S141 A disintegrin and metalloprotease (ADAM) 33 protein in patients with pulmonary sarcoidosis

Abstract

cellular matrix, resulting in loss of airway or alveolar function. Murine lineage tracing experiments have suggested that these fibroblasts may, in part, be derived from lung epithelium via epithelial to mesenchymal transition (EMT). Transforming Growth Factor-b1 (TGF-b1) has been shown to drive EMT both in vitro and in vivo. Previous work in our lab has shown that inflammatory stimuli such as Tumour Necrosis Factor-a (TNFa) can accentuate TGF-b1 driven EMT in primary bronchial epithelial cells (PBEC). This crosstalk between inflammatory and TGF-b1 signalling may have implications in the pathogenesis of fibrotic disorders of the lung including Bronchiolitis Obliterans Syndrome and Idiopathic Pulmonary Fibrosis. We hypothesised that TGF-b activated kinase-1 (TAK-1), a protein phosphorylated in response to both TGF-b1 and TNFa, may be a key convergent point and assessed its role in inflammatory accentuation of EMT. Methods PBEC isolated from stable lung transplant recipients were treated with TGF-b1 (10 ng/ml) and/or TNFa (20 ng/ml) and the phosphorylation and localisation of signalling molecules SMAD-3 and TAK-1 assessed (western blot, confocal microscopy). TAK-1 function was blocked using a selective inhibitor and by siRNA knockdown; effects on EMT marker expression were assessed (western blot, confocal microscopy). Results TAK-1 is phosphorylated in response to both TGF-b1 (161% increase) and TNFa (145%) with an accentuated phosphorylation upon co-treatment (516%, n¼3). Co-treatment also resulted in pronounced nuclear localisation of phosphorylated TAK-1, which has not previously been reported. Chemical Inhibition of TAK-1 phosphorylation significantly reduced TGF-b1 driven down-regulation of E-cadherin (22% inhibition) and up-regulation of Vimentin (78%) and Fibronectin (39%, p<0.05, n¼4). Furthermore, TAK-1 inhibition significantly reduced TNFa accentuated TGF-b1 driven down-regulation of E-cadherin (39%) and up-regulation of Fibronectin (87%) and Vimentin (258%, p<0.05, n¼4). These findings were independently validated via siRNA knockdown of TAK-1. Conclusions These findings suggest that TAK-1 is an important convergent point for inflammatory and TGF-b1 signalling, controlling the accentuation of EMT in primary human airway epithelium. Examination of TAK-1 activation and function in animal models of lung fibrosis may provide information on the potential for TAK-1 inhibition as a therapeutic target.