Inhibition of Allergen-Induced Airway Remodeling in Smad 3-Deficient Mice

Abstract

Intracellular signaling pathways that converge on Smad 3 are used by both TGF-β and activin A, key cytokines implicated in the process of fibrogenesis. To determine the role of Smad 3 in allergen-induced airway remodeling, Smad 3-deficient and wild-type (WT) mice were sensitized to OVA and challenged by repetitive administration of OVA for 1 mo. Increased levels of activin A and increased numbers of peribronchial TGF-β1+ cells were detected in WT and Smad 3-deficient mice following repetitive OVA challenge. Smad 3-deficient mice challenged with OVA had significantly less peribronchial fibrosis (total lung collagen content and trichrome staining), reduced thickness of the peribronchial smooth muscle layer, and reduced epithelial mucus production compared with WT mice. As TGF-β and Smad 3 signaling are hypothesized to mediate differentiation of fibroblasts to myofibroblasts in vivo, we determined the number of peribronchial myofibroblasts (Col-1+ and α-smooth muscle actin+) as assessed by double-label immunofluorescence microscopy. Although the number of peribronchial myofibroblasts increased significantly in WT mice following OVA challenge, there was a significant reduction in the number of peribronchial myofibroblasts in OVA-challenged Smad 3-deficient mice. There was no difference in levels of eosinophilic airway inflammation or airway responsiveness in Smad 3-deficient compared with WT mice. These results suggest that Smad 3 signaling is required for allergen-induced airway remodeling, as well as allergen-induced accumulation of myofibroblasts in the airway. However, Smad 3 signaling does not contribute significantly to airway responsiveness.

Attenuation of Chronic Pulmonary Inflammation in A2B Adenosine Receptor Knockout Mice

“…Although a direct stimulation of TGF-b 1 secretion by adenosine has not been described in any respiratory cell, it is possible that, in this chronic model, A 2B receptors induce TGF-b 1 up-regulation indirectly through their effects on immune cell differentiation in the lung. Because TGF-b 1 has been implicated in smooth muscle and goblet cell hyperplasia/hypertrophy in mouse models of chronic airway allergen exposure (22,32), this may explain the observed decrease in thickness of the peribronchial smooth muscle layer and airway mucus expression in allergenchallenged A 2B KO mice compared with WT animals.…”

“…In the current study, we used an established mouse model of chronic pulmonary inflammation associated with Th2 cytokine expression, eosinophilic infiltration, and airway remodeling (21,22). Because the effects of A 2B receptor gene ablation on acute inflammatory responses were originally described in C57Bl/6 mice (19,20) and reproduced in our recent studies employing the same mouse strain (14, 15), we used animals on C57Bl/6 genetic background to model chronic pulmonary inflammation.…”

“…Therefore, we sought to determine if genetic removal of A 2B receptors would dampen a chronic inflammation associated with increased interstitial adenosine concentrations. For this purpose, we chose an established mouse model of allergen-induced chronic airway inflammation characterized by predominantly a Th2 type of immune response with eosinophilic infiltrations and increased airway mucus production (21,22). We initially documented that this model of chronic airway inflammation indeed results in increased extracellular adenosine levels in the mouse lungs.…”

“…To evaluate airway smooth muscle hyperplasia, lung sections were immunostained with diaminobenzideneperoxidase detection reagents with antibodies against a-smooth muscle actin (a-SMA) (clone 1A-4; Sigma) and smooth muscle myosin heavy chain (SMM) (Biomedical Technologies, Stoughton, MA), as previously described (27). Peribronchial smooth muscle layer thickness was estimated by dividing the area of a-SMA immunostaining by the length of bronchiolar basement membrane (22). Image analysis of digital photomicrographs (original magnification, 403) was performed with Image-Pro Plus software (Media Cybernetics, Bethesda, MD).…”

Pin1 Protein Regulates Smad Protein Signaling and Pulmonary Fibrosis

“…These results also suggest that the role of Pin1 in TGF-␤1 signaling probably shows cell-specific differences. Pin1 Ϫ/Ϫ Fb Are Defective in TGF-␤/Smad Signaling-Smad2 and -3 mediate TGF-␤1-induced ECM expression, play a criti- cal role in Fb differentiation and tissue fibrosis (23), and show persistent activation in BLM-exposed lung tissue (24). Therefore, we evaluated Smad-dependent transcription 3 days after infecting primary wild-type and knock-out Fb with lentivirus containing a Smad2/3 response element upstream of a luciferase reporter.…”