Targeting the hedgehog-glioma-associated oncogene homolog pathway inhibits bleomycin-induced lung fibrosis in mice.

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-ß, connective tissue growth factor, plasminogen activator inhibitor 1, vascular endothelial growth factor-A) and proinflammatory mediators (IL-1ß) 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.

The hedgehog system machinery controls transforming growth factor-ß-dependent myofibroblastic differentiation in humans: involvement in idiopathic pulmonary fibrosis.

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)-ß1. We showed that the hedgehog pathway was reactivated in the IPF lung. Importantly, we deciphered the cross talk between the hedgehog and TGF-ß pathway in human lung fibroblasts. TGF-ß1 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-ß1-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-ß1 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 periplakin as a new target for autoreactivity in idiopathic pulmonary fibrosis.

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.

Acylase 1 expression in rat intestinal crypt--villus axis.

Acylase 1 was investigated at the cellular level in the rat small intestine along the enterocyte-differentiation axis. As confirmed by microscopic analysis, villus tip cells and crypt cells of rat jejunal mucosa were successfully separated using the Weiser method. The proliferating undifferentiated crypt cells showed much higher ACY 1 activity levels than the villus cells, with a 6.4-fold decrease as the cells migrated and differentiated along the crypt-villus axis. RT-PCR studies on mRNA extracted from isolated cells showed that ACY 1 mRNA was mainly expressed in crypt cells, reaching levels that were 12-fold higher than those recorded in other cell types along the whole enterocyte differentiation axis. It was concluded that the expression of ACY 1 in the intestinal crypt cells is regulated at the mRNA level. Immunohistochemistry revealed the expression of ACY 1 in the absorbing lineage cells from the ileal and colonic crypts and the absence of ACY 1 in the mucus producing goblet cells. These findings proposed ACY 1 as a new marker transcript for absorbing cells of intestinal crypt, which can be used to monitor the process of intestinal N-alpha-acetylated protein metabolism.