BARD1 mediates TGF-β signaling in pulmonary fibrosis

Background Idiopathic pulmonary fibrosis (IPF) is a rapid progressive fibro-proliferative disorder with poor prognosis similar to lung cancer. The pathogenesis of IPF is uncertain, but loss of epithelial cells and fibroblast proliferation are thought to be central processes. Previous reports have shown that BARD1 expression is upregulated in response to hypoxia and associated with TGF-β signaling, both recognized factors driving lung fibrosis. Differentially spliced BARD1 isoforms, in particular BARD1β, are oncogenic drivers of proliferation in cancers of various origins. We therefore hypothesized that BARD1 and/or its isoforms might play a role in lung fibrosis. Methods We investigated BARD1 expression as a function of TGF-β in cultured cells, in mice with experimentally induced lung fibrosis, and in lung biopsies from pulmonary fibrosis patients. Results FL BARD1 and BARD1β were upregulated in response to TGF-β in epithelial cells and fibroblasts in vitro and in vivo. Protein and mRNA expression studies showed very low expression in healthy lung tissues, but upregulated expression of full length (FL) BARD1 and BARD1β in fibrotic tissues. Conclusion Our data suggest that FL BARD1 and BARD1β might be mediators of pleiotropic effects of TGF-β. In particular BARD1β might be a driver of proliferation and of pulmonary fibrosis pathogenesis and progression and represent a target for treatment

Canonical wnt signaling activity in early stages of chick lung development

Wnt signaling pathway is an essential player during vertebrate embryonic development which has been associated with several developmental processes such as gastrulation, body axis formation and morphogenesis of numerous organs, namely the lung. Wnt proteins act through specific transmembrane receptors, which activate intracellular pathways that regulate cellular processes such as cell proliferation, differentiation and death. Morphogenesis of the fetal lung depends on epithelial-mesenchymal interactions that are governed by several growth and transcription factors that regulate cell proliferation, fate, migration and differentiation. This process is controlled by different signaling pathways such as FGF, Shh and Wnt among others. Wnt signaling is recognized as a key molecular player in mammalian pulmonary development but little is known about its function in avian lung development. The present work characterizes, for the first time, the expression pattern of several Wnt signaling members, such as wnt-1, wnt-2b, wnt-3a, wnt-5a, wnt-7b, wnt-8b, wnt-9a, lrp5, lrp6, sfrp1, dkk1, β-catenin and axin2 at early stages of chick lung development. In general, their expression is similar to their mammalian counterparts. By assessing protein expression levels of active/total β-catenin and phospho-LRP6/LRP6 it is revealed that canonical Wnt signaling is active in this embryonic tissue. In vitro inhibition studies were performed in order to evaluate the function of Wnt signaling pathway in lung branching. Lung explants treated with canonical Wnt signaling inhibitors (FH535 and PK115-584) presented an impairment of secondary branch formation after 48 h of culture along with a decrease in axin2 expression levels. Branching analysis confirmed this inhibition. Wnt-FGF crosstalk assessment revealed that this interaction is preserved in the chick lung. This study demonstrates that Wnt signaling is crucial for precise chick lung branching and further supports the avian lung as a good model for branching studies since it recapitulates early mammalian pulmonary development.Rute S. Moura was supported by a grant of ON.2 SR&TD Integrated Program (N-01-01-0124-01-07), ref: UMINHO/BPD/31/2013. The funders had no role in study design, data collection and analysis

From arginine methylation to ADMA: A novel mechanism with therapeutic potential in chronic lung diseases

Protein arginine methylation is a novel posttranslational modification regulating a diversity of cellular processes, including protein-protein interaction, signal transduction, or histone function. It has recently been shown to be dysregulated in chronic renal, vascular, and pulmonary diseases, and metabolic products originating from protein arginine methylation have been suggested to serve as biomarkers in cardiovascular and pulmonary diseases

Functional Wnt Signaling Is Increased in Idiopathic Pulmonary Fibrosis

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease, characterized by distorted lung architecture and loss of respiratory function. Alveolar epithelial cell injury and hyperplasia, enhanced extracellular matrix deposition, and (myo)fibroblast activation are features of IPF. Wnt/beta-catenin signaling has been shown to determine epithelial cell fate during development. As aberrant reactivation of developmental signaling pathways has been suggested to contribute to IPF pathogenesis, we hypothesized that Wnt/beta-catenin signaling is activated in epithelial cells in IPF. Thus, we quantified and localized the expression and activity of the Wnt/beta-catenin pathway in IPF. METHODOLOGY/PRINCIPAL FINDINGS: The expression of Wnt1, 3a, 7b, and 10b, the Wnt receptors Fzd1-4, Lrp5-6, as well as the intracellular signal transducers Gsk-3beta, beta-catenin, Tcf1, 3, 4, and Lef1 was analyzed in IPF and transplant donor lungs by quantitative real-time (q)RT-PCR. Wnt1, 7b and 10b, Fzd2 and 3, beta-catenin, and Lef1 expression was significantly increased in IPF. Immunohistochemical analysis localized Wnt1, Wnt3a, beta-catenin, and Gsk-3beta expression largely to alveolar and bronchial epithelium. This was confirmed by qRT-PCR of primary alveolar epithelial type II (ATII) cells, demonstrating a significant increase of Wnt signaling in ATII cells derived from IPF patients. In addition, Western blot analysis of phospho-Gsk-3beta, phospho-Lrp6, and beta-catenin, and qRT-PCR of the Wnt target genes cyclin D1, Mmp 7, or Fibronectin 1 demonstrated increased functional Wnt/beta-catenin signaling in IPF compared with controls. Functional in vitro studies further revealed that Wnt ligands induced lung epithelial cell proliferation and (myo)fibroblast activation and collagen synthesis. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that the Wnt/beta-catenin pathway is expressed and operative in adult lung epithelium. Increased Wnt/beta-catenin signaling may be involved in epithelial cell injury and hyperplasia, as well as impaired epithelial-mesenchymal cross-talk in IPF. Thus, modification of Wnt signaling may represent a therapeutic option in IPF

Vessel-Associated Transforming Growth Factor-Beta1 (TGF-β1) Is Increased in the Bronchial Reticular Basement Membrane in COPD and Normal Smokers

BACKGROUND: Transforming growth factor-beta1 (TGF-β1) is a multipotential cytokine with angiogenic activity. There are only limited data about its role in airway remodeling in COPD. We have previously shown that the reticular basement membrane (Rbm) is hypervascular in the airways of current smokers either with or without chronic obstructive pulmonary disease (COPD). This study evaluated TGF-β1 immunostaining in the Rbm and its relationship to vascularity in smokers with or without COPD. METHODOLOGY/PRINCIPAL FINDINGS: Bronchial biopsies from 15 smokers with normal lung function, 19 current and 14 ex-smokers with COPD were immunostained for TGF-β1 antibody and compared to 17 healthy controls. The percentage area of tissue and also number and area of vessels staining positively for TGF-β1 were measured and compared between groups. Some bronchial biopsies from current smoking COPD subjects were also stained for phosphorylated (active) Smad2/3. Epithelial TGF- β1 staining was not different between COPD current smokers and normal controls. TGF-β1 stained vessels in the Rbm were increased in smokers with normal lung function, current smoking COPD and ex-smokers with COPD compared to controls [median (range) for number of vessels/mm Rbm 2.5 (0.0-12.7), 3.4 (0.0-8.1) and 1.0 (0.0-6.3) vs. 0.0 (0.0-7.0), p<0.05]. Percentage of vessels stained was also increased in these clinical groups. Preliminary data suggest that in current smoking COPD subjects endothelial cells and cells in the Rbm stain positively for phosphorylated Smad2/3 suggesting TGF-β1 is functionally active in this situation. CONCLUSIONS/SIGNIFICANCE: Vessel-associated TGF-β1 activity is increased in the bronchial Rbm in smokers and especially those with COPD

Down-Regulation of the Canonical Wnt β-Catenin Pathway in the Airway Epithelium of Healthy Smokers and Smokers with COPD

Background: The Wnt pathway mediates differentiation of epithelial tissues; depending on the tissue types, Wnt can either drive or inhibit the differentiation process. We hypothesized that key genes in the Wnt pathway are suppressed in the human airway epithelium under the stress of cigarette smoking, a stress associated with dysregulation of the epithelial differentiated state. Methodology/Principal Findings: Microarrays were used to assess the expression of Wnt-related genes in the small airway epithelium (SAE) obtained via bronchoscopy and brushing of healthy nonsmokers, healthy smokers, and smokers with COPD. Thirty-three of 56 known Wnt-related genes were expressed in the SAE. Wnt pathway downstream mediators b-catenin and the transcription factor 7-like 1 were down-regulated in healthy smokers and smokers with COPD, as were many Wnt target genes. Among the extracellular regulators that suppress the Wnt pathway, secreted frizzled-related protein 2 (SFRP2), was up-regulated 4.3-fold in healthy smokers and 4.9-fold in COPD smokers, an observation confirmed by TaqMan Real-time PCR, Western analysis and immunohistochemistry. Finally, cigarette smoke extract mediated up-regulation of SFRP2 and down-regulation of Wnt target genes in airway epithelial cells in vitro. Conclusions/Significance: Smoking down-regulates the Wnt pathway in the human airway epithelium. In the context that Wnt pathway plays an important role in differentiation of epithelial tissues, the down-regulation of Wnt pathway ma

Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis

The transforming growth factor-β (TGF-β) signalling pathway is a key mediator of fibroblast activation that drives the aberrant synthesis of extracellular matrix in fibrotic diseases. Here we demonstrate a novel link between transforming growth factor-β and the canonical Wnt pathway. TGF-β stimulates canonical Wnt signalling in a p38-dependent manner by decreasing the expression of the Wnt antagonist Dickkopf-1. Tissue samples from human fibrotic diseases show enhanced expression of Wnt proteins and decreased expression of Dickkopf-1. Activation of the canonical Wnt pathway stimulates fibroblasts in vitro and induces fibrosis in vivo. Transgenic overexpression of Dickkopf-1 ameliorates skin fibrosis induced by constitutively active TGF-β receptor type I signalling and also prevents fibrosis in other TGF-β-dependent animal models. These findings demonstrate that canonical Wnt signalling is necessary for TGF-β-mediated fibrosis and highlight a key role for the interaction of both pathways in the pathogenesis of fibrotic diseases

ALMS1-Deficient Fibroblasts Over-Express Extra-Cellular Matrix Components, Display Cell Cycle Delay and Are Resistant to Apoptosis

Alström Syndrome (ALMS) is a rare genetic disorder (483 living cases), characterized by many clinical manifestations, including blindness, obesity, type 2 diabetes and cardiomyopathy. ALMS is caused by mutations in the ALMS1 gene, encoding for a large protein with implicated roles in ciliary function, cellular quiescence and intracellular transport. Patients with ALMS have extensive fibrosis in nearly all tissues resulting in a progressive organ failure which is often the ultimate cause of death. To focus on the role of ALMS1 mutations in the generation and maintenance of this pathological fibrosis, we performed gene expression analysis, ultrastructural characterization and functional assays in 4 dermal fibroblast cultures from ALMS patients. Using a genome-wide gene expression analysis we found alterations in genes belonging to specific categories (cell cycle, extracellular matrix (ECM) and fibrosis, cellular architecture/motility and apoptosis). ALMS fibroblasts display cytoskeleton abnormalities and migration impairment, up-regulate the expression and production of collagens and despite the increase in the cell cycle length are more resistant to apoptosis. Therefore ALMS1-deficient fibroblasts showed a constitutively activated myofibroblast phenotype even if they do not derive from a fibrotic lesion. Our results support a genetic basis for the fibrosis observed in ALMS and show that both an excessive ECM production and a failure to eliminate myofibroblasts are key mechanisms. Furthermore, our findings suggest new roles for ALMS1 in both intra- and extra-cellular events which are essential not only for the normal cellular function but also for cell-cell and ECM-cell interactions

Wnt3a mitigates acute lung injury by reducing P2X7 receptor-mediated alveolar epithelial type I cell death

Acute lung injury (ALI) is characterized by pulmonary endothelial and epithelial cell damage, and loss of the alveolar-capillary barrier. We have previously shown that P2X7 receptor (P2X7R), a cell death receptor, is specifically expressed in alveolar epithelial type I cells (AEC I). In this study, we hypothesized that P2X7R-mediated purinergic signaling and its interaction with Wnt/B-catenin signaling contributes to AEC I death. We examined the effect of P2X7R agonist 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP) and Wnt agonist Wnt3a on AEC I death in vitro and in vivo. We also assessed the therapeutic potential of Wnt3a in a clinically relevant ALI model of intratracheal lipopolysaccharide (LPS) exposure in ventilated mice. We found that the activation of P2X7R by BzATP caused the death of AEC I by suppressing Wnt/B-catenin signaling through stimulating glycogen synthase kinase-3B (GSK-3B) and proteasome. On the other hand, the activation of Wnt/B-catenin signaling by Wnt3a, GSK-3B inhibitor, or proteasome inhibitor blocked the P2X7R-mediated cell death. More importantly, Wnt3a attenuated the AEC I damage caused by intratracheal instillation of BzATP in rats or LPS in ventilated mice. Our results suggest that Wnt3a overrides the effect of P2X7R on the Wnt/B-catenin signaling to prevent the AEC I death and restrict the severity of ALI.Peer reviewedPhysiological Science