TRPA1 function in human pulmonary fibroblasts

Abstract

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective Ca2+ permeable cation channel. While TRPA1 was mostly studied in neuronal cells, it was originally cloned from cultured human lung fibroblasts. TRPA1-mediated Ca2+ entry is evoked by exposure to several chemicals including allyl isothiocyanate (AITC) and inhaled components of cigarette smoke, suggesting a possible role of TRPA1 in progression of lung disease. However, a protective effect of TRPA1 activation in the development of cardiac fibrosis was proposed. Yet, the function of TRPA1 in transforming growth factor β1 (TGF-β1)-driven fibroblast to myofibroblast differentiation as well as the development of pulmonary fibrosis remains elusive. TRPA1 expression and function was analyzed in cultured primary human pulmonary fibroblasts (HPF) obtained from three different donors before and after incubation with TGF-β1. Transcriptome sequencing, qRT-PCR and immunoblotting were used to identify target genes on mRNA and protein levels. TRPA1 specific siRNAs were established to assess the role of the channel in cell function during fibroblast myofibroblasts differentiation. TRPA1 mRNA is highly expressed in HPF and levels were significantly reduced after adding TGF-β1. Expression of fibrosis markers, e.g. alpha smooth muscle actin (αSMA), plasminogen activator inhibitor (PAI), fibronectin (FN1) and collagen (Col1a1), was increased after down-regulation of TRPA1-mRNA by siRNA targeting in HPF. Moreover, TGF-β1 treatment of HPFs reduced Ca2+ entry evoked by the TRPA1 activator AITC, while increasing barrier function and contractile characteristics of the cells. Effects of siRNA-mediated knock-down of TRPA1 on cell contraction, migration and resistance of HPF will be quantified and compared to TGF-β1-induced fibroblast myofibroblast differentiation. Our results suggest an inhibitory function of TRPA1 channels in TGF-β1-driven fibroblast to myofibroblast differentiation. Therefore, activation of TRPA1 channels might protect from the development of pulmonary fibrosis in patients