Reemergence of Hedgehog Mediates Epithelial–Mesenchymal Crosstalk in Pulmonary Fibrosis

Abstract

Hedgehog signaling plays important roles in cell development and differentiation. In this study, the ability of Sonic Hedgehog (SHH) to induce myofibroblast differentiation was analyzed in isolated human lung fibroblasts, and its in vivo significance was evaluated in rodent bleomycin-induced pulmonary fibrosis. The results showed that SHH could induce myofibroblast differentiation in human lung fibroblasts in a Smo- and Gli1-dependent manner. Gel shift analysis, chromatin immunoprecipitation assay, and site-directed mutagenesis revealed that a Gli1 binding consensus in the α-SMA gene promoter was important for mediating SHH-induced myofibroblast differentiation. Analysis of Hedgehog reemergence in vivo revealed that of all three Hedgehog isoforms, only SHH was significantly induced in bleomycin-injured lung along with Gli1. The induction of SHH was only noted in epithelial cells, and its expression was undetectable in lung fibroblasts or macrophages. transforming growth factor (TGF)-β induced SHH significantly in cultured alveolar epithelial cells, whereas SHH induced TGF-β in lung fibroblasts. Pulmonary fibrosis and α-smooth muscle actin (α-SMA) expression were significantly reduced in mice that were Smo deficient only in type I collagen-expressing cells. Thus, the reemergence of SHH in epithelial cells could result in induction of myofibroblast differentiation in a Smo-dependent manner and subsequent Gli1 activation of the α-SMA promoter.

Sonic hedgehog signaling in kidney fibrosis: a master communicator

“…In vitro , it has been well documented that recombinant Shh protein induces an activated phenotype of cultured lung fibroblasts and hepatic stellate cells, as manifested by an augmented cell proliferation, migration, excessive ECM production (Hu et al, 2015; Yang et al, 2008). Shh signaling is upregulated in animal models and patients with idiopathic pulmonary fibrosis (IPF), other interstitial lung diseases and nonalcoholic fatty liver disease (NAFLD), with nuclear accumulation of Gli1, Gli2 in the fibrotic areas and an increased expression of the Shh downstream target genes.…”

“…In lung, Shh is predominantly expressed in type II like epithelial cells of terminal bronchioles and alveoli (Bolanos et al, 2012). Shh activation causes aberrant epithelium-fibroblast interactions and directly triggers pulmonary fibrosis after injury (Hu et al, 2015; McGowan and McCoy, 2013). Unlike lung, Shh is found in both ballooned hepatocyte and hepatic stellate cells in the liver of NAFLD, which eventually leads to liver fibrosis or cirrhosis (Hirsova and Gores, 2015; Rangwala et al, 2011).…”

Mesenchymal Deficiency of Notch1 Attenuates Bleomycin-Induced Pulmonary Fibrosis

“…After the tamoxifen treatment regimen, the Notch1 CKO and control mice were injected endotracheally with 2 U/kg body weight of bleomycin (Blenoxane; Mead Johnson, Ramsey, NJ) dissolved in sterile phosphate-buffered saline (PBS) as before. 44,45 The control group received the same volume of sterile PBS only. Daily tamoxifen treatment continued until they were sacrificed at the indicated time points after bleomycin treatment.…”

“…44,45,49 Cells cultured between passages 3 and 5 after primary culture were used in experiments as indicated.…”

“…44,45 Briefly, 21 days after the endotracheal injection of saline or bleomycin, mice were euthanized and the lungs were thoroughly perfused with saline to remove blood from the lung vascular beds. The lungs were then removed from the thoracic cavity, inflated, and fixed in formalin, followed by paraffin embedding, dividing into sections, and staining with trichrome staining.…”