Pirfenidone exerts anti-fibrotic effects through Inhibition of GLI transcription factors

Objective: Pirfenidone is an anti-fibrotic drug approved for the treatment of idiopathic pulmonary fibrosis (IPF) patients. Although pirfenidone exhibits anti-inflammatory, anti-oxidant and anti-fibrotic properties, the molecular mechanism underlying its protective effects still remains largely unknown. Results: We demonstrate that Pirfenidone decreases Hedgehog (Hh) pathway activity in IPF patients and in patient-derived primary lung fibroblasts. Exposure of human lung fibroblasts (HLF) to SAG, a Hh pathway agonist, increased expression of Hh target genes and stimulated cell migration and trans-differentiation into myofibroblasts. These effects were inhibited by co-treatment of the cells with pirfenidone. Depletion of the negative regulators of the Hh pathway such as Ptch1, SuFu and Gli3 revealed that pirfenidone directly interferes with the stability of glioma-associated oncogene homolog (GLI)2 transcription factor. Interestingly, Hh-triggered TGF-β1 expression potentiated Hh responsiveness of primary lung fibroblasts by elevating the available pool of GLI1/GLI2. In line with this notion, global gene expression analysis of lung fibroblasts revealed that Pirfenidone causes significant perturbation of GLI1 target genes, including Smad3, EGF, HGF receptor, and TDRD7. In selected patients, plasma samples prior to and during pirfenidone treatment were investigated and demonstrated decreased Hh activity during treatment. Conclusion: Pirfenidone exerts its clinically beneficial effects through dual Hh/TGF-β inhibition by targeting GLI proteins.