AITC inhibits fibroblast-myofibroblast transition via TRPA1-independent MAPK and NRF2/HO-1 pathways and reverses corticosteroids insensitivity in human lung fibroblasts

Abstract

Background Little is known on the role of transient receptor potential ankyrin 1 (TRPA1) in fibroblast—myofibroblast transition (FMT) that can lead to airway remodeling which is a major problem for severe asthma and fibrosis. Thus, this study investigated the effect of TRPA1 modulators on transforming growth factor beta 1(TGF-β1) -treated lung fibroblasts. Methods MRC-5 cells were preincubated with TGF-β1 for 24 h. TRPA1 agonist or antagonist were added and further incubated for 24 h. The changes in TRPA1 and alpha-smooth muscle actin (α-SMA) expressions by stimuli were evaluated using qRT-PCR, western blot and immunohistochemical analyses. Statistical significance was determined by using one- or two-way ANOVA, followed by Bonferroni’s post hoc analysis for comparison of multiple groups and paired 2-tailed Student’s t-test between 2 groups. Results MRC-5 cells treated by TGF-β1 significantly upregulated α-SMA mRNA expressions (P < 0.01), but downregulated TRPA1 gene expression (P < 0.001). Post-treatment of TRPA1 activator, allyl isothiocyanate (AITC), after TGF-β1 significantly downregulated the α-SMA gene induction (P < 0.01 at 24 h), protein expression (P < 0.05) and immunoreactivity with stress fibers (P < 0.05). On the other hand, TRPA1 antagonist HC-030031 did not prevent this effect, and instead tended to facilitate the suppressive effect of AITC when co-stimulated. AITC significantly increased phosphorylated- extracellular signal-regulated kinase (ERK) 1/2 and heme oxygenase (HO)-1 protein expressions (P < 0.05) in TGF-β1-treated cells. Combined inhibition with ERK1/2 mitogen-activated protein kinase (MAPK) and nuclear factor erythroid 2-related factor (NRF2) almost completely reversed AITC-induced α-SMA suppression (P < 0.05). Dexamethasone was not able to inhibit the upregulated α-SMA induction by TGF-β1. However, AITC improved dexamethasone-insensitive myodifferentiation in the presence of the corticosteroid (P < 0.01). Conclusion We found that AITC exerts protective effect on TGF-β1-induced α-SMA induction by activating ERK1/2 MAPK and NRF2/HO-1 pathways in lung fibroblasts. It also overcomes corticosteroids insensitivity in TGF-β1-induced α-SMA induction. TRPA1 antagonist modulates the suppressive effect, but not prevent it. AITC and TRPA1 antagonist may be therapeutic agents in treating chronic respiratory diseases.

Transient Receptor Potential (TRP) Channels in Airway Toxicity and Disease: An Update

“…The Si-RNA-mediated knock-down of TRPA1 increased the expression of profibrotic genes, while the stimulation of TRPA1 by its specific activator (allylisothiocyanate (AITC)) reduced transcriptional activity, most probably by an inhibitory linker phosphorylation of SMAD 2 (see , accessed date: 14 September 2022). Similar results were obtained in MRC5 cells and human myofibroblasts, where TRPA1 channel activation reduced the induction of fibrotic genes or induced cell death, respectively [ 136 , 137 ]. Thus, TRPA1 activity seems to inhibit fibroblast-to-myofibroblast differentiation, a hallmark of lung fibrosis development in clear contrast to TRPV4 and TRPC6 channels, which support fibrotic processes.…”

An Inhibitory Function of TRPA1 Channels in TGF-β1-driven Fibroblast to Myofibroblast Differentiation

“…In a recent publication, a similar effect was shown in MRC-5 and HF19 cell lines, which are both isolated from lungs of human fetuses and resemble some of the characteristics of human fibroblasts (41). TGF-β1 significantly down-regulated TRPA1 expression and AITC enhanced α-SMA gene and protein expression in these cell lines (41). HC-030031 as a TRPA1 antagonist however, failed to suppress the AITC-induced down-regulation of α-SMA and seemed to work synergistically with AITC (41).…”

Investigation of the Role of the TRPA1 Ion Channel in Conveying the Effect of Dimethyl Trisulfide on Vascular and Histological Changes in Serum-Transfer Arthritis

“…Others attributed protective outcomes to TRPA1 activation in mesenchymal cells. The TRPA1 agonist allyl isothiocyanate ameliorated the expression of α-SMA [35]. Activation of TRPA1 by polygodial in rheumatoid arthritis FLS led to necrosis of the cells and reduced proliferation [36].…”