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

Cluster-Randomized Studies Part 25 of a Series on Evaluating Scientific Publications

Background: Cluster-randomized trials (CRT) are needed to compare interventions that are allocated to entire groups of subjects, rather than to individuals. Publications about CRT have become steadily more common over the past decade. Readers of such publications should be able to categorize and interpret the findings of CRT correctly while considering the methodological requirements applicable to this type of study. Methods: This review is based on a selection of pertinent literature and on the authors' expertise. CRT-specific methodological aspects of the planning, performance, and interpretation of studies are discussed. Results: Readers of publications on CRT should check whether due consideration has been given to correlations within and between the clusters during the planning of the study. These correlations enable the determination whether persons within a cluster resemble each other more closely, or respond more similarly to the study intervention, than persons drawn from different clusters. It should also be checked whether the randomization for the study has been carried out with such methods as stratification and covariate-adjusted randomization. CRT can be analyzed on either the individual or the cluster level. The rationale for the choice of a cluster-randomized design should be explained, and intracluster correlation coefficients (ICC) should be reported as an aid to the planning of future studies. Particular requirements are also described in an extended version of the CONSORT guidelines that has been developed specifically for CRT. Conclusion: Readers of publications on CRT should be aware of the special requirements mentioned above with respect to the design, performance, and analysis of this type of study as opposed to individually randomized studies. If no special techniques are applied in the design, performance, and analysis of a CRT, or if the assumptions underlying each of these steps have not been properly checked, then the findings of the study may well be misleading

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

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

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