Regulation of a disintegrin and metalloprotease-33 expression by transforming growth factor-β.

Abstract

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)-β 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-β 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-β(2) to induce myofibroblast differentiation. ADAM33 expression was assessed using quantitative RT-PCR and Western blotting. To examine the mechanisms whereby TGF-β(2) affected ADAM33 expression, quantitative methylation-sensitive PCR, chromatin immunoprecipitation, and nuclear accessibility assays were conducted on the ADAM33 promoter. We found that TGF-β(2) caused a time- and concentration-dependent reduction in ADAM33 mRNA expression in normal and asthmatic fibroblasts, affecting levels of splice variants similarly. TGF-β(2) also induced ADAM33 protein turnover and appearance of a cell-associated C-terminal fragment. TGF-β(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-β(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-β(2)-induced ectodomain shedding of ADAM33.