TGF-ß-induced profibrotic signaling is regulated in part by the WNT receptor Frizzled-8.

TGF-ß is important in lung injury and remodeling processes. TGF-ß and Wingless/integrase-1 (WNT) signaling are interconnected; however, the WNT ligand-receptor complexes involved are unknown. Thus, we aimed to identify Frizzled (FZD) receptors that mediate TGF-ß-induced profibrotic signaling. MRC-5 and primary human lung fibroblasts were stimulated with TGF-ß1, WNT-5A, or WNT-5B in the presence and absence of specific pathway inhibitors. Specific small interfering RNA was used to knock down FZD8. In vivo studies using bleomycin-induced lung fibrosis were performed in wild-type and FZD8-deficient mice. TGF-ß1 induced FZD8 specifically via Smad3-dependent signaling in MRC-5 and primary human lung fibroblasts. It is noteworthy that FZD8 knockdown reduced TGF-ß1-induced collagen Iα1, fibronectin, versican, α-smooth muscle (sm)-actin, and connective tissue growth factor. Moreover, bleomycin-induced lung fibrosis was attenuated in FZD8-deficient mice in vivo Although inhibition of canonical WNT signaling did not affect TGF-ß1-induced gene expression in vitro, noncanonical WNT-5B mimicked TGF-ß1-induced fibroblast activation. FZD8 knockdown reduced both WNT-5B-induced gene expression of fibronectin and α-sm-actin, as well as WNT-5B-induced changes in cellular impedance. Collectively, our findings demonstrate a role for FZD8 in TGF-ß-induced profibrotic signaling and imply that WNT-5B may be the ligand for FZD8 in these responses.-Spanjer, A. I. R., Baarsma, H. A., Oostenbrink, L. M., Jansen, S. R., Kuipers, C. C., Lindner, M., Postma, D. S., Meurs, H., Heijink, I. H., Gosens, R., Königshoff, M. TGF-ß-induced profibrotic signaling is regulated in part by the WNT receptor Frizzled-8.

Noncanonical WNT-5B signaling induces inflammatory responses in human lung fibroblasts.

COPD is a progressive chronic lung disease characterized by pulmonary inflammation. Several recent studies indicate aberrant expression of WNT ligands and Frizzled receptors in the disease. For example, WNT-5A/B ligand expression was recently found to be increased in lung fibroblasts of COPD patients. However, possible effects of WNT-5A and WNT-5B on inflammation have not been investigated yet. In this study, we assessed the regulation of inflammatory cytokine release in response to WNT-5A/B signaling in human lung fibroblasts. Primary human fetal lung fibroblasts (MRC-5), and primary lung fibroblasts from COPD patients and non-COPD controls were treated with recombinant WNT-5A or WNT-5B to assess IL-6 and CXCL8 cytokine secretion and gene expression levels. Following WNT-5B, and to a lesser extent WNT-5A stimulation, fibroblasts showed increased IL-6 and CXCL8 cytokine secretion and mRNA expression. WNT-5B-mediated IL-6 and CXCL8 release was higher in fibroblasts from COPD patients than in non-COPD controls. In MRC-5 fibroblasts, WNT-5B-induced CXCL8 release was mediated primarily via the Frizzled-2 receptor and TAK1 signaling, whereas canonical ß-catenin signaling was not involved. In further support of noncanonical signaling, we showed activation of JNK, p38, and p65 NF-κB by WNT-5B. Furthermore, inhibition of JNK and p38 prevented WNT-5B-induced IL-6 and CXCL8 secretion, whereas IKK inhibition prevented CXCL8 secretion only, indicating distinct pathways for WNT-5B-induced IL-6 and CXCL8 release. WNT-5B induces IL-6 and CXCL8 secretion in pulmonary fibroblasts. In summary, WNT-5B mediates this via Frizzled-2 and TAK1. As WNT-5 signaling is increased in COPD, this WNT-5-induced inflammatory response could represent a therapeutic target.

A pro-inflammatory role for the Frizzled-8 receptor in chronic bronchitis.

RATIONALE: We have previously shown increased expression of the Frizzled-8 receptor of the Wingless/integrase-1 (WNT) signalling pathway in COPD. Here, we investigated if the Frizzled-8 receptor has a functional role in airway inflammation associated with chronic bronchitis. METHODS: Acute cigarette-smoke-induced airway inflammation was studied in wild-type and Frizzled-8-deficient mice. Genetic association studies and lung expression quantitative trait loci (eQTL) analyses for Frizzled-8 were performed to evaluate polymorphisms in FZD8 and their relationship to tissue expression in chronic bronchitis. Primary human lung fibroblasts and primary human airway epithelial cells were used for in vitro studies. RESULTS: Cigarette-smoke-exposure induced airway inflammation in wild-type mice, which was prevented in Frizzled-8-deficient mice, suggesting a crucial role for Frizzled-8 in airway inflammation. Furthermore, we found a significant genetic association (p=0.009) between single nucleotide polymorphism (SNP) rs663700 in the FZD8 region and chronic mucus hypersecretion, a characteristic of chronic bronchitis, in a large cohort of smoking individuals. We found SNP rs663700 to be a cis-eQTL regulating Frizzled-8 expression in lung tissue. Functional data link mesenchymal Frizzled-8 expression to inflammation as its expression in COPD-derived lung fibroblasts was regulated by pro-inflammatory cytokines in a genotype-dependent manner. Moreover, Frizzled-8 regulates inflammatory cytokine secretion from human lung fibroblasts, which in turn promoted MUC5AC expression by differentiated human airway epithelium. CONCLUSIONS: These findings indicate an important pro-inflammatory role for Frizzled-8 and suggest that its expression is related to chronic bronchitis. Furthermore, our findings indicate an unexpected role for fibroblasts in regulating airway inflammation in COPD.

Interleukin-1α drives the dysfunctional cross-talk of the airway epithelium and lung fibroblasts in COPD.

Chronic obstructive pulmonary disease (COPD) has been associated with aberrant epithelial-mesenchymal interactions resulting in inflammatory and remodelling processes. We developed a co-culture model using COPD and control-derived airway epithelial cells (AECs) and lung fibroblasts to understand the mediators that are involved in remodelling and inflammation in COPD.AECs and fibroblasts obtained from COPD and control lung tissue were grown in co-culture with fetal lung fibroblast or human bronchial epithelial cell lines. mRNA and protein expression of inflammatory mediators, pro-fibrotic molecules and extracellular matrix (ECM) proteins were assessed.Co-culture resulted in the release of pro-inflammatory mediators interleukin (IL)-8/CXCL8 and heat shock protein (Hsp70) from lung fibroblasts, and decreased expression of ECM molecules (e.g. collagen, decorin) that was not different between control and COPD-derived primary cells. This pro-inflammatory effect was mediated by epithelial-derived IL-1α and increased upon epithelial exposure to cigarette smoke extract (CSE). When exposed to CSE, COPD-derived AECs elicited a stronger IL-1α response compared with control-derived airway epithelium and this corresponded with a significantly enhanced IL-8 release from lung fibroblasts.We demonstrate that, through IL-1α production, AECs induce a pro-inflammatory lung fibroblast phenotype that is further enhanced with CSE exposure in COPD, suggesting an aberrant epithelial-fibroblast interaction in COPD.

A large lung gene expression study identifying fibulin-5 as a novel player in tissue repair in COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a progressive, incurable lung disease characterised by abnormal tissue repair causing emphysema and small airways fibrosis. Since current therapy cannot modify this abnormal repair, it is crucial to unravel its underlying molecular mechanisms. Unbiased analysis of genome-wide gene expression profiles in lung tissue provides a powerful tool to investigate this. METHODS: We performed genome-wide gene expression profiling in 581 lung tissue samples from current and ex-smokers with (n=311) and without COPD (n=270). Subsequently, quantitative PCR, western blot and immunohistochemical analyses were performed to validate our main findings. RESULTS: 112 genes were found to be upregulated in patients with COPD compared with controls, whereas 61 genes were downregulated. Among the most upregulated genes were fibulin-5 (FBLN5), elastin (ELN), latent transforming growth factor ß binding protein 2 (LTBP2) and microfibrillar associated protein 4 (MFAP4), all implicated in elastogenesis. Our gene expression findings were validated at mRNA and protein level. We demonstrated higher ELN gene expression in COPD lung tissue and similar trends for FBLN5 and MFAP4, and negative correlations with lung function. FBLN5 protein levels were increased in COPD lung tissue and cleaved, possibly non-functional FBLN5 protein was present. Strong coexpression of FBLN5, ELN, LTBP2 and MFAP4 in lung tissue and in silico analysis indicated cofunctionality of these genes. Finally, colocalisation of FBLN5, MFAP4 and LTBP2 with elastic fibres was demonstrated in lung tissue. CONCLUSIONS: We identified a clear gene signature for elastogenesis in COPD and propose FBLN5 as a novel player in tissue repair in COPD.

Focal adhesion kinase regulates collagen I-induced airway smooth muscle phenotype switching.

Increased extracellular matrix (ECM) deposition and airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma. Recently, we demonstrated that the ECM protein collagen I, which is increased surrounding asthmatic ASM, induces a proliferative, hypocontractile ASM phenotype. Little is known, however, about the signaling pathways involved. Using bovine tracheal smooth muscle, we investigated the role of focal adhesion kinase (FAK) and downstream signaling pathways in collagen I-induced ASM phenotype modulation. Phosphorylation of FAK was increased during adhesion to both uncoated and collagen I-coated culture dishes, without differences between these matrices. Nor were any differences found in cellular adhesion. Inhibition of FAK activity by overexpression of the FAK deletion mutants FAT (focal adhesion targeting domain) and FRNK (FAK-related nonkinase) attenuated adhesion. After attachment, FAK phosphorylation increased in a time-dependent manner in cells cultured on collagen I, whereas no activation was found on an uncoated plastic matrix. In addition, collagen I increased in a time- and concentration-dependent manner the cell proliferation, which was fully inhibited by FAT and FRNK. Similarly, the specific pharmacologic FAK inhibitor PF-573228 [6-((4-((3-(methanesulfonyl)benzyl)amino)-5-trifluoromethylpyrimidin-2-yl) amino)-3,4-dihydro-1H-quinolin-2-one] as well as specific inhibitors of p38 mitogen-activated protein kinase (MAPK) and Src also fully inhibited collagen I-induced proliferation, whereas partial inhibition was observed by inhibition of phosphatidylinositol-3-kinase (PI3-kinase) and mitogen-activated protein kinase kinase (MEK). The inhibition of cell proliferation by these inhibitors was associated with attenuation of the collagen I-induced hypocontractility. Collectively, the results indicate that induction of a proliferative, hypocontractile ASM phenotype by collagen I is mediated by FAK and downstream signaling pathways.

Activation of WNT/ß-catenin signaling in pulmonary fibroblasts by TGF-ß1 is increased in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by abnormal extracellular matrix (ECM) turnover. Recently, activation of the WNT/ß-catenin pathway has been associated with abnormal ECM turnover in various chronic diseases. We determined WNT-pathway gene expression in pulmonary fibroblasts of individuals with and without COPD and disentangled the role of ß-catenin in fibroblast phenotype and function. METHODS: We assessed the expression of WNT-pathway genes and the functional role of ß-catenin, using MRC-5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. RESULTS: Pulmonary fibroblasts expressed mRNA of genes required for WNT signaling. Stimulation of fibroblasts with TGF-ß1, a growth factor important in COPD pathogenesis, induced WNT-5B, FZD8, DVL3 and ß-catenin mRNA expression. The induction of WNT-5B, FZD6, FZD8 and DVL3 mRNA by TGF-ß1 was higher in fibroblasts of individuals with COPD than without COPD, whilst basal expression was similar. Accordingly, TGF-ß1 activated ß-catenin signaling, as shown by an increase in transcriptionally active and total ß-catenin protein expression. Furthermore, TGF-ß1induced the expression of collagen1α1, α-sm-actin and fibronectin, which was attenuated by ß-catenin specific siRNA and by pharmacological inhibition of ß-catenin, whereas the TGF-ß1-induced expression of PAI-1 was not affected. The induction of transcriptionally active ß-catenin and subsequent fibronectin deposition induced by TGF-ß1 were enhanced in pulmonary fibroblasts from individuals with COPD. CONCLUSIONS: ß-catenin signaling contributes to ECM production by pulmonary fibroblasts and contributes to myofibroblasts differentiation. WNT/ß-catenin pathway expression and activation by TGF-ß1 is enhanced in pulmonary fibroblasts from individuals with COPD. This suggests an important role of the WNT/ß-catenin pathway in regulating fibroblast phenotype and function in COPD.