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.

Introduction. Idiopathic Pulmonary Fibrosis (IPF) has been associated with aberrant reactivation of developmental pathways, namely the Hedgehog (HH) pathway, which is involved in epithelial cells-fibroblasts interaction. We reported that the activation of the HH pathway, from Smoothened (SMO, the mandatory transducer of the pathway) to the transcription factors GLI in the nucleus is required for the TGF-β1 induced myofibroblastic differentiation in human lung fibroblasts (Cigna et al. Am J Pathol 2012;181:2126). Aims. We hypothesized that inhibition of the HH pathway could decrease bleomycin-induced fibrosis in mice. Methods. After intratracheal injection of bleomycin, C57Bl6 mice were treated either with cyclopamine (CYC, an inhibitor of SMO) or with GANT-61 (an inhibitor of GLI transcription factors in the nucleus). At day 14, whole lung homogenates were obtained for morphological analysis, collagen quantification and evaluation of profibrotic (TGF-β1, CTGF, PAI-1, VEGF) and inflammatory mediators (IL-1β). Results. We showed that the HH pathway was activated in bleomycin-induced lung fibrosis, with increased mRNA expression and nuclear localization of GLI1 and GLI2 on day 14. Inhibition of SMO with CYC increased lung fibrosis and induced a profibrotic and proinflammatory environment. By contrast, the inhibition of GLI activity with GANT-61 decreased fibrosis histological score, collagen content, collagen expression and promoted an anti-fibrotic and anti-inflammatory environment. Conclusions. Inhibition of the HH pathway targeting GLI transcriptional activity may be seen as a potential therapeutic target in IPF.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease of unknown cause. Key signaling developmental pathways are aberrantly expressed in IPF. The hedgehog pathway plays a key role during fetal lung development and may be involved in lung fibrogenesis. We determined the expression pattern of several Sonic hedgehog (SHH) pathway members in normal and IPF human lung biopsies and primary fibroblasts. The effect of hedgehog pathway inhibition was assayed by lung fibroblast proliferation and differentiation with and without transforming growth factor (TGF)-β1. We showed that the hedgehog pathway was reactivated in the IPF lung. Importantly, we deciphered the cross talk between the hedgehog and TGF-β pathway in human lung fibroblasts. TGF-β1 modulated the expression of key components of the hedgehog pathway independent of Smoothened, the obligatory signal transducer of the pathway. Smoothened was required for TGF-β1-induced myofibroblastic differentiation of control fibroblasts, but differentiation of IPF fibroblasts was partially resistant to Smoothened inhibition. Furthermore, functional hedgehog pathway machinery from the primary cilium, as well as GLI-dependent transcription in the nucleus, was required for the TGF-β1 effects on normal and IPF fibroblasts during myofibroblastic differentiation. These data identify the GLI transcription factors as potential therapeutic targets in lung fibrosis.

Identification of new therapeutic targets in idiopathic pulmonary fibrosis (IPF) is required. We hypothesized that, in the adult lung, SHH pathway plays a critical role during the alveolar repair process and lung fibrogenesis.Methods: We determined the expression pattern of several members of the SHH pathway in lung biopsies and primary cultured fibroblasts from controls and IPF patients. We also examined the expression profile of SHH pathway after TGF-β stimulation in fibroblasts. We characterized the action of recombinant SHH and cyclopamine, an inhibitor of SHH pathway, on the effect of TGF-β on fibroblast differentiation markers.Results: In lung biopsies, the ligand SHH, the receptor Patched (PTC), the transmembrane protein Smoothened (SMO) and the transcription factors GLI 1-2-3 were detected in both control and IPF tissues. Compared to controls, PTC was decreased in IPF, both in epithelial cells and fibroblasts. SMO was maintained and GLI2 was localized in the nuclei in IPF. In vitro, there was no difference between control and IPF fibroblasts in the expression of HH signalling components. After TGF-β stimulation, PTC, SMO and GLI3 mRNA were strongly inhibited while GLI2 was increased. Cyclopamine inhibited TGF-β induced expression of myofibroblast differentiation markers in control fibroblasts. IPF fibroblasts were less sensitive to cyclopamine treatment. Though, addition of recombinant SHH had no effect.Conclusions: Our results show that the signaling pathway downstream of SMO is activated in IPF. SMO activity is also necessary to TGF-β induced myofibroblastic differentiation. These data support a pro-fibrotic action of the SHH pathway in IPF.

RATIONALE:Injury to alveolar epithelial cells is central to the pathophysiology of idiopathic pulmonary fibrosis (IPF). An abnormal autoimmune response directed against antigens of the alveolar epithelium may contribute to the disease.OBJECTIVES:To detect circulating autoantibodies (autoAbs) directed against epithelial structures.METHODS:We performed immunoblot by separating human placental amnion extract or alveolar epithelial cell (A549 cell line) proteins on polyacrylamide gels, blotting on nitrocellulose membranes, and incubating with serum from patients with IPF (n = 40) or healthy subjects (n = 40). Proteomic analysis and mass spectrometry characterized the target protein. Inhibition experiments performed with the correspondent recombinant protein confirmed our results.MEASUREMENTS AND MAIN RESULTS:We identified IgG autoAbs recognizing a 200-kD protein in the serum of patients with IPF. Proteomic analysis identified this protein as human periplakin (PPL), a component of desmosomes. Anti-PPL Abs were found by immunoblot in both serum and bronchoalveolar lavage in patients with IPF: 16/40 (40%) of them were positive versus none of the control subjects. Immunohistochemistry revealed that PPL was strongly expressed in bronchial and alveolar epithelium, but that PPL exhibited changes in intracellular localization among normal and fibrotic alveolar epithelium. In an alveolar epithelial wound repair assay, an anti-PPL IgG decreased cell migration. Recombinant PPL induced bronchoalveolar lavage T lymphocyte proliferation. Patients with IPF with anti-PPL Abs had a more severe respiratory disease, despite no difference in survival.CONCLUSIONS:We found a new circulating autoAb directed against PPL in patients with IPF, associated with a more severe disease.