The canonical or β-catenin/Tcf dependent Wnt pathway was discovered first, studied most and as a result reviewed frequently 56. Briefly, in the absence of Wnt signalling, glycogen synthase kinase (GSK-3) is active and phosphorylates β-catenin in the scaffolding protein complex of adenomatous polyposis coli (APC) and axin 78. The phosporylated β-catenin is targeted for ubiquitination and 26S proteasome-mediated degradation, thereby decreasing the cytosolic level of β-catenin 910 (Figure 1). A Wnt-Fz-LRP6 complex is formed in the presence of Wnt-s that leads to the phosphorylation of three domains of Dishevelled (Dvl), which is a family of cytosolic signal transducer molecules 11. Activation of Dvl ultimately leads to phosphorylation and consequently inhibition of GSK-3. This process is summarised in Figure 2. Inhibition of GSK3 results in stabilisation and consequently cytosolic accumulation of β-catenin (Figure 2). The accumulated β-catenin translocates to the nucleus, where it forms an active transcription complex with members of the T Cell Factor (LEF1, TCF1, TCF3, TCF4) transcription factor family 1213 and transcription initiator p300 14. Successful assembly of the transcription complex leads to target gene activation. Target genes of the canonical β-catenin pathway include matrix metalloproteinases (MMP2, MMP3, MMP7, and MMP9) 15, cyclin D1 1617, Cox-2 18, c-myc 19, c-jun 20, Fra-1 20, VEGFR 21, etc. (For a recent update see Nusse's Wnt website: http://www.stanford.edu/~rnusse/wntwindow.html).

The non-canonical Wnt pathways, the JNK/AP1 dependent, PCP and the PKC/CAMKII/NFAT dependent Ca2+ pathway (just like the canonical Wnt pathway) become activated following Wnt-Fz receptor binding 2223. The non-canonical pathways differ from the β-catenin pathway in their dependency on the type of G-proteins 24 they require for activation. Further downstream, Dvl is critical for signal transduction in both 25 but in contrast to canonical Wnt signalling, phosphorylation of all three domains of Dvl, is not a requirement 26. Although the Dvl family has long been accepted as cytosol based signal transducers for the three Wnt-pathways, recent studies have revealed the ability of Dvl to translocate into the nucleus where it regulates intranuclear stability of β-catenin 2728. How this new function of Dvl fits into the more traditional role of the molecule awaits further investigation.

Nevertheless, downstream of the cytosolic Dvl, the two non-canonical Wnt pathways can activate different signalling cascades and trigger the transcription of different gene-sets, although cross-pathway activation, signal integration, and consequently gene expression modification via complex formation between NFAT and AP1 29 can also occur. The noncanonical pathways are summarised in figure 3 and 4.

Modulation of Wnt expression in embryonic and adult mouse lung suggests that Wnt pathways are important for cell fate decisions and differentiation of lung cell types. The involvement of canonical Wnt signalling in lung development has been proven by several ways. A TCF promoter-LacZ based reporter system has shown, that canonical Wnt signalling is active throughout lung development in mouse embryos 59. β-catenin, a central molecule of canonical Wnt signalling, has been shown to localize in the cytoplasm, and often also the nucleus of the undifferentiated primordial epithelium (PE), differentiating alveolar epithelium (AE), and adjacent mesenchyme 60. Using a conditional knockout system for β-catenin in mice has also revealed that β-catenin dependent signalling is central to the formation of the peripheral airways of the lungs, responsible for conducting gas exchange, but is dispensable for the formation of the proximal airways 61. Constitutive activation of the canonical Wnt pathway using a β-catenin-Lef1 fusion protein, produced a similar effect 59. Although proximal airways developed, the lung was reduced in size and lacked alveoli 59.

Recent studies have related particular Wnt production to specific lung cell types. Wnt2 62 for example has been mapped predominantly to the mesenchyme, Wnt11 to both epithelium and mesenchyme 63, while Wnt7b was exclusively expressed in the lung epithelium 64. Additional studies have revealed that Wnt7b promoter activity is regulated by a homeodomain transcription factor, TTF1, which is essential to the differentiation of lung epithelium, being especially important for the highly specialised Type II alveolar epithelial cells 65. Since the TTF1 null mice have a lethal lung phenotype with increased epithelial and mesenchymal proliferation, which at the neonatal stage contains abundant mesenchyme and no functional alveoli 65, it is likely that the lack of functional alveoli is a result of dysregulated Wnt7b signalling 64.

Apart from β-catenin and Wnt-s, mRNA of Fz-1, -2 and -7 and several intracellular signalling molecules including Tcf-1, -3, -4, Lef1, and secreted Fz related proteins (sFrp-1, -2 and -4) have been found to be expressed in the developing lung 60 in specific, spatio-temporal patterns 60. Wnt signalling has also been reported to be important in the regulation of spatial and distal branching of the lung 61.

While the importance of canonical Wnt signalling in lung development is well established, the role of non-canonical Wnt signalling is less clear. Wnt5a knock-out studies have shown, however, that non-canonical Wnt signalling is also important. In Wnt5a-/- animals the lung is morphologically smaller than in the wild type 66 and has thickened mesenchyme. Furthermore, alveolar development is delayed, although not prevented 66. Lungs of Wnt5a knock-out animals also have increased expression of FGF10 and Shh 6667 suggesting that the morphological changes might be related to dysregulation of other signalling pathways modulated by Wnt signalling (see below for further details).

Primary lung tissue and cell lines, derived from adult lung tissue, express a wide range of Wnt-s including Wnt-3, -4, -5a, -7a, -7b, -10b, and -11 68, as well as Fz-3, -6 and -7 68, Dvl 69, and Dkk 70. Since, generally, Wnt signalling retains cells in a low differentiation state, the role of Wnt signalling in adult tissue may not be immediately clear. If we assume that the maintenance of adult organs is stem cell dependent and that stem cells rely on β-catenin and Tcf/Lef signalling to be maintained in the required low differentiation level, the role of Wnt signals in adult tissue becomes understandable. Stem cell niches in proximal and distal airways exist 7172, similarly to intestine, hair follicle and dermis, and would need Wnt signalling to be able to fulfill their role in maintenance of adult lung structure.

While lung cancer is one of the leading causes of cancer deaths worldwide 7374 data regarding the role of Wnt pathways in human lung cancer is still limited. The most studied pathway mutations in cancer are the inherited and sporadic mutations in the tumour suppressor adenomatous polyposis coli (APC) and β-catenin. Since APC is part of the degradation scaffold for β-catenin, mutations of APC can result in reduced degradation and increased nuclear accumulation of β-catenin leading to activation of target genes such as oncogenes cyclin D1 and c-myc 75. Degradation resistant β-catenin has similar effect on target gene activation 59. Although increased levels of β-catenin have been reported in different types of lung cancers 7677, mutations of APC 78 and β-catenin 7980 are rare in lung cancers. However, proof of dysregulation of specific Wnt molecules leading to oncogenic signalling has emerged. While frequent loss of Wnt7a mRNA was demonstrated in some studies in lung cancer cell lines and primary tumours 81, elevated levels of Wnt1 82 and Wnt2 83 have been reported in non small cell lung cancer. Decreased levels of Wnt7a indicates that Wnt7a may function as a tumour suppressor in lung cancer. In support this concept, non-small-cell lung cancer cells transformed with Wnt7a showed inhibition of anchorage independent growth 68. Although member of the canonical group, Wnt7a inhibits proliferation and induces differentiation via the JNK/AP1 dependent PCP signalling pathway 68. The role of non-canonical Wnt signalling in the development of lung cancer remains controversial despite recent findings. Although the non-canonical pathway activator Wnt5a is an important regulator of lung development, and generally is an inhibitor of canonical Wnt signalling, elevated levels of Wnt5a in lung metastases of human sarcoma 84 has been reported and thus questions the role of non-canonical Wnt signalling as a general inhibitor of lung cancer. In metastatic stage of any tumours including human lung carcinomas, epithelial-mesenchymal transformation (EMT) is typical 85 and generally linked to increased β-catenin dependent signalling 86. As β-catenin mutations in lung cancers are relatively rare 798087, another possible mechanism might be at place which regulates EMT and consequently tumour metastasis in the lung. Certainly, non-canonical Wnt5a the very molecule which has recently been reported to regulate fibroblast growth factor (FGF) 10 and sonic hedgehog (Shh) expression 67 has been found elevated in lung metastases 84. Both FGF-s and the hedgehog family are well-known modulators of epithelial-mesenchymal interactions 88 and epithelial-mesenchymal transformations (EMT) 899091. Dysregulation of FGF and Shh signalling certainly raises the possibility that Wnt5a and perhaps non-canonical Wnt signalling in general, is indirect regulator of lung tumour metastasis.

Lung developmental studies have also provided support for the involvement of canonical Wnt signalling in lung cancer. Constitutive activation of the canonical pathway in the developing lung resulted in a non-differentiated lung phenotype resembling cancer 59. Target genes of the canonical and PCP Wnt pathways include matrix metalloproteinases, which are essential for tissue remodelling and are elevated in invasive cancer 9293, thus providing additional evidence for the involvement of Wnt signalling in lung cancer.

Overexpression of Dvl, a positive regulator of Wnt signalling pathways has been reported in 75% of non-small-cell-lung-cancer samples compared with autologous matched normal tissue 94. Downregulation of Wnt pathway antagonists like Dkk3 70, WIF 9596 and sFRP 97 have also been reported in various types of lung cancers providing further evidence of the role of this complex pathway.

To date there is no direct evidence for the involvement of Wnt signalling in inflammation of the central airways. However, based on the general features of inflammatory diseases and evidence for Wnt regulated signalling in inflammation in the joint 34, we have addressed the potential involvement of Wnt signalling in inflammatory diseases of the lung.

Increased levels of pro-inflammatory and inflammatory cytokines such as IL1, IL6, IL8, and IL15, monocyte chemotactic protein-1 (MCP-1), TNFα and intercellular adhesion molecule-1 (ICAM-1) are general features of inflammation. The elevated expression of ICAM in the epithelium is important in leukocyte recruitment, adhesion and retention 98, while IL8 secreted by the bronchial epithelium 99, is thought to be central to the attraction of neutrophils. Neutrophils together with macrophages contribute to the pathogenesis of inflammatory tissue injury by reactive oxygen metabolites and proteinase release. Increased levels of tissue matrix metalloproteinases (MMP-s) are a feature of inflammatory conditions and may contribute to the overall evolution of the inflammation-induced tissue destruction. Several pulmonary cells including resident alveolar macrophages, neutrophils, parenchymal cells (including interstitial fibroblasts), type II epithelial cells and vascular endothelial cells are capable of elaborating MMPs 100, and numerous MMP-s, including MMP3 and MMP9, have been considered to have important pro-inflammatory roles in acute lung inflammation 101. Activation of MMP gene transcription has been attributed to both pro-inflammatory cytokines 102103 and canonical Wnt signalling 15, but it is still not clear whether they act in competition or in close connection to regulate the transcription of MMP genes. Certainly, the canonical pathway activator Wnt-1 has been linked to stimulation of pro-MMP3 transcription 104, which is implicated in lung inflammation 105. Understanding of signalling pathway interaction is thus of importance in the study of pathogenic processes and hence disease modulation.

Studies of rheumatoid arthritis have accumulated evidence that Wnt5a-Fz5 mediated signalling can contribute significantly to the production of pro-inflammatory cytokines (IL6, IL8, IL15) 33 and that overexpression of Wnt5a leads to increased pro-inflammatory cytokine levels. Furthermore, dominant negative and antisense Wnt5a and anti-Fz-5 antibody block Wnt5-Fz5 signalling leading to decreased cytokine production 33.

Additionally, the inflammatory cytokine inducing Wnt5a has also been implicated in the down-regulation of Shh levels in the lung 67. Elevated Shh signalling is well established in the regulation of inflammatory and fibrotic processes of the gut and lung 91. This suggests a role for Wnt5a but further investigation would be necessary to clarify this in the central airways- in pulmonary inflammation.

Lung diseases resulting in tissue damage activate a defence mechanism to repair the lesions. Tissue damage can result from several acute and chronic stimuli including inflammation caused by infections, autoimmune reactions (asthma, allergic alveolitis), and drugs and toxins (bleomycin, asbestos) or mechanical injury (surgery, and irradiation). Any tissue repair involves coordinated cellular infiltration together with extracellular matrix deposition and where appropriate, re-epitheliasation. In the first regenerative step, injured cells are replaced by cells of the same type, then normal parenchyma is replaced by connective tissue leading to fibrosis. Usually both steps are required for healing, however, when the fibrotic step becomes uncontrolled and pathogenic, the process can lead to organ failure and death. The interstitial lung disease (ILD) includes a wide range of disorders in which pulmonary inflammation and fibrosis are the final common pathway.

Generally, any activated state of tissue repair requires the stimulation of signalling pathways involved in proliferation, cell migration and differentiation. It is therefore understandable that the fibrotic process is influenced by a combination of growth factors (such as TGFβ, FGF), and cell adhesion molecules (such as integrins). Modulation of growth factor expression, loss of E-cadherin and activation of β-catenin dependent gene transcription leads to epithelial-mesenchymal transition (EMT) which is also an important feature of the fibrotic process. Direct involvement of canonical Wnt signalling in EMT has been confirmed in studies using Wnt1 and Lef-1 overexpression 106. Furthermore, during cellular migration, which is an important factor in tissue repair, proteolytic degradation of the extracellular matrix is necessary to enable fibroblasts to migrate through the extracellular matrix to the site of the lesion. Proteolytic degradation of the extracellular matrix requires plasminogen and matrix metalloproteinases 107108. Gene transcription of MMP-s is regulated by Wnt signalling of both canonical and non-canonical pathways. Metalloproteinase matrilysin (MMP7), a target gene of the canonical Wnt signalling pathway 109, has recently been identified as a key regulator of pulmonary fibrosis 110111. In many cases of idiopathic pulmonary fibrosis, the levels of nuclear β-catenin are elevated 112, as are the levels of β-catenin target genes, cyclin D1 and MMP-s 112.

As Wnt-s have also been implicated in the modulation of proliferation and differentiation of many lung cells 596066, the role of Wnt signalling in regulating cell proliferation and differentiation during idiopathic pulmonary fibrosis, is likely to be central rather than a consequence of the disease.

In summary, Wnt signalling may also be central to all causes of pulmonary fibrosis and requires further evaluation.