary phenomena, developing late in the disease process as a consequence of persistent inflammation. We discuss an alternative view of asthma pathogenesis by emphasizing the importance of the airway microenvironment (the epithelial mesenchymal trophic unit) in the origins of the disease. Our proposals are supported by the recent identification of ADAM33 as an asthma susceptibility gene, the expression of which is abundant in airway fibroblasts and smooth muscle but absent from T lymphocytes or inflammatory cells that infiltrate the airway wall in patients with asthma.
