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

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

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

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

Characteristics of IPF patients with UIP pattern.

<p>VC = vital capacity, TLC = total lung capacity, DL_CO/VA = diffusing capacity of the lung for CO per unit of alveolar volume (all in % predicted), Pa_O2/CO2 = partial pressure of O₂/CO₂ in the arterial blood.</p

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

Myofibroblast activation and collagen deposition in response to Wnt3a.

<p>(a) The mRNA levels of the Wnt target gene Cyclin D1, or the myofibroblast activation markers smooth muscle actin (Acta2) and fibroblast-specific protein (Fsp) 1 were analyzed by qRT-PCR. Results are derived from 3 independent experiments and presented as mean±s.e.m., * p<0.05. (b) The total collagen content of NIH-3T3 fibroblasts stimulated with Wnt3a (100 ng/ml) or TGF-β1 (2 ng/ml) for 24 h was quantified using the Sircol collagen assay. Results are derived from 5 independent experiments and presented as mean±s.e.m., * p<0.05. (c) Fibroblast collagen expression and localisation after Wnt3a stimulation for 24 h was also assessed by immunofluorescent detection of collagen type 1 (red). Nuclei were visualized by DAPI staining (blue). Control negative immunostainings using iso-type matched IgG instead of a specific primary antibody are demontrated in the inlets of the left panels. Data are representative for at least three independent experiments.</p