Abstract
The WNT family of signaling proteins is essential to organ development in general and lung morphogenesis in particular. Originally identified as a developmentally active signaling pathway, the WNT pathway has recently been linked to the pathogenesis of important lung diseases, in particular lung cancer and pulmonary fibrosis. This review summarizes our current understanding about WNT signaling in lung development and disease, and is structured into three chapters. The first chapter presents an introduction to WNT signaling, outlining WNT proteins, their receptors and signaling intermediates, as well as the regulation of this complex pathway. The second chapter focuses on the role of WNT signaling in the normal embryonic and adult lung, and highlights recent findings of altered WNT signaling in lung diseases, such as lung cancer, pulmonary fibrosis, or pulmonary arterial hypertension. In the last chapter, we will discuss novel data and ideas about the biological effects of WNT signaling on the cellular level, highlighting pleiotropic effects induced by WNT ligands on distinct cell types, and how these cellular effects may be relevant to the pathogenesis of the aforementioned diseases.
This review summarizes our current understanding about WNT signaling in lung development and disease. We discuss novel data and ideas about the biological effects of WNT signaling on the cellular level, highlighting pleiotropic effects induced by WNT ligands on distinct cell types, and how these cellular effects may be relevant to the pathogenesis of the aforementioned diseases.
The WNT family constitutes a large family of secreted glycoproteins with highly conserved cysteine residues (1–4). The name “WNT” is derived from a synthesis of the two genes wingless and int-1. The int-1 gene was first described by Nusse and Varmus as a proto-oncogene that caused mammary tumors, when the mouse mammary tumor virus (MMTV) was integrated in the int-1 locus (5, 6). At about the same time, Nüsslein-Volhard and colleagues (7) described that fruit flies (Drosophila melanogaster) lacking the gene wingless were unable to develop wings. In the following years, wingless and int-1 were found to encode identical proteins, and the new name, WNT, was formed (8, 9). To date, 19 different WNT proteins have been identified in humans, most of which have been shown to be expressed in a tightly regulated spatiotemporal manner. Many WNT proteins share a high amino acid sequence homology—in particular, those that are grouped within similar subfamilies (e.g., WNT7a and WNT7b). The array of WNT genes identified and characterized thus far suggests that different WNT ligands can functionally compensate a loss of function of a distinct WNT ligand. Secreted WNT proteins are usually quite insoluble and hydrophobic due to extensive palmitoylation of the mature protein (10). This post-translational modification is critical for proper WNT signaling, but, at the same time, has also delayed the purification, production, and subsequent analysis of recombinant WNT proteins for a long time (11). New insights into the production, secretion, or transportation of WNT ligands have only recently been presented, which are covered in detail by excellent reviews (1, 12).
WNT signaling cascades can be divided into at least the three following distinct pathways. First, the “canonical” WNT/β-catenin pathway involves WNT ligand binding to cell surface receptors and cytosolic stabilization and nuclear translocation of β-catenin for target gene expression. Second, the WNT/Ca2+ pathway signals via calmodulin kinase II and protein kinase C. Third, the WNT/JNK or planar cell polarity pathway signals through small GTPases, and is implicated in cytoskeletal organization and epithelial cell polarity. Some WNT ligands are known to activate both the canonical and the noncanonical pathway, whereas others, such as WNT5a, appear to be specific for noncanonical signaling (1, 3, 10).
The best-characterized WNT signaling pathway is the β-catenin–dependent WNT signaling pathway (Figure 1). Here, in the absence of active WNT ligands, β-catenin is bound to the scaffold proteins Axin and adenomatosis polyposis coli (APC), and constitutively phosphorylated at four N-terminal residues via interaction with casein kinase I and glycogen synthase kinase (GSK)-3β. Phosphorylated β-catenin is a target for subsequent ubiquitination and proteosomal degradation via the β-transducin repeat containing protein.
In the presence of WNT ligands, the membrane receptors, frizzled (FZD) and low-density lipoprotein receptor–related protein (LRP) 5 or 6, are activated upon ligand binding. The FZDs are seven transmembrane spanning receptors, and represent the primary receptor for WNT ligand binding, whereas LRP5/6 are single-pass transmembrane receptors, and represent WNT coreceptors. Up to now, 10 FZDs have been described and investigated (4, 13). In addition, alternative WNT receptors, such as Ryk, have also been identified lately (14). The number and diversity of different WNT ligands and receptors is remarkable, and clearly emphasizes the system's complexity, as well as the ongoing challenge to identify specific ligand–receptor interactions that activate distinct intracellular signaling pathways. This multiplicity of WNT ligand–receptor interactions have recently been covered in great detail (4, 12, 13, 15), and is not within the scope of this review.
In the canonical WNT/β-catenin pathway, WNT ligand binding to FZD and LRP leads to the phosphorylation of LRP6 by GSK-3β and casein kinase γ in its cytoplasmic region, which leads to the recruitment of the cytosolic proteins, Dishevelled (DVL) 1–3 and Axin (Figure 1). Subsequently, β-catenin phosphorylation is inhibited and its degradation attenuated. Accumulated β-catenin then undergoes nuclear translocation, and regulates target gene expression via interaction with members of the T cell–specific transcription factor (TCF)/lymphoid enhancer–binding factor (LEF) family (1, 3). In the absence of β-catenin, TCF and Groucho form a constitutive repressor complex. After its nuclear translocation, β-catenin disrupts this interaction of TCF and Groucho, and regulates gene transcription by binding to its target sequence. The transcriptional profile of the WNT/β-catenin pathway is diverse, and the number of cell-specific, as well as general WNT target genes, is steadily increasing (16–18). WNT target genes also include WNT signaling components themselves, such as Dickkopf (DKK) 1 (19) or Axin2 (20), indicating feedback regulation of this system, as well as several oncogenes (i.e., Cyclin D or c-myc [21, 22]).
The WNT signaling pathway is highly regulated, and different WNT inhibitors have been described thus far (23–25). It has not yet been fully determined, in most cases, whether these proteins consistently inhibit or facilitate WNT signaling, which depends on their expression levels and the cellular context. We therefore prefer to use the term “WNT regulators” rather than “WNT inhibitors.” In general, WNT regulators can be divided into two different groups. The first include secreted FZD receptor proteins (sFRPs) 1, 2, 4, and 5, as well as WNT inhibitory factor (WIF). The sFRPs represent extracellular WNT regulators that directly bind WNT ligands, thereby preventing ligand binding to the signaling receptors, FZD and LRP. The sFRPs exhibit a high degree of homology with the ligand-binding domains of FZD, which explains their binding capacity for WNT ligands (23, 26). Similar mechanisms have been proposed for WIF (27).
The second group of WNT regulators include DKK proteins and Wise. These extracellular proteins use a fundamentally different mechanism to modulate WNT signaling, as DKK and Wise bind to the WNT-binding domain of LRP6 (25, 28). Four different DKK proteins have thus far been discovered, which all share conserved cysteine-rich domains. DKK1 is the founding member of the family, which was originally identified as an embryonic head inducer and WNT inhibitor in Xenopus (29). Whereas DKK1, -2, and -4 share a high degree of sequence homology, DKK3 is less related to the other three DKKs. DKK1 is classically known as a WNT inhibitor, whereas DKK2 can function as either an activator or an inhibitor of WNT signaling, depending on its cellular context (30, 31). Recently, it has been shown that DKK1 and -2 also exhibit a high binding affinity for a second class of receptors, termed Kremen (KRM) (32–34). The formation of a ternary complex of DKK, KRM, and LRP6 is thought to lead to endocytosis of the whole receptor complex, thereby blocking WNT signaling (35–37). Although KRM1 and -2 have been shown to potentiate the ability of DKK to modulate WNT signaling, it is likely that DKK can even modulate WNT signaling in the absence of KRM.
Another WNT regulator (Wise) has recently been identified, which has been shown to interact with LRP6, with the ability to activate or inhibit WNT signaling in a context-dependent manner (28). Most WNT regulators, such as sFRP or DKK, are highly regulated on the transcriptional level, and present direct feedback control of WNT stimulation (19, 20). Most recently, novel WNT regulators have been identified, such as the R-Spondin family (38, 39). R-Spondins are secreted proteins capable of promoting WNT signaling, probably due to binding to FZD and LRP receptors; their precise mechanism of action, however, remains elusive (14).
In addition to the aforementioned regulatory mechanisms, the interactions of WNT proteins with other signaling pathways potentially control WNT action. Several studies have demonstrated that the WNT pathway interacts with other pathways, such as the transforming growth factor (TGF)-β, connective tissue growth factor (CTGF), sonic hedgehog (SHH), or Notch pathway (40–45). These cross-pathway communications play an essential role for the spatiotemporal activity of WNT signaling, and direct evidence of single-pathway interactions will be discussed in the following chapters.
The number of studies investigating WNT signaling in the lung has increased exponentially in recent years. The first evidence of WNT expression in the lung was published in 1990 (46). Subsequent to this finding, the first years of WNT research in the lung have largely focused on lung cancer and lung development. More recently, WNT signaling in lung fibrosis and lung stem cells developed as a promising research area. In the following chapters, we will highlight these studies and summarize the current understanding of WNT signaling in lung development and disease.
The lung arises from a small diverticulum in the anterior foregut endoderm at the laryngotracheal groove. The respiratory epithelium then invades the surrounding mesenchyme, followed by the process of branching morphogenesis (43, 47). This process depends on the precise coordination of epithelial–mesenchymal interactions involving cell–cell and cell–matrix interactions, controlled by an orchestra of secreted growth factors (48). Multiple signaling pathways have been implicated in the control of branching morphogenesis, these include, among others, the fibroblast growth factor, TGF-β, bone-morphogenic proteins (BMPs), SHH, or WNT (47, 49).
WNT signaling is known to regulate epithelial and mesenchymal cell biology in an autocrine and paracrine fashion (48, 50). Several WNT ligands, receptors, and components of the canonical pathway are expressed in a highly cell-specific fashion in the developing lung. For instance, WNT2 is highly expressed in the distal mesenchyme (51), whereas WNT7b is expressed predominantly in the epithelium (52). In addition, WNT5a is expressed in both cell types (53). Despite early and strong expression of WNT2 in the developing lung, transgenic deletion of WNT2 did not result in any detectable defects of lung development and function (54), probably due to functional redundancy of WNT2 proteins. Shortly after this initial study, WNT2b, another WNT2 family member, was identified and characterized; this may have compensated for the loss of WNT2 seen in the initial study (55).
Transgenic deletion of WNT7b, by replacement of the first exon with a lacZ-coding region (WNT7blacZ), causes perinatal death due to respiratory failure. WNT7blacZ mice are characterized by impaired mesenchymal growth and vascular development due to defective autocrine and paracrine WNT signaling in airway epithelium (52). The observed defects in vascular smooth muscle development suggest a role of WNT7b signaling in vascular lung diseases, such as pulmonary arterial hypertension (PAH) (52, 56). In another study, a conditional null allele of the WNT7b gene was generated by removal of exon 3 and, in contrast to the previous report, these mice demonstrated a reduction in the proliferation of both epithelium and mesenchyme, as well as normal vascular smooth muscle development (57). It has been shown that WNT7b consists of two different isoforms generated by two initiating exons. The later study proposed a hypomorphic allele in the WNT7blacZ mice due to the presence of an alternative exon 1, which may lead to partial loss of function, and thus may explain the aforementioned effects (57). On the cellular level, it has been shown that WNT7b is expressed by lung epithelium, and activates the canonical, but not the noncanonical, WNT pathway in vascular smooth muscle cells through FZD1 in a paracrine fashion. Furthermore, WNT7b exhibits autocrine signaling activity via binding to FZD10, which is expressed on the surface of epithelial cells (56).
Deletion of WNT5a by homologous recombination in mice leads to overexpansion of distal airways and expanded interstitium, accompanied by increased SHH expression (53). In addition, transgenic mice with epithelium-specific overexpression of WNT5a exhibited reduced epithelial branching morphogenesis and distal air space enlargement. WNT5a overexpression led to increased mesenchymal fibroblast growth factor signaling and concomitant decreased epithelial SHH signaling (58). This strongly suggests that WNT proteins interact with other signaling pathways in lung development.
Active WNT signaling in lung development has also been demonstrated using transgenic WNT reporter mice and nuclear β-catenin staining. TOPGAL or BATGAL mice, both of which harbor a β-galactosidase gene under the control of a LEF/TCF–inducible promoter fragment (TOP for Tcf-optimal-promoter and BAT for beta-catenin-activated transgene), revealed active canonical WNT signaling early throughout the epithelium and the mesenchyme adjacent to proximal airways at Embryonic Days (E) 10.5–12.5. This signaling disappeared first in the mesenchyme, and was subsequently reduced in the epithelium at E13.5–E18.5 (50, 59, 60).
Epithelial cell–specific expression of constitutively active β-catenin leads to epithelial cell dysplasia and ectopic differentiation of alveolar epithelial type II cells in the conducting airways during embryonic development. Postnatally, these mice exhibit air space enlargement and develop pulmonary tumors (61). Lung epithelial cell–specific deletion of β-catenin, in contrast, results in disruption of branching morphogenesis, with distorted differentiation of the peripheral lung. The mice died neonatally due to respiratory failure (62). Because β-catenin functions as a key regulator of WNT signaling, as well as cell adhesion processes, disruption of β-catenin may not only result in distorted WNT signaling, but also dysregulated cell adhesion (63).
Further evidence for this key role of canonical WNT signaling in epithelial function was provided by targeted overexpression of the WNT regulator, DKK1, in lung epithelium, which resulted in disruption of proximal–distal patterning via inhibition of active WNT signaling (60). DKK1 treatment of mouse lung organ cultures also led to impaired branching morphogenesis and defects in the formation of the pulmonary vascular network (64). In this study, inhibition of fibronectin using a neutralizing antibody led to similar branching defects as observed by DKK1 induction. In contrast, addition of exogenous fibronectin rescued the DKK1-mediated phenotype, emphasizing the importance of extracellular matrix formation in these processes (64). It has to be pointed out, however, that, in an above-mentioned study (58), epithelium-specific overexpression of WNT5a led to an inhibition of epithelial branching morphogenesis.
The fact that some of the above-mentioned studies have resulted in apparent opposite results further underlines the complexity of the WNT signaling system, which is subjected to a tightly regulated spatiotemporal expression pattern in the lung. Further studies—most importantly, in human lung development—are required to clarify these issues. Overall, however, these studies have demonstrated a critical role of properly regulated WNT signaling for normal epithelial–mesenchymal interaction during lung morphogenesis, and emphasized the deleterious impact of distorted WNT signaling on proper lung development.
In the adult lung, most WNT components, including canonical and noncanonical WNT ligands, receptors, regulators, and intracellular signal transducers are expressed at detectable levels. Recently, we have demonstrated that WNT1 and -3a mainly localized to bronchial and alveolar epithelium, along with expression of WNT1 in pulmonary endothelial and smooth muscle cells (65). Quantification of the mRNA levels of canonical WNT/β-catenin signaling components in lung tissue samples of transplant donors revealed that the WNT ligands, WNT1, -2, -3a, and -7b, were expressed at similar levels in normal adult lung tissue, whereas WNT10b exhibited low expression. The most abundant receptors in the human lung were FZD1/4 and LRP5/6. The main canonical WNT signal transducers, GSK-3β and β-catenin, as well as members of the TCF/LEF family, were expressed, with the exception of TCF1 (65). In support of this, Winn and colleagues (66) demonstrated the expression of several WNT proteins in lung epithelial cell lines. Thus, it is evident that the adult lung expresses all required components for WNT signaling, but functional studies thereof are missing thus far.
The role of WNT signaling in various types of cancer is well established, and excellent reviews have been published on this topic (67, 68). This chapter will focus on studies of lung cancer, which is the leading cause of death among cancer patients worldwide. Lung cancer can be separated in to two major forms: non–small cell lung cancer (NSCLC) and small cell lung cancer, which account for 80 and 20% of all lung carcinomas, respectively (69). A multistep oncogenic process, involving tumor suppressors as well as oncogenes, seems to trigger the transition of normal epithelial cells to metaplastic cells, and, subsequently, carcinoma cells (70). Dysregulated WNT signaling in cancer has been primarily found in colon cancer, and more recently, studies focused on lung cancer—in particular, NSCLC (69, 71). It has been shown that mutations in key WNT signaling genes, such as APC or β-catenin, are frequently associated with colon cancer, whereas such mutations seem to be rare in lung cancer (72–74).
Several WNT proteins are differentially expressed in NSCLC specimens, including WNT1, -2, and -7a. WNT1 is overexpressed in NSCLC samples, and cancer cells expressing WNT1 are resistant to apoptotic therapies (75, 76). Conversely, inhibition of WNT1 led to apoptosis in human cancer cells, and reduced tumor growth in vivo and in vitro (75). Likewise, WNT2 was overexpressed in NSCLC, and blockade of WNT2 induced apoptosis in cancer cells (77). Huang and colleagues (78) reported that WNT5a gene expression was higher in squamous cell carcinoma compared with adenocarcinoma, suggesting that WNT5a expression is responsible for more aggressive forms of NSCLC. Interestingly, WNT7a is decreased in NSCLC. Forced overexpression of WNT7a and FZD9 reversed cellular transformation by inhibiting cell growth and inducing epithelial cell differentiation in NSCLC (66). These antitumorigenic effects were mediated by activation of peroxisome proliferator–activated receptor γ, suggesting a noncanonical WNT signaling pathway involved in tumorigenesis (79).
Uematsu and colleagues (80) provided evidence that active WNT signaling in NSCLC is mediated by overexpression of the intracellular signal transducer, DVL. Specifically, DVL3 was overexpressed in microdissected NSCLC samples, and inhibition of DVL decreased β-catenin expression and cell growth (80). The WNT regulator, WIF, as well as sFRP1 and DKK3, are down-regulated in several cancer types, including NSCLC, due to transcriptional silencing via hypermethylation of their promoters (81–85). It has also been reported that promoter hypermethylation of WIF and sFRP1 was more frequent in colorectal metastases compared with the corresponding primary lung adenocarcinomas, whereas APC promoter methylation was significantly more common in primary lung adenocarcinomas (86). This differential methylation pattern may thus allow easier discrimination of primary lung tumors and lung metastases of different primary tumors.
In contrast to the above-mentioned results, the role of β-catenin in lung cancer remains to be fully elucidated. Mutations in the β-catenin gene are rare in lung cancer cell lines or primary lung carcinomas. Several groups have observed reduced expression of β-catenin in lung carcinoma, which was associated with poorer prognosis (87, 88), possibly due to proliferative cellular activity and undifferentiation in NSCLC (89). Conversely, Hommura and colleagues (90) reported that increased β-catenin expression is associated with enhanced cell proliferation and a beneficial prognosis. These results are most probably due to multiple roles of β-catenin in transducing canonical WNT pathways, as well as in regulating cellular adhesion. In sum, increased expression of WNT proteins, along with decreased expression of WNT regulators, is likely involved in the initial phase of tumorigenesis, as well as in the ongoing, multistep process of lung cancerogenesis (Figure 2, left panel).
Aberrant activation of a developmental pathway is a common and well investigated feature of cancer, but this mechanism is largely underappreciated in nonmalignant diseases. New technologies, such as high-throughput whole-genome expression or proteomic profiling, have recently contributed to our understanding of pathways that are involved in disease progression.
In pulmonary fibrosis, studies in animal models, as well as human disease, have provided evidence for the reactivation of developmental programs, including the WNT signaling pathway, which was recently summarized in an excellent review (91). Idiopathic pulmonary fibrosis (IPF), the most common form of idiopathic interstitial pneumonias, represents a progressive and lethal disorder with limited responsiveness to currently available therapies (92–94). It is well accepted that repetitive injury and subsequent repair of alveolar epithelial type II (ATII) cells, in the presence or absence of local inflammation, represent a key pathogenic mechanism in IPF (95–98). This, in turn, leads to aberrant growth factor activation, and perpetuation of fibrotic transformation and aggregation of activated myofibroblasts (fibroblast foci), which promote excessive extracellular matrix (ECM) deposition (99). Fibroblast foci occur in subepithelial layers, close to areas of alveolar epithelial cell injury and repair, suggesting that impaired epithelial–mesenchymal crosstalk contributes to the pathobiology of IPF (95, 100). Unbiased microarray screens have revealed the overexpression of WNT genes, including WNT2 and -5a, the receptors FZD7 and -10, and WNT regulators, such as sFRP1 and -2, in IPF lungs compared with normal lungs or those with other interstitial lung diseases (91, 101–103). In addition, several WNT target genes, such as matrix metalloproteinase 7 (104), osteopontin (105), or WNT1-inducible signaling protein (WISP) 1, were recently identified in experimental and idiopathic lung fibrosis (102, 106, 107). Chilosi and colleagues (108) reported nuclear localization of β-catenin in ATII cells and interstitial fibroblasts in IPF lungs, indicative of activated WNT signaling (109). In addition, we have recently reported that several components of the canonical WNT signaling pathway are overexpressed in IPF (65), as well as in experimental lung fibrosis (107). Canonical WNT signaling components (including WNT ligands, β-catenin, or GSK-3β) localized mainly to the bronchial and alveolar epithelium, as observed by immunohistochemistry and gene expression analysis of ATII cells (65). Importantly, increased activity of the WNT pathway in IPF was documented by increased phosphorylation of LRP6 and GSK-3β (65), which has recently been demonstrated to be a sensitive indicator of WNT activity in tissue sections (110, 111). In addition, we recently demonstrated the activation of canonical WNT signaling in experimental lung fibrosis in vivo, along with increased expression of the WNT target gene, WISP1, in hyperplastic ATII cells. Most importantly, inhibition of WISP1 led to the attenuation of experimental lung fibrosis in vivo (107).
In contrast, the role of WNT signaling in lung inflammation is largely unexplored. Recently, Lewis and colleagues (106) provided a comparison of the gene expression profile of 12 different mouse models of infection, allergy, or lung injury. The authors reported regulation of the WNT signaling pathway only in the mouse model of bleomycin-induced lung fibrosis, but not in any other inflammatory lung disease models. In another study, Douglas and colleagues (112) investigated the resolution phase after lung injury, which is characterized by a decrease of inflammation, re-epithelialization, and matrix remodeling. The authors used a mouse model of oxidant-induced injury with subsequent hyperoxic exposure, and demonstrated increased nuclear β-catenin during the fibroproliferative phase after acute lung injury. The authors showed a concomitant increase in E-cadherin expression and epithelial cell proliferation, supporting a noncanonical cadherin–catenin axis involved in the resolution of inflammation. Both studies strongly suggest a role for WNT signaling in the resolution and regeneration phase after lung injury. It is therefore likely that this resolution process is distorted in IPF (Figure 2, right panel). This is further supported by studies in rheumatoid arthritis, in which WNT proteins induced the expression of cytokines, and appeared to be responsible for an activated phenotype of fibroblast-like synovial cells that exhibit characteristics of immature mesenchymal cells. These cells are involved in long-standing disease, with joint destruction and attempted regeneration (113).
Of interest, WNT signaling crosstalk with other profibrotic growth factors, such as TGF-β, has recently been reported. TGF-β is a key mediator of IPF disease progression, inducing epithelial-to-mesenchymal transition (EMT), fibroblast-to-myofibroblast activation, and extracellular matrix production. With respect to fibrosis, activation of β-catenin by TGF-β in hypertrophic scars has been reported (41). Thus, crosstalk between WNT and TGF-β in lung fibrosis is of major interest that needs to be explored in much more detail. Another growth factor known to be involved in lung fibrosis, CTGF, binds to the WNT coreceptor, LRP6, revealing additional ways of action for CTGF in the fibrotic process (40). Furthermore, expression of SHH is increased in IPF, but not in other interstitial lung diseases (114). In a mouse model of fibrotic disease, but not in nonfibrotic inflammation, SHH and TGF-β were up-regulated in epithelial cells (115).
In sum, WNT signaling seems to be reactivated in response to an as-yet unknown injurious stimulus in IPF, most likely as “attempted regeneration” of lung epithelium. This may explain the findings of hyperplastic epithelium and EMT in IPF lungs (Figure 2, right panel). Unfortunately, this “attempted regeneration” is not only insufficient to restore normal lung architecture in IPF, but, moreover, further drives fibrogenesis and IPF progression by paracrine actions on fibroblasts (65, 107).
PAH is a devastating disorder, characterized by cellular and structural changes in pulmonary arteries (116, 117). Recently, a gene expression analysis of pulmonary arterial resistance vessels revealed differentially regulated canonical and noncanonical WNT genes in PAH (118). One distinct feature of PAH is the increased abundance of α-smooth muscle actin–positive cells in the vessel wall. The possible origin of these cells include: (1) proliferation of resident vascular smooth muscle cells or adventitial fibroblasts; (2) recruitment of vascular progenitor cells; or (3) the transition of endothelial cells into a mesenchymal phenotype, a process that has been suggested to play an important role during cardiovascular development (116–119). The last concept is similar to the process of EMT, which is known to be regulated by WNT signaling, and will be discussed in a separate chapter.
A pathological hallmark of PAH is the “plexiform lesion,” which consists of conglomerates of cells obliterating the vascular lumen. These conglomerates are composed of endothelial cells, smooth muscle cells, mast cells, or lymphocytes (116, 117). In a recent article, Rai and colleagues (120) hypothesized that plexiform lesions reflect complex vascular lesions exhibiting neoplastic features. The authors reported nuclear β-catenin staining and loss of WNT7a in endothelial cells of plexiform lesions in PAH. A recent study demonstrated that BMP2, via its receptor, BMPRII, led to pulmonary arterial endothelial cell proliferation and survival via activation of the WNT signaling pathway (121). Mutations in BMPRII are associated with PAH, suggesting that impaired BMP–WNT signaling is involved in PAH pathogenesis (121).
Although studies investigating WNT signaling in the lung have increased over the past years, our knowledge about the mechanisms and functions of WNT signaling on the cellular level in the lung remains limited. In the following section, we highlight current biological effects of WNT signaling in distinct lung cell types, underlining novel ideas on how this may contribute to the development of chronic lung disease.
Most of the internal surface area of the lung is lined by ATII and type I (ATI) cells, which play a multifunctional role in lung homeostasis, and exhibit a high degree of cell plasticity (122–124). These cells are involved in cell–cell adhesion, ATII-to-ATI cell transition, proliferation, and EMT. WNT signaling has been shown to regulate many of these cellular functions, and, if dysregulated, may contribute to the initiation and progression of several lung diseases—particularly, lung cancer and fibrosis.
EMT is defined as a reversible phenotypic switching of epithelial-to-fibroblast-like cells. EMT has been described in the process of embryonic development, as well as in oncogenic progression and metastasis (125, 126). The orchestrated series of events during EMT involve changes in cell polarity, loss of epithelial cell markers, such as E-cadherin, remodeling of epithelial cell–cell and cell–matrix adhesion contacts, reorganization of the actin cytoskeleton, induction of mesenchymal gene expression, or enhanced cell migration (126, 127).
In lung cancer, EMT is thought to promote cancer invasion and metastasis, and it has been proposed to be a survival strategy for cells to avoid induction of apoptosis by drug treatment (128, 129). Barr and colleagues (130) reported increased resistance to therapies, such as epidermal growth factor receptor inhibition by erlotinib treatment, in cancer cells exhibiting an EMT phenotype. The impact of WNT signaling as a regulator of EMT has been demonstrated by the direct participation of β-catenin in these processes (131, 132), as well as by induction of EMT by WNT ligands in vitro (133). Evidence for the involvement of WNT signaling in the process of EMT in lung cancer in vivo, however, is missing.
In organ fibrosis, accumulating evidence suggests EMT as a possible mechanism that increases the (myo-)fibroblast pool, thereby influencing the process of repair and remodeling after epithelial injury in a number of tissues (134). In pulmonary fibrosis, it was recently demonstrated that TGF-β induces EMT in alveolar epithelial cells in vitro and in vivo (135–137), and that β-catenin is involved in the process of EMT in alveolar epithelial cells (138). Furthermore, epithelial as well as mesenchymal markers colocalize to hyperplastic ATII cells in lung tissue of patients with IPF, suggesting that EMT represents a significant mechanism contributing to IPF pathogenesis (136). Recent studies revealed that the WNT target gene, WISP1, induces EMT in vitro, further highlighting the role of WNT signaling in the process of EMT in lung fibrosis (107).
A key feature of all epithelial cells is the generation of tight cell–cell adhesions. In several lung diseases, the normal alveolar epithelial cell layer is disrupted, and epithelial cells exhibit a proliferative or migratory phenotype, facilitating processes, such as epithelial cell transformation, EMT, or metastasis (125, 127, 139). Cadherins are cell adhesion receptors, such as E-cadherin in epithelial cells, which interact with β-catenin (140, 141). This is of particular interest, as β-catenin is one of the key signaling molecules in WNT signaling. This dual role of β-catenin emphasizes the close connection between cell adhesion and WNT signaling (142–144). This was highlighted in two recent studies reporting different conformational changes of β-catenin that allow its discrimination of WNT signaling and cell adhesion participation (145, 146). It is thought that only a minority of the cellular β-catenin content in epithelial cells contributes to WNT signaling, whereas the majority of β-catenin molecules is present within the cytosol and at cell–cell contacts (144, 147). Future studies are needed to understand how β-catenin is able to differentiate between these two pathways—in particular, in lung epithelium.
In NSCLC, enhanced expression of WNT1 and -2 drives epithelial cell proliferation, and inhibition thereof induces epithelial cell apoptosis (75, 77). In contrast, WNT7a expression is decreased in NSCLC, and gain-of-function studies of WNT7a in NSCLC cell lines revealed a reversal of cell transformation and growth (66, 79). Furthermore, WNT7a induces the expression of E-cadherin, also suggesting an involvement of WNT7a in the prevention/reversal of EMT (148).
In IPF, initial alveolar epithelial cell damage and subsequent impaired repair are thought to represent key pathogenic features (95, 97, 149). Hyperplastic, proliferating alveolar epithelial cells are frequently found in pulmonary fibrosis, and alveolar epithelial cell proliferation may be involved in this impaired repair (107, 150–152). Recently, we reported increased proliferation of alveolar epithelial cells, in concert with up-regulation of WNT target genes, in response to canonical WNT activation, suggesting a role of active WNT signaling in impaired epithelial cell repair mechanism (65, 107). It has to be highlighted that an increased incidence of lung cancer in IPF suggests a link between epithelial cell hyperplasia, impaired repair, and carcinogenesis (153–155). While further research is clearly required to elucidate these associations, studies thus far indicate active WNT signaling as a common molecular mechanism linking alveolar epithelial cell transformation and hyperplasia with fibrosis or cancer.
Proliferation and activation of (myo-)fibroblasts is a key feature in both lung cancer and lung fibrosis (156, 157). The (myo-)fibroblasts contribute to tumor stroma and fibroblast accumulation in IPF by enhanced production of ECM and secretion of growth factors. Recent studies have demonstrated the capability of lung fibroblasts to respond to WNT stimulation (65, 107, 158–160). Gene expression profiling of human lung fibroblasts treated with WNT3a revealed several new WNT-induced genes in fibroblasts, among these genes that play a role in fibroblast-to-myofibroblast differentiation (158). In addition, Chen and colleagues (160) reported differences in the expression of genes involved in cell motility/adhesion, cell cycle, or cytoskeletal formation, in WNT3a-stimulated NIH-3T3 cells. Furthermore, functional studies on NIH-3T3 and lung fibroblasts revealed activation and increased collagen deposition in response to canonical WNT signaling (65) or WISP1 (107). Taken together, these data support a role of WNT signaling on profibrotic phenotypes of lung fibroblasts—in particular, via paracrine effects of epithelium-derived WNT ligands.
An emerging area of research is dedicated to the role of WNT signaling in somatic or adult stem cell maintenance and differentiation. Stem cells were first identified as human bone marrow cells that exhibit the potential for proliferation, self-renewal, and differentiation into an array of different cell types. These properties favored stem cells for a range of therapeutic applications and, in the last years, have attracted huge scientific interest. Several studies have characterized stem cell populations and their ability to repair damaged organs. WNT proteins, for the most part, have been proposed to regulate stem cell maintenance and self-renewal (11, 69, 161, 162).
In the lung, local stem cell niches in the airway epithelium have been described (163–165). A number of cells at the bronchoalveolar duct junction exhibit features of stem cells. These “bronchoalveolar stem cells” (BASCs) coexpress Clara and alveolar epithelial cell marker proteins, and appear to be refractory to injury due to naphthalene treatment (166). BASCs divide after induction of damage, which results in repair of epithelial tissue. Different from other tissue stem cells, only rare BASCs may be activated to participate in epithelial repair after depletion of so-called “transit-amplifying cells,” cells characterized as progenies of tissue stem cells with proliferative capacity (69, 165, 167). Recently, the involvement of WNT signaling in lung stem cell maintenance and activation has been pointed out (168–170). Zhang and colleagues (168) demonstrated that the transcription factor, GATA6, influences the temporal appearance and number of BASC in the lung by regulating WNT signaling. Loss of GATA6 led to increased WNT signaling and BASC expansion, with decreased differentiation. Furthermore, Reynolds and colleagues (169) provide evidence that β-catenin affects stem cell maintenance within the slowly renewing epithelium of the lung. Conditional potentiation of β-catenin signaling in the embryonic lung results in amplification of rather undifferentiated airway stem cells. In contrast, a recent study from the same group suggests that β-catenin signaling is not essential for stem cell maintenance in the adult lung, suggesting important differences in the regulation of β-catenin in the developing and adult lung (171).
Along these lines, it has been suggested that BASCs may give rise to cancer stem cells in lung cancer and be involved in epithelial cell transformation, thus fostering cancer development (166). Lately, in addition to BASC, the role of mesenchymal stem cells (MSCs) in the lung has become of interest (172, 173). Importantly, it has been reported that MSCs exhibit a distinct WNT profile, which takes part in lineage specification (174). In lung cancer, a contribution of MSCs was shown by in vitro migration studies that demonstrated an enhanced migration of MSC toward tumor cells. Furthermore, MSCs that expressed the TNF-related apoptosis-inducing ligand inhibited tumor growth when applied to an experimental lung cancer model (175), underlining the potential of MSCs as a novel therapeutic strategy. In other cancers, however, the application of MSCs has been shown to promote tumor progression and invasion (176).
In lung fibrosis, fibroblasts were classically thought to be of resident tissue origin; however, recent experiments in mouse models, as well as human fibrosis, revealed increased recruitment of bone marrow–derived cells at sites of lung fibrosis (177–181). Ortiz and colleagues (178) reported that systemically administered MSCs home to the lung in response to injury, adopt an epithelial-like phenotype, and reduce the amount of fibrosis. On the other hand, circulating fibrocytes, derived from hematopoietic stem cells in the blood, have been described to contribute to the fibroblast pool in lung fibrosis, thus promoting fibrogenesis (180).
In addition to bone marrow–derived MSCs, a recent study of cells derived from human lung allografts revealed the existence of a multipotent mesenchymal cell population, which is locally resident in the adult human lung and exhibits extended life span in vivo (182). Future studies are clearly needed to improve our knowledge about the origin and contribution of different stem cells in chronic lung diseases—in particular, with respect to WNT signaling in these processes.
It is safe to state at this point in time that the WNT signaling pathway is involved in chronic lung disease. Modulation of the WNT pathway may, therefore, present as a suitable and promising therapeutic strategy in the future. Successful implementation of therapeutic modulation of WNT signaling, however, clearly requires further elucidation of the following disease-specific aspects. In lung carcinoma, increased expression of WNT proteins, along with decreased expression of WNT regulators, is likely involved in the initial phase of tumorigenesis, as well as in the ongoing multistep process of lung cancerogenesis, thus representing an initial “failure signal” in epithelial cells. In lung fibrosis, studies thus far suggest that WNT signaling is activated after an initial injury, thus, most likely representing a regeneration signal of the damaged epithelium. Unfortunately, this “attempted regeneration” is not only insufficient to restore normal lung architecture, but, moreover, drives fibrogenesis and IPF progression by, for example, epithelial cell transformation or EMT, fibroblast proliferation and activation, or interaction with other profibrotic growth factors, altogether perpetuating fibrogenesis.
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