gdc-0449 and Pulmonary-Fibrosis

Hedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways.. The GLI inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the SMO inhibitor vismodegib was used to selectively target canonical hedgehog signalling. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-β (TGF-β) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-β receptor I.. TGF-β upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBR. Our data demonstrate that hedgehog pathways and TGF-β signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.

Topics: Adult; Aged; Anilides; Animals; Cells, Cultured; Collagen Type I; Connective Tissue Growth Factor; Female; Fibroblasts; Fibrosis; Gene Knockout Techniques; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Male; Mice; Mice, Knockout; Mice, Transgenic; Middle Aged; Plasminogen Activator Inhibitor 1; Protein Serine-Threonine Kinases; Pteridines; Pulmonary Fibrosis; Pyridines; Pyrimidines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recombinant Proteins; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Skin; Smad3 Protein; Smoothened Receptor; Transforming Growth Factor beta; Young Adult; Zinc Finger Protein Gli2

Idiopathic pulmonary fibrosis has been associated with the reactivation of developmental pathways, notably the Hedgehog-Glioma-associated oncogene homolog (GLI) pathway. In this study, we determined whether the Hedgehog pathway was activated in bleomycin-induced lung injury in mice, and whether targeting the Hedgehog-Gli pathway could decrease bleomycin-induced lung fibrosis. After intratracheal injection of bleomycin on Day 0, C57Bl6 mice received GDC-0449 (an inhibitor of Smoothened, the transducer of the pathway), or 2,2'-[[Dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N dimethylbenzenamine (GANT61; an inhibitor of GLI transcription factors in the nucleus), from Day 7 to Day 13. At Day 14, whole-lung homogenates were obtained for morphological analysis, assessment of cell apoptosis and proliferation, collagen quantification, and evaluation of profibrotic (transforming growth factor-β, connective tissue growth factor, plasminogen activator inhibitor 1, vascular endothelial growth factor-A) and proinflammatory mediators (IL-1β) expression. We showed that the Hedgehog pathway was activated in bleomycin-induced lung fibrosis on Day 14 after injury, with an increased lung expression of the ligand, Sonic Hedgehog, and with increased messenger RNA expression and nuclear localization of GLI1 and GLI2. Inhibition of Smoothened with GDC-0449 did not influence the development of bleomycin-induced lung fibrosis. By contrast, the inhibition of GLI activity with GANT61 decreased lung fibrosis and lung collagen accumulation, and promoted an antifibrotic and anti-inflammatory environment. Our results identify the hedgehog-Gli pathway as a profibrotic pathway in experimental fibrosis. Inhibition of the Hedgehog-Gli pathway at the level of GLI transcriptional activity could be a therapeutic option in fibrotic lung diseases.

Topics: Anilides; Animals; Antibiotics, Antineoplastic; Apoptosis; Bleomycin; Blotting, Western; Cell Proliferation; Collagen; Fluorescent Antibody Technique; Glioma; Hedgehog Proteins; Immunoenzyme Techniques; Kruppel-Like Transcription Factors; Male; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Pyridines; Pyrimidines; Real-Time Polymerase Chain Reaction; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smoothened Receptor; Transforming Growth Factor beta; Zinc Finger Protein GLI1