'WNT-er is coming': WNT signalling in chronic lung diseases.

Chronic lung diseases represent a major public health problem with only limited therapeutic options. An important unmet need is to identify compounds and drugs that target key molecular pathways involved in the pathogenesis of chronic lung diseases. Over the last decade, there has been extensive interest in investigating Wingless/integrase-1 (WNT) signalling pathways; and WNT signal alterations have been linked to pulmonary disease pathogenesis and progression. Here, we comprehensively review the cumulative evidence for WNT pathway alterations in chronic lung pathologies, including idiopathic pulmonary fibrosis, pulmonary arterial hypertension, asthma and COPD. While many studies have focused on the canonical WNT/ß-catenin signalling pathway, recent reports highlight that non-canonical WNT signalling may also significantly contribute to chronic lung pathologies; these studies will be particularly featured in this review. We further discuss recent advances uncovering the role of WNT signalling early in life, the potential of pharmaceutically modulating WNT signalling pathways and highlight (pre)clinical studies describing promising new therapies for chronic lung diseases.

WISP1 mediates IL-6-dependent proliferation in primary human lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. IPF is characterized by epithelial cell injury and reprogramming, increases in (myo)fibroblasts, and altered deposition of extracellular matrix. The Wnt1-inducible signaling protein 1 (WISP1) is involved in impaired epithelial-mesenchymal crosstalk in pulmonary fibrosis. Here, we aimed to further investigate WISP1 regulation and function in primary human lung fibroblasts (phLFs). We demonstrate that WISP1 is directly upregulated by Transforming growth factor ß1 (TGFß1) and Tumor necrosis factor α (TNFα) in phLFs, using a luciferase-based reporter system. WISP1 mRNA and protein secretion increased in a time- and concentration-dependent manner by TGFß1 and TNFα in phLFs, as analysed by qPCR and ELISA, respectively. Notably, WISP1 is required for TGFß1- and TNFα-dependent induction of interleukin 6 (IL-6), a mechanism that is conserved in IPF phLFs. The siRNA-mediated WISP1 knockdown led to a significant IL-6 reduction after TGFß1 or TNFα stimulation. Furthermore, siRNA-mediated downregulation or antibody-mediated neutralization of WISP1 reduced phLFs proliferation, a process that was in part rescued by IL-6. Taken together, these results strongly indicate that WISP1-induced IL-6 expression contributes to the pro-proliferative effect on fibroblasts, which is likely orchestrated by a variety of profibrotic mediators, including Wnts, TGFß1 and TNFα.

Wnt pathway activation in long term remnant rat model.

Progression of chronic kidney disease (CKD) is characterized by deposition of extracellular matrix. This is an irreversible process that leads to tubulointerstitial fibrosis and finally loss of kidney function. Wnt/ ß-catenin pathway was reported to be aberrantly activated in the progressive damage associated with chronic organ failure. Extensive renal ablation is an experimental model widely used to gain insight into the mechanisms responsible for the development of CKD, but it was not evaluated for Wnt/ ß-catenin pathway. This study aimed to elucidate if the rat 5/6 renal mass reduction model (RMR) is a good model for the Wnt/ ß-catenin activation and possible next modulation. RMR model was evaluated at 12 and 18 weeks after the surgery, when CKD is close to end-stage kidney disease demonstrated by molecular and histological studies. Wnt pathway components were analyzed at mRNA and protein level. Our results demonstrate that Wnt pathway is active by increase of ß-catenin at mRNA level and nuclear translocation in tubular epithelium as well as some target genes. These results validate the RMR model for future modulation of Wnt pathway, starting at shorter time after the surgery.

Terguride ameliorates monocrotaline-induced pulmonary hypertension in rats.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterised by vasoconstriction and remodelling of the pulmonary vasculature. The serotonin (5-hydroxytryptamine (5-HT)) pathway has been shown to play a major role in the pathogenesis of PAH, but pharmacological modulation of this pathway for treatment of PAH is, to date, at a pre-clinical level. Terguride is a 5-HT receptor (5-HTR) antagonist that is well tolerated and clinically approved for ovulation disorders. Immunohistochemistry against 5-HTR(2A/B) on human lungs revealed their localisation to the vascular smooth muscle layer and quantitative RT-PCR showed 5-HTR(2B) upregulation in pulmonary artery smooth muscle cells (PASMC) isolated from PAH patients. Proliferation and migration of cultured primary human PASMC were dose-dependently blocked by terguride. Therapeutic 5-HT signalling inhibition was 1) demonstrated in isolated, ventilated and perfused rat lungs and 2) by chronic terguride treatment of rats with monocrotaline (MCT)-induced pulmonary hypertension in a preventive or curative approach. Terguride inhibited proliferation of PASMCs and abolished 5-HT-induced pulmonary vasoconstriction. Chronic terguride treatment prevented dose-dependently the development and progression of MCT-induced PAH in rats. Thus, terguride represents a valuable novel therapeutic approach in PAH.

Dickkopf proteins influence lung epithelial cell proliferation in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with unknown pathogenesis. The WNT/ß-catenin pathway has recently been reported to be operative in epithelial cells in IPF. Dickkopf (DKK) proteins are known to regulate WNT signalling via interaction with Kremen (KRM) receptors, yet their expression and role in the adult lung and in IPF has not been addressed. We analysed the expression, localisation and function of DKK and KRM proteins in IPF lungs using Western blotting, quantitative RT-PCR, immunohistochemistry, ELISA and functional in vitro studies. Enhanced expression of DKK1 and DKK4 and KRM1 was detected in lung homogenates of IPF patients compared with transplant donors. Immunohistochemistry revealed that DKK1 was predominantly localised in basal bronchial epithelial cells. Furthermore, prominent expression of all proteins was observed in hyperplastic alveolar epithelial cells in IPF. Quantitative measurement of DKK1 revealed enhanced protein expression in the bronchoalveolar lumen of IPF patients. Finally, functional studies using human bronchial and alveolar epithelial cell lines demonstrated that WNT-induced epithelial cell proliferation is regulated by DKK1 in a dose-dependent fashion. In summary, DKK proteins are expressed in the lung epithelium in IPF. DKK proteins influence epithelial cell proliferation and may, therefore, be suitable therapeutic targets for IPF.

SNAI transcription factors mediate epithelial-mesenchymal transition in lung fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterised by accumulation of activated (myo)fibroblasts and excessive extracellular matrix deposition. The enhanced accumulation of (myo)fibroblasts may be attributed, in part, to the process of transforming growth factor beta1 (TGFbeta1)-induced epithelial-mesenchymal transition (EMT), the phenotypic switching of epithelial to fibroblast-like cells. Although alveolar epithelial type II (ATII) cells have been shown to undergo EMT, the precise mediators and mechanisms remain to be resolved. The objective of this study is to investigate the role of SNAI transcription factors in the process of EMT and in IPF. METHODS: Using quantitative reverse transcription-PCR (RT-PCR), immunofluorescence, immunohistochemistry, western blotting, as well as gain- and loss-of-function studies and functional assays, the role of SNAI1 and SNAI2 in TGFbeta1-induced EMT in ATII cells in vitro was assessed; and the expression of SNAI transcription factors was analysed in experimental and human IPF in vivo. RESULTS: TGFbeta1 treatment increased the expression and nuclear accumulation of SNAI1 and SNAI2, in concert with induction of EMT in ATII cells. SNAI overexpression was sufficient to induce EMT, and small interfering RNA (siRNA)-mediated SNAI depletion attenuated TGFbeta1-induced ATII cell migration and EMT. SNAI expression was elevated in experimental and human IPF and localised to hyperplastic ATII cells in vivo. CONCLUSIONS: The results demonstrate that TGFbeta1-induced EMT in ATII cells is essentially controlled by the expression and nuclear translocation of SNAI transcription factors. Increased SNAI1 and SNAI2 expression in experimental and human IPF in vivo suggests that SNAI-mediated EMT may contribute to the fibroblast pool in idiopathic pulmonary fibrosis.

Shroom expression is attenuated in pulmonary arterial hypertension.

Shroom is a PDZ-domain protein involved in the regulation and maintenance of cytoskeletal architecture by binding to actin. Hypertrophy and altered actin organisation of pulmonary arterial smooth muscle cells (PASMC) is a hallmark of pulmonary arterial hypertension (PAH). The aim of the present study was to localise and characterise Shroom expression in the lung in experimental and idiopathic PAH (IPAH). Shroom expression and localisation in hypoxia-induced PAH in mice and IPAH in humans, in vivo, as well as in primary PASMC, in vitro, was assessed by quantitative RT-PCR, immunofluorescence, laser-assisted microdissection and immunohistochemistry. Shroom localised exclusively to PASMC (both bronchial and vascular) in mouse and human lungs. Both in vivo and in primary PASMC, in vitro, Shroom exhibited spatially similar expression with alpha-smooth muscle actin (alpha-SMA). Shroom expression was significantly reduced in the mouse model of PAH, in primary murine PASMC exposed to hypoxia, and in primary PASMC isolated from patients with IPAH. The ratio between Shroom and alpha-SMA RNA expression further confirmed Shroom downregulation in both mouse and human PASMC. In summary, Shroom localises exclusively to pulmonary smooth muscle cells. Shroom downregulation in pulmonary arterial hypertension suggests a link between Shroom expression and pulmonary arterial smooth muscle cell hypertrophy in pulmonary arterial hypertension.