Search

Subepithelial fibrosis is one of the common pathological features of asthmatic airway remodeling. During subepithelial fibrosis, type I collagen becomes the most abundant extracellular protein component. Studies have shown that Notch signaling participates in the progression of fibrosis; however, whether Notch signaling is involved in regulating type I collagen expression in airway fibroblasts remains unclear. The aim of the present study was to examine whether Notch signaling can regulate type I collagen expression in airway fibroblasts and to explore the underlying molecular mechanisms. Here, the expression of Notch signaling components was examined in mouse L929 cells and human MRC-5 cells. After upregulating or downregulating Notch signaling in these cell lines, col1α1 and col1α2 expression was examined. Using gene reporter assays, site-directed mutagenesis, and ChIP assays, the role of Hes1 binding sites in both the mouse and human COL1A1 and COL1A2 promoters was investigated. This study revealed that Notch signaling-related molecules (including Notch1, Hes1, and others) are expressed in L929 and MRC-5 cells and that Notch signaling regulates the expression of col1α1 and col1α2 in both cell lines. Additionally, over-expression of the Notch intracellular domain resulted in activation of the COL1A1 and COL1A2 promoters, and site-directed mutagenesis reporter assays revealed that Hes1 proteins might augment both mouse and human COL1A1 and COL1A2 promoter activity. Furthermore, ChIP assays confirmed that Hes1 binds to the COL1A1 and COL1A2 promoters in both L929 and MRC-5 cells. Therefore, it is reasonable to assume that Notch signaling can directly upregulate COL1A1 and COL1A2 promoter activity through a Hes1-dependent mechanism, which could serve as a possible target for pharmacotherapy of airway subepithelial fibrosis.

Rationale Genistein is a dietary isoflavone and a broad-spectrum tyrosine kinase inhibitor contained in soy products. An epidemiologic study of asthma revealed that subjects with high consumption of dietary soy isoflavones had better lung function than those with lower intake. In a guinea pig model of allergic asthma, genistein reduced methacholine-induced bronchoconstriction. To explore the mechanisms underlying these observations, we tested genistein9s ability to block lung myofibroblast differentiation, a key phenotypic change in asthmatic airway remodeling. Methods Human fetal lung fibroblasts (IMR-90) were grown to subconfluence in DMEM containing 10% FBS, serum-deprived for 24 hours, and treated with genistein (10 μM) for an additional 24 hurs. Cells were then stimulated with TGF-b1 (2 ng/mL) for 24 hours, and a-smooth muscle actin (aSMA) expression, a marker of the myofibroblast phenotype, was assessed by both immunoblot and immunofluorescence microscopy. To determine the intracellular mechanism of inhibitory actions by genistein, we also assessed phosphorylation of Smad2 by immunoblot in the presence and absence of genistein after 30-minute stimulation with TGF-b1. Results Treatment with genistein resulted in 54.9 ± 17.1% reduction in TGF-b1 induced expression of aSMA. Immunofluorescence microscopy revealed a decrease in aSMA staining intensity and stress fiber formation in genistein-treated cells. In addition, Smad2 phosphorylation was inhibited by 71.1 ± 16.7% in genistein-treated cells. Conclusions These results demonstrate that genistein interferes with TGF-b1-stimulated myofibroblast differentiation and suggest that tyrosine kinase inhibition may have a role in modulation of asthmatic airway remodeling. Funded by NIH RO1HL072891, T32HL076139, The CHEST Foundation-GSK Clinical Research Trainee Award.