Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation

Abstract

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Several profibrotic factors drive pulmonary fibrosis, with transforming growth factor-beta (TGF-beta) being the most established. TGF-beta promotes transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. Anoctamin-1 (ANO1), also known as TMEM16A, is a calcium-activated chloride channel. We found that TGF-beta robustly upregulates ANO1 expression in human lung fibroblasts (HLF) at mRNA and protein levels. Consistent, ANO1 was readily detected in fibrotic areas of IPF lungs. TGF-beta treatment of HLF resulted in a significant increase in steady state accumulation of intracellular chloride concentration, which was prevented by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 or ANO1 siRNA significantly inhibited TGF-beta-induced myofibroblast differentiation as determined by the expression of smooth muscle alpha-actin, collagen-1 and fibronectin. Mechanistically, pharmacological or knockdown-mediated inhibition of ANO1 did not have an effect on the initial TGF-beta signaling (Smad2 phosphorylation), but it blocked downstream TGF-beta signaling including Rho pathway (assessed by phosphorylation of myosin light chain) and AKT activation. Together, these data demonstrate that ANO1 is a TGF-beta-inducible chloride channel that largely contributes to the increase in intracellular chloride concentration in TGF-beta-treated cells. Furthermore, ANO1 mediates TGF-beta-induced myofibroblast differentiation, at least partially through activation of Rho pathway and of AKT.