TRPML1 agonist ML-SA5 attenuates pulmonary fibroblast activation by suppressing mTOR and restoring autophagic flux.

Modulating autophagic processes has shown promise as a treatment for pulmonary fibrosis (PF) with recent studies identifying transient receptor potential mucolipin-1 (TRPML1), a lysosomal ion channel, as a novel regulator involved in PF. However, the mechanism by which TRPML1 modulates autophagy in the context of PF remains incompletely understood. Here we establish in vitro PF models by exposing human embryonic lung fibroblasts (MRC-5) or primary lung fibroblasts to transforming growth factor (TGF-β1). We then assessed the effects of the TRPML1 agonist ML-SA5 on migratory capacity, the levels of fibrosis-related proteins and autophagy markers, and autophagic flux. Our findings show that TGF-β1 stimulation reduced TRPML1 protein levels, while ML-SA5 treatment suppressed TGF-β1-induced cell migration, reduced the expression of fibrotic markers, increased LC3II levels, and restored autophagic flux in lung fibroblasts. Finally, investigating the role of mammalian target of rapamycin (mTOR) signaling in these effects using rapamycin (an mTOR inhibitor) and MHY1485 (an mTOR activator), we found ML-SA5 inhibited mTOR signaling. Specifically, the anti-fibrotic effects of ML-SA5 were enhanced by co-treatment with rapamycin and reversed by MHY1485. Thus, we conclude that ML-SA5 attenuates TGF-β1-induced migration and collagen synthesis in pulmonary fibroblasts, at least in part through suppression of mTOR signaling and restoration of autophagic flux.