Introduction. The mesenchymal differentiation of alveolar epithelial cells induced by Transforming Growth Factor-beta1 (TGF-beta1), also called Epithelial Mesenchymal Transition (EMT), may contribute to Idiopathic Pulmonary Fibrosis (IPF). The Sonic Hedgehog (SHH) pathway is involved in epithelial cells-fibroblasts interaction during fetal lung development and lung fibrogenesis in adult lung. Previously, our laboratory has demonstrated that the SHH pathway is necessary to the action of TGF-beta 1 in human pulmonary fibroblasts (Cigna et al. in revision). Aims. We hypothesized that the SHH pathway could play a role in mesenchymal differentiation of alveolar epithelial cells induced by TGF-beta 1. Methods. The A549 cell line or primary alveolar epithelial murine cells are pre-treated 1h with agonists (recombinant SHH, Smoothened Agonist) or with inhibitors of the pathway (Cyclopamine, HPI-4, GANT61) in the absence or presence of TGF-beta 1 (1-5 ng/ml) for 48h in serum-free medium. The expression of E-Cadherin, N-Cadherin, and fibronectin is evaluated by real-time PCR, Western blotting and immunocytochemistry. The migratory capacity of A549 is also measured in these conditions. Results. Inhibition of the pathway via SMO/GLI abolishes the effect of TGF-beta 1 on the migration of epithelial cells but do not influence the effect of TGF-beta 1 on cell differentiation. By contrast, the inhibition of the HH pathway in the primary cilium with HPI-4 prevents and reverts the effect of TGF-beta 1 on epithelial cell differentiation. Conclusions. Our results indicate that the primary cilium controls the effect of TGF-beta 1 on A549 cells in vitro.
La Fibrose Pulmonaire Idiopathique (FPI) est une maladie devastatrice, d’etiologie inconnue, qui reste pour le moment incurable. Cette maladie est caracterisee par l’accumulation de fibroblastes et de proteines de la matrice extracellulaire dans les espaces aeriens distaux aboutissant a une destruction alveolaire et a une alteration des proprietes mecaniques du poumon. La physiopathologie de la FPI est mal connue mais de nombreuses etudes suggerent que la reactivation des voies impliquees dans le developpement contribue a l’accumulation de la matrice extra-cellulaire et au comportement anormal des cellules epitheliales et des fibroblastes.La voie Hedgehog (HH) joue un role crucial dans le developpement embryonnaire. Dans le developpement pulmonaire fœtal, la voie HH est impliquee dans les interactions epithelium-fibroblaste et controle la proliferation et la differenciation du mesenchyme. La voie HH a ete impliquee dans la fibrogenese, notamment dans le foie et le rein.L’objectif de cette these a ete de caracteriser la voie HH dans la fibrose pulmonaire chez l’homme et dans un modele de fibrose induite par la bleomycine chez la souris.Nous avons demontre que la voie HH est reactivee dans les tissus pulmonaires de patients atteints de FPI et dans le modele de fibrose pulmonaire chez la souris. Nous avons montre que le TGF-β1 activait la voie HH dans les fibroblastes pulmonaires humains et que l’inhibition pharmacologique de la voie HH au niveau des facteurs GLI inhibait l’effet du TGF-β1 in vitro. Par contre, ces inhibiteurs ne protegent pas les cellules epitheliales alveolaires de la transition epithelio-mesenchymateuse induite par le TGF-β1. In vivo, chez la souris, nous avons montre que le traitement par des inhibiteurs de Smoothened ne protegeait pas du developpement de la fibrose tandis que le GANT61, un inhibiteur de l’interaction des GLI avec l’ADN, inhibait la fibrose.En conclusion, nos resultats demontrent l’implication de la voie HH dans la fibrose pulmonaire et ouvrent des perspectives therapeutiques nouvelles.
de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
Induction of heme oxygenase-1, a stress-inducible enzyme with anti-inflammatory activity, reduces the immunogenicity of therapeutic factor VIII in experimental hemophilia A. In humans, heme oxygenase-1 expression is modulated by polymorphisms in the promoter of the heme oxygenase-1-encoding gene (HMOX1). We investigated the relationship between polymorphisms in the HMOX1 promoter and factor VIII inhibitor development in severe hemophilia A. We performed a case-control study on 99 inhibitor-positive patients and 263 patients who did not develop inhibitors within the first 150 cumulative days of exposure to therapeutic factor VIII. Direct sequencing and DNA fragment analysis were used to study (GT)n polymorphism and single nucleotide polymorphisms located at −1135 and −413 in the promoter of HMOX1. We assessed associations between the individual allele frequencies or genotypes, and inhibitor development. Our results demonstrate that inhibitor-positive patients had a higher frequency of alleles with large (GT)n repeats (L: n≥30), which are associated with lesser heme oxygenase-1 expression (odds ratio 2.31; 95% confidence interval 1.46–3.66; P
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.
