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

Role of the WNT/beta-catenin signal pathway in idiopathic and experimental pulmonary fibrosis

Human idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease of unknown origin, which is refractory to any currently available therapy. It is characterised by initial alveolar epithelial cell injury and hyperplasia, enhanced fibroblast/myofibroblast proliferation and activation and increased deposition of extracellular matrix (ECM) in the lung interstitium. These key features, ultimately lead to architectural distortion of the normal lung parenchyma and due to severe loss of function, to respiratory failure. However, the molecular mechanisms underlying alveolar epithelial type II (ATII) cell dysfunction are still poorly understood. This study is based on the hypothesis that the WNT/beta-catenin signalling pathway, which is essential for organ development, is aberrantly activated in ATII cells in idiopathic and experimental pulmonary fibrosis. The role of canonical WNT signalling was elucidated by determining the expression, function and activity of the pathway. In summary, this study shows that WNT signalling is expressed and localised in idiopathic and experimental pulmonary fibrosis, in particular in alveolar ATII cells. Reversal and/or inhibition predominantly of the WNT/beta-catenin signalling pathway, but equally the use of neutralising antibodies or inhibitors of IL1beta/IL6 may represent a valid therapeutic option in human lung fibrosis.Die häufigste und schwerwiegendste Form der idiopathischen interstitiellen Pneumonien (IIP) beim Menschen ist die idiopathische pulmonale Fibrose (IPF). Es handelt sich um eine therapierefraktäre, progressiv und tödlich verlaufende Lungenerkrankung mit unbekannter Ätiologie. Die initiale Schädigung des Alveolarepithels und eine daraus resultierende abnormale Wundheilung charakterisieren diese Erkrankung. Eine verstärkte Proliferation und Aktivierung von Fibroblasten/Myofibroblasten und die vermehrte Ansammlung von extrazellulärer Matrix (ECM) im Lungeninterstitium sind zusätzliche Begleiterscheinungen. Dies führt letztendlich zu einem kompletten Umbau bzw. einer Zerstörung des normalen Lungengewebes und aufgrund des funktionellen Verlustes zum respiratorischen Versagen. Die molekularen Mechanismen, die den veränderten Schädigungs- und Reparaturprozessen des Alveolarepithels zugrunde liegen und demnach für die Entwicklung von IPF verantwortlich sind, sind allerdings größtenteils noch unklar. Der kanonische WNT Signaltransduktionsweg, benannt nach dem Liganden „WNT“, hat in der Organentwicklung eine besondere Bedeutung, spielt aber auch eine essentielle Rolle bei verschiedenen Erkrankungen. Das Signalprotein „WNT“ fungiert als lokaler Mediator, und der Name setzt sich zusammen aus den Genen Wingless (Wg) und Integration 1 (Int-1). Die vorliegende Arbeit basiert auf der Hypothese, dass der WNT/beta-catenin Signalweg in alveolaren Epithelzellen Typ II (ATII) in der idiopathischen sowie experimentellen pulmonalen Fibrose differenziell reguliert und aktiviert vorliegt. In dieser Studie wurden neben humanem und murinem Lungenhomogenat, zusätzlich ATII Zellen aus gesunden bzw. fibrotischen murinen Lungen isoliert und untersucht. Zusammenfassend konnte gezeigt werden, dass der WNT Signalweg bei der idiopathischen und experimentellen Lungenfibrose, vornehmlich in ATII Zellen lokalisiert sowie exprimiert wird. Demnach spielt dieser Signaltransduktionsweg eine potentiell erhebliche Rolle im Rahmen der Pathogenese der Erkrankung. Eine generelle Hemmung des WNT/beta-catenin Signalweges, aber auch die spezifische Verwendung von neutralisierenden IL1beta/IL6 Antikörpern, könnte einen möglichen therapeutischen Ansatz in der Behandlung der IPF darstellen

Proliferative effect induced by Wnt3a in alveolar epithelial cells.

<p>(a) A549 lung epithelial cell were transiently transfected with FOPflash or TOPflash Wnt reporter constructs (FOP and TOP, respectively), and stimulated with Wnt3a or Wnt7a (at 100 ng/ml each), as indicated. Luciferase expression is plotted as fold activation over unstimulated controls. Results are derived from six independent experiments and presented as mean±s.e.m., * p<0.05. (b) Proliferation of A549 cells was assessed by cell counting 24 h after stimulation with Wnt3a (100 ng/ml). All experiments were performed in quadruplicate, with each condition counted at least three times. Results are presented as mean±s.e.m., * p<0.05.</p

Expression and localization of Wnt1 in lung tissues of donor and IPF patients.

<p>Immunohistochemical staining was performed on tissue sections of donor (a) or IPF lungs (b). Representative pictures with focus on the bronchial (upper panel) or alveolar epithelium (lower panel) are given. Stainings are representative of two independent experiments using at least three different donor or IPF lung tissues (magnification as indicated). Arrowhead indicates positive endothelial cells.</p

The mRNA expression profile of canonical Wnt signaling components in primary human ATII cells.

<p>Primary human ATII cells were isolated from lung tissues of donor and IPF patients as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002142#s4" target="_blank">Material and Methods</a>. The mRNA levels of Wnt1, 3a, 7b, and 10b (a), the receptors Fzd2 and 3, and Lrp5 and 6 (b), and Gsk-3β, β-catenin, and Lef1 (c) in ATII cells were assessed by qRT-PCR. Results are derived from 3 different cell isolations each and presented as mean±s.e.m., * p<0.05.</p

The mRNA expression profile of canonical Wnt signaling components in IPF.

<p>The mRNA levels of the Wnt ligands Wnt1, 2, 3a, 7b, and 10b (a), the receptors frizzled (Fzd) 1–4, low density lipoprotein-related protein (Lrp) 5 and 6 (b), and the intracellular signal transducers glycogen synthase kinase (Gsk)-3β, β-catenin, T-cell-specific transcription facor (Tcf) 3, Tcf 4, lymphoid enhancer-binding factor (Lef) 1 (c) were assessed in donor and IPF lung specimen by quantitative real-time PCR (qRT-PCR). Results are derived from 12 donors and 12 IPF patients and presented as mean±s.e.m., * p<0.05.</p

Expression and localization of total β-catenin in lung tissues of donor and IPF patients.

<p>Immunohistochemical staining was performed on tissue sections of donor (a) or IPF lungs (b). Representative pictures with focus on the bronchial (upper panel) or alveolar epithelium (lower panel) are given. Stainings are representative of two independent experiments using at least three different donor or IPF lung tissues (magnification as indicated). Arrow indicates nuclear staining of β-catenin. Arrowhead indicates positive endothelial cells.</p

Activity of the canonical Wnt signal pathway in lung homogenates of donor and IPF patients.

<p>(a) The expression of active Wnt components in lung homogenates of donor and IPF patients was analyzed by immunoblotting of phosphorylated Gsk-3β and Lrp6, total β-catenin, and the Wnt target gene Cyclin D1. Blotting of total Gsk-3β, Lrp6, and lamin A/C served as loading controls. Immunoblotting of smooth muscle actin (ActA2) was used as a positive control for IPF specimen. (b) The mRNA levels of Fn 1, Mmp 7, and Cyclin D1 were assessed by qRT-PCR. Results are derived from 6 donors and 6 IPF patients and presented as mean±s.e.m., * p<0.05.</p