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University of Groningen
WNT signaling in airway remodeling in asthmaKumawat, Kuldeep
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General introduction
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General introduction
Kuldeep Kumawat
1
Chapter 1
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General introduction
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1.1 Asthma
Asthma is a heterogeneous chronic obstructive disease of the airways inflicting
approximately 300 million people worldwide and imposing a substantial burden on patients
and the healthcare system [1]. A precise definition and diagnosis of asthma is still unclear,
mainly due to the existence of a number of different asthma phenotypes such as allergic,
non-allergic, nocturnal and occupational asthma. Often, these phenotypes co-exist and act
synergistically in patients albeit with different underlying mechanisms. The Global Initiative
for Asthma (GINA) has defined asthma as “a chronic inflammatory disorder of the airways
in which many cells and cellular elements play a role. The chronic inflammation causes an
associated increase in the airway hyperresponsiveness that leads to recurrent episodes of
wheezing, breathlessness, chest tightness and coughing particularly at night or in early
morning. These episodes are usually associated with widespread but variable airflow
obstruction that is often reversible either spontaneously or with treatment” [2].
Asthma is a multifaceted manifestation of (epi)genetic and environmental factors that
contribute to the evolution of the disease from childhood and often actively regulate the
course of disease and its management in later stages. Allergic asthma, in particular,
frequently starts in early childhood and can be identified by the allergen-specific symptoms
with a positive skin prick test and presence of allergen-specific serum IgE along with other
respiratory symptoms such as wheezing and cough [3].
Exposure to the inhaled stimuli such as allergens or respiratory viruses triggers exaggerated
response in asthmatic airways inducing airway constriction leading to episodes of
breathlessness and wheezing. Asthma can be effectively managed in mild asthma patients
using short-acting β2 adrenoreceptor agonists alone (e.g. Albuterol) and if needed using a
corticosteroid (e.g. Fluticasone, Budesonide), with or without a long-acting β2
adrenoreceptor agonist (e.g. Salmeterol, Formoterol) thereby providing substantial relief
from the episodic breathlessness. However, despite the most effective current therapies, a
subset of severe asthma patients remain poorly controlled even at the highest doses of
asthma medication [4,5]. Increasing our understanding of asthma pathophysiology and
contribution of its various components to the disease severity and management by current
therapies would help develop new drugs that target the patient subsets more effectively.
1.2 Pathophysiology
Asthma is characterized by the presence of chronic airway inflammation, airway
hyperresponsiveness (AHR), reversible airflow obstruction, extensive structural changes in
the airways termed as airway remodeling and decline in lung function in severe disease [6].
The internal milieu of asthmatic airways is highly heterogeneous due to presence of plethora
of cytokines, chemokines and growth factors released by the inflammatory cells and
structural components of the affected airways [7,8]. Thus, asthma has an intricate
multicomponent pathophysiology. Some of the key features are discussed here.
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1.2.1 Airway Inflammation
Chronic inflammation in asthmatic airways is characterized by the presence of activated
allergen-specific type 2 T helper (Th2) cells, eosinophils, mononuclear cells such as
lymphocytes and macrophages and IgE production [6,9,10]. Bronchial provocation by
inhaled allergens leads to an asthmatic response in patients which is divided into two phases-
early and late asthmatic reaction. The early reaction, as the name suggests, is an immediate
response to the allergen, driven by local mast cell activation, release of mediators and prompt
decrease in bronchial airflow constituting as an acute asthmatic attack. The early phase is
followed by a more severe late asthmatic reaction which is driven by the infiltration of
inflammatory cells with persistent decline in bronchial airflow for prolonged periods [10].
Many of these inflammatory mediators also worsen development of AHR in asthmatics.
The onset of the chronic inflammation is believed to result from inappropriate Th2 responses
to common environmental agents and is maintained in later stages by a complex interplay
of the host immune and structural components. A plethora of cytokines and chemokines
have been implicated in asthmatic inflammation [8,11]. Most abundant among them are
Th2-derived cytokines such as Interleukin (IL) -4, -5, -9, -13 and -25. IL-4 and IL-13 are
crucial for driving IgE production whereas IL-5 drives eosinophilic inflammation. IL-9 and
IL-13 are involved in AHR [12].
Type 2 polarization of CD4+ T-helper cells is influenced by two prominent cytokines - IL-33
and thymic stromal lymphopoietin (TSLP) which are contributed primarily by airway
structural cells [13-17]. Increased expression of IL-33 has been shown in endobronchial
biopsies and bronchoalveolar lavage (BAL) fluid of asthmatics [13,14]. Further, deficiency of
the IL-33 receptor- ST2 impedes the development of Th2 responses as demonstrated in a
mouse model of pulmonary granuloma [18] whereas blocking of IL-33 using a soluble
receptor (ST2) isoform attenuates the release of Th2 cytokines from splenocytes obtained
from allergen-challenged mice [19] underlining the importance of IL-33 in Th2 polarization
in allergic airway inflammation. Similarly, TSLP abundance is increased in asthma
[17,20,21] and a single nucleotide polymorphism in the TSLP gene has been associated with
asthma susceptibility [22,23] underlining its vital role in asthma pathophysiology. Lung
epithelium-specific transgenic expression of TSLP under the surfactant protein C (Sp-C)
driver induces airway inflammation and AHR with production of Th2-specific cytokines and
increased serum IgE levels [24]. In line with its role in asthma, deficiency of the TSLP
receptor protects against the development of allergic airway disease [24,25], primarily due
to an impaired Th2 response against inhaled allergens [25].
In addition to the type 2 polarized T helper cells, other subsets of T cells have recently been
identified as new players in asthma pathology. Th17 is a subset of CD4+ T cells, primarily
derived in response to IL-23 but can also be generated by TGF-β and IL-6 stimulation
[26,27]. Th17 cells release IL-17 which can recruit neutrophils either directly by IL-8
production or indirectly via release of colony stimulating factors and other peptides [26].
Neutrophilic inflammation has been linked with fixed airflow obstruction in severe asthma
[28], sudden-onset fatal asthma [29], occupational [30] and nocturnal asthma [31].
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Additionally, IL-17 can also promote release of profibrotic cytokines such as TGF-β, IL-11
and IL-6 from eosinophils [32] and fibroblasts [33] aggravating airway remodeling.
Regulatory T cells (Tregs) have recently been associated with asthma pathology [12].
Reduced number of Tregs have been reported in the BAL fluid from children with asthma as
compared to healthy subjects [12,34]. Interestingly, the percentage of Tregs in BAL
correlated positively with forced expiratory volume in one second (FEV1) in asthmatic
children underlining a crucial role for Tregs in asthma pathobiology [34]. Tregs are a subset
of CD4+ T cells which also express CD25. These CD4+ CD25+ T cells do not proliferate and
produce cytokines. Of note, Tregs suppress the proliferation and inflammatory response by
other T cells including Th2 cells [12]. In line with that, Tregs have been shown to suppress
established AHR and airway inflammation in animal models of allergic airway disease [35].
Thus, reduced number of Tregs would lead to augmentation of T cell-driven inflammation
in asthma.
1.2.2 Airway Remodeling
Airway remodeling is a hallmark pathological feature of individuals with asthma and is
associated with airway obstruction [37], AHR [38] and declining lung function in severe
disease [39]. The tissue repair response that is activated during remodeling is normally
associated with lung development and response to tissue injury, where it is appropriate and
regulated. However, aberrant airway remodeling, as observed in chronic airway diseases
such as asthma and chronic obstructive pulmonary disease, is pathological and has
detrimental consequences for the patient. Persistent airway remodeling along with chronic
inflammation leads to compromised lung function [40]. Airway remodeling is associated
with the severity of disease. For instance, in fatal asthma, the entire airway tree is massively
remodeled whereas in non-fatal asthma, remodeling is less prominent and afflicts mainly
small airways [40]. Similarly, the thickness of the remodeled airway wall also correlates with
the severity of the disease [40-43]. Airway remodeling is characterized by extensive
structural changes in the airway wall which include airway smooth muscle (ASM) cell
hypertrophy and hyperplasia, subepithelial fibrosis, mucus hypersecretion,
neovascularization and increased and altered extracellular matrix (ECM) expression, leading
to airway wall thickening [40] (Figure 1).
1.2.3 Reticular Basement Membrane Thickening
Reticular basement membrane (RBM) thickening is a predominant feature in asthma and
contributes to the airway wall thickening mainly by deposition of extracellular matrix
proteins such as collagen I, III and IV and laminins [40]. Endobronchial examinations have
revealed a tremendous increase in the thickness of the collagen layer below the airway
epithelium. Contrasting reports exist about the impact of RBM thickening on airway function
in asthma. While studies have suggested a correlation between airway distensibility and
RBM thickening, other studies have found asthmatics without RBM thickening and non-
asthmatics with RBM thickening [40]. The significance of this event in airway remodeling
and asthma needs further investigation.
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Figure 1. Airway remodeling in asthma. (A) Schematic representation of cross-sectional view of
the healthy and asthmatic airways. Asthmatic airways show features of airway remodeling such as
airway wall fibrosis, airway smooth muscle thickening, increased vasculature, epithelial thickening
and increased presence of mucus in airway lumen. (B) Major biological events involved in airway
remodeling. In asthmatic patients, exposure to various inhaled triggers such as viruses, allergens and
environmental triggers leads to damaged epithelium with goblet cell hyperplasia, inflammation,
airway smooth muscle (ASM) hypertrophy and hypertrophy, fibroblast activation and altered
extracellular (ECM) composition. (Image B is taken from Prakash YS (2013) [36]
A
B
Airway wall Fibrosis
smooth muscle
Blood vessel
epithelium
Mucus
Healthy airway Asthmatic airway
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1.2.4 Vasculature
Neovascularization and expansion of existing airway vasculature is widely observed in
airway remodeling. Morphometric analysis of postmortem lung tissues and endobronchial
biopsies from asthmatics have shown multifold increase in total number of vessels and in
the vascular area of the airways in comparison to healthy subjects and correlated to asthma
severity [44]. Increased airway vasculature and associated hyperpermeability are believed to
contribute to the clinical manifestations of asthma as both these alterations could increase
tissue swelling thereby decreasing the airway dispensability and increasing the airway
narrowing. Increased vasculature could also promote chronic inflammation, provide
increased access of various pathological mediators to the airway components, thus,
supporting airway remodeling. In addition, it could assist rapid and increased availability of
various medications to the airways and fast-track the clearance of spasmogens supporting
disease managment but it could also promote rapid clearance of medication thereby
hampering the therapeutic intervention [44].
1.2.5 Airway mesenchymal cells
Hypertrophy and hyperplasia of airway mesenchymal cells is an important feature of airway
remodeling. Myofibroblasts and ASM cells constitute an important source of various
cytokines and growth factors in airways and can contribute to the asthma pathophysiology
[45-47]
1.2.5.1 Myofibroblasts
Myofibroblasts are specialized cells derived from the differentiation of either fibroblasts or
smooth muscle cells and possess features of both the fibroblast and myocyte lineage and are
basically associated with repair processes. Increased myofibroblast population is suggested
in the asthmatic airways, particularly in the submucosa [45,48]. The source of
myofibroblasts in asthmatic airways remains unclear but various growth factors such as
TGF-β can induce differentiation of fibroblasts into myfibroblasts and hence, could
contribute to the observed myofibroblast population in asthma [49,50]. In addition,
myofibrobalsts could be derived from circulating fibrocytes or from a less defined precursor
already present in the asthmatic airways such as epithelial cells as they appear quickly in the
airways post allergen challenge [47,51,52]. Myofibroblasts are a rich source of ECM proteins
and as such can contribute to the fibrotic component of airway remodeling.
1.2.5.2 Airway smooth muscle cells
Increased ASM mass is a predominant feature of airway remodeling in asthma and correlates
with the severity of disease [48,53,54]. Both hypertrophy and hyperplasia can contribute to
the increased volume of ASM in asthmatics, although the relative contribution of individual
processes remains unclear [53]. While a study observed two different phenotypes on analysis
of ASM bundles in fatal asthma- one with predominant hyperplasia and another with
predominant hypertrophy [53,55], another found only hyperplasia and no hypertrophy in
the ASM layer of asthmatics [54]. Hyperplasia is believed to be a major source of increased
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ASM mass in asthmatics. This could be, in part, due to increased proliferation or increased
survival of existing ASM. Indeed, asthmatic ASM show increased proliferation in
comparison to healthy ASM cells in vitro [56]. Similarly, ASM bundle in endobronchial
biopsies from severe asthma patients show higher population of proliferative cells in
comparison to the moderately asthmatic and healthy non-asthmatic subjects, providing an
evidence for hyperplastic ASM cells in vivo [57]. Interestingly, migration of fibroblasts and
myofibroblasts may also lead to the increased thickening of ASM bundle as demonstrated by
the induced migration of mesenchymal cells towards the ASM bundle in response to
chemotactic agents such as platelet derived growth factor (PDGF) and CC chemokine ligand
19 released by ASM and mast cells [58,59]. In addition, new ASM cells can also be derived
from differentiation of mesenchymal stem cell (MSCs). Tissue-resident MSCs are a common
feature of many tissues where they play critical roles in repair and regeneration. The role of
lung resident MSCs, however, is unclear. Increased abundance of MSCs has been
demonstrated in the lungs of mouse model of chronic airway inflammatory disease [60].
Interestingly, MSCs have been shown to attain a myofibroblast phenotype, however, a direct
evidence for their contribution to ASM hyperplasia is absent [61].
Mesenchymal cells can also arise from the epithelial cells and fibroblasts [62] and may
contribute to the increased ASM mass. Epithelial cells can transdifferentiate to lose adhesion
and attain mesenchymal characteristics like the presence of vimentin and α-smooth muscle
actin (α-SMA), enhanced motility and production of ECM, in a process known as epithelial-
to-mesenchymal transition (EMT). TGF-β is considered as a master inducer of EMT in
various organs including lung epithelial cells [63]. A study demonstrated that TGF-β can
induce extensive EMT throughout the bronchial epithelium derived from the asthmatic
subjects as compared to rather localized EMT specific to basal cells in non-asthmatic
epithelium [64]. Another study provided in vivo evidence for EMT in a mouse model of
allergic airway inflammation where intranasal administration of house dust mite (HDM)
extract led to loss of epithelial markers and gain of mesenchymal markers in airway epithelial
cells and their subsequent migration to the subepithelial regions [52]. While cellular
transdifferentiation can generate mesenchymal cells, the relative contribution of such
processes in increased ASM mass in airway remodeling requires further investigation.
Mathematical modeling studies have suggested that increased ASM mass is one of the key
functional manifestations of airway remodeling in the asthmatic airways leading to airflow
obstruction, assuming the force generated by the ASM bundle is proportional to its mass.
Studies have indicated that asthmatic ASM cells contract with greater velocity and maximum
shortening capacity in comparison to the healthy ASM cells [46,65,66]. This difference could
be, in part, attributed to the increased abundance of various contractile proteins such as
smooth muscle-myosin light-chain kinase (sm-MLCK), smooth muscle-specific SM22 and
smooth muscle-myosin heavy chain (sm-MHC) in asthmatic ASM cells in addition to
increased ASM mass [46]. Interestingly, bronchial thermoplasty has been shown to improve
asthma control in patients [67] which might be attributed to the reduced ASM mass
following this treatment. However, bronchial thermoplasty might also damage the neural
circuits in the treated area. The relative contribution of damaged innervation and reduced
ASM mass to these beneficial effects of thermoplasty remains unclear. Thus, multiple
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mechanisms could contribute to the increase in ASM mass in asthmatics which can have
tremendous implications for the pathophysiology of asthma.
Primarily involved in the maintenance and regulation of bronchial tone due to their
contractile properties, ASM cells have emerged as a major source of proinflammatory and
proremodeling factors [46,68,69]. For instance, ASM cells in culture release RANTES
(regulated upon activation normal T-cell expressed and secreted) in response to tumor
necrosis factor-α (TNF-α) [70] and eotaxin when stimulated with IL-1β, TNF-α or platelet-
activating factor (PAF) [71-74]. Both RANTES and eotaxin are potent eosinophil
chemoattractant contributing to the airway inflammation in asthma [75]. Similarly,
stimulation of ASM cells with IL-1β, TNF-α and interferon γ (IFNγ) induces the release of
granulocyte-macrophage colony-stimulating factor (GM-CSF) [76] and prostaglandin E2
(PGE2) in culture [77]. Whereas PGE2 is believed to have protective effects in allergen-
induced airway responses and airway inflammation in asthma [78,79], GM-CSF promotes
survival of eosinophils [80]. ASM cells are also an important source of Th2-polarizing
cytokines TSLP [16,17] and IL-33 [13]. In addition, ASM cells can contribute plethora of
other proinflammatory mediators such as IL-1β, IL-6, leukemia inhibitory factor and IL-8
thereby actively participating in the chronic airway inflammation in asthma [68]. Further,
ASM cells are a rich source of various ECM proteins and matrixmetalloproteases
contributing to airway remodeling [46].
1.2.6 Extracellular Matrix
ECM is an intricate network of macromolecules composed of a variety of proteoglycans and
fibrous proteins produced and deposited locally by various mesenchymal cells in the airway
including fibroblasts and airway smooth muscle cells [81,82]. Altered and enhanced ECM
protein deposition within and surrounding the smooth muscle bundle has been observed in
asthmatic airways and contributes to the pathology [46,83,84]. Asthmatic airways show
increased deposition of collagen (I, II and V), fibronectin, hyaluronan, biglycan, versican,
tenascin and laminin α2/β2 whereas abundance of collagen IV, elastin and decorin is
decreased [46,83]. Alterations in ECM composition in asthmatic airways denote the
disruption of ECM homeostasis and may modify the mechanical and functional properties
of embedded structural components such as airway smooth muscle cells [85-87].
1.3 TGF-β: key regulator of airway remodeling
TGF-β is a pleiotropic mediator involved in many biological functions in the lungs, including
the regulation of inflammatory cells, the differentiation and proliferation of resident
structural cells and regulation of angiogenesis [88]. Enhanced abundance of TGF-β is found
in BAL fluid and lungs of asthmatic subjects [89-91]. While TGF-β is contributed by almost
all the structural and inflammatory cells, eosinophils constitute the major source of TGF-β
in asthmatic lungs [92]. Preformed and newly synthesized TGF-β can activate several
pathways leading to both transcriptional and post-transcriptional regulation of factors that
are involved in airway remodeling (Figure 2). Therefore, understanding TGF-β biology and
mechanisms associated with its cellular effects is an important step in elucidating the
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pathophysiology of airway remodeling as well as for the development of novel therapeutic
approaches.
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1.3.1 TGF-β Signaling
The TGF-β family comprises of three structurally related isoforms (β1, β2 and β3) which share
high sequence homology and have non-redundant but sometimes overlapping functions
[94,95]. TGF-β1 is the most extensively studied isoform in airway remodeling and other
diseases. Each isoform is synthesized as a large precursor molecule from its mRNA
containing a signal peptide with a mature form of TGF-β at the C-terminal. On proteolytic
cleavage of the signal peptide, mature TGF-β is secreted as an inactive homodimer bound to
latency-associated peptide (LAP). The release of TGF-β from this inactive complex is a tightly
regulated process and can be catalyzed via several mechanisms such as transient changes in
pH, cleavage by proteases (integrin αvβ6, MMP-2 and -9, plasmin and calpains) or
conformational rearrangements (thrombospondin). Active TGF-β exerts its cellular effects
via a receptor complex comprised of three structurally related transmembrane
serine/threonine kinase proteins - TβR-I, TβR-II and TβR-III. The TGF-β homodimer binds
to the receptor concluding a tetrameric complex along with TβR-I and TβR-II. TβR-I
phosphorylates TβR-II in its cytosolic domain leading to its activation. The phosphorylated
TβR-I, in turn, initiates the intracellular signaling cascades by interacting with and
phosphorylating SMAD proteins- SMAD2 and 3. Phosphorylated SMAD2/3 form a
heteromeric complex with SMAD4 and translocate to the nucleus where they bind to the
SMAD-binding elements in the TGF-β-target promoters via their DNA-binding domains.
SMADs can also partner with a wide array of transcriptional factors and cofactors exercising
a broad modulation of TGF-β-activated transcriptional outcomes [94,95] (Figure 2A).
In addition to the activation of SMAD-dependent canonical signaling, TGF-β/TβR complex
can also activate multiple SMAD-independent signaling cascades including MAPKs like
Figure 2. The TGF-β signaling pathway. Diagrammatic representation of the TGF-β signaling
pathway. Binding of dimeric TGF-β ligand leads to the formation of a ternary heteromeric membrane
complex composed of TGF-β and TGF-β receptors- TβRI and TβRII with subsequent phosphorylation
of TβRI by TβRII. TGF-β may activate SMAD-dependent or –independent signaling cascades
downstream of receptor activation. (A) SMAD-dependent pathway. The activated receptor complex
phosphorylates R-SMADs- SMAD2 and SMAD3 which, in turn, form heteromeric complex with co-
SMAD- SMAD4. The R-SMAD-SMAD4 complex translocate to the nucleus and associates with the
genomic SMAD-binding element (SBE) in a sequence-specific manner. Additionally, the R-SMAD–co-
SMAD complex interacts with other transcription factors that can bind to distinct sequences adjacent
to the SBE and allows for high-affinity binding to the SBE elements. SKI and SNO (also known as
SKIL) are nuclear antagonists of SMADs. SMAD7, an inhibitory SMAD, antagonizes TGF-β signaling
at the receptor level by inducing the degradation of TβRI and/ or by inhibiting phosphorylation of R-
SMADs or it can inhibit the formation of the R-SMAD–co-SMAD complex. In addition to regulating
transcription, TGF-β signaling can also participate in microRNA (miRNA) biogenesis by mediating
the processing of primary miRNA into precursor miRNA in the nucleus in an R-SMAD-dependent and
co-SMAD-independent process. 'mG' and 'AAAAA' represent 5′ capping and 3′ polyadenylation of
mRNAs, respectively. (B) SMAD-independent pathway. The activated TGF-β-TβRI-TβRII complex
transmits downstream signaling via activation of various pathways as depicted in the figure. (Images
shown in panel A and B are taken from Akhurst and Hata (2012) [93])
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extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal Kinase (JNK) and p38,
TGF-β-activated kinase 1 (TAK1) and WNT/β-catenin signaling in various cell types
including ASM cells [96,97]. Rapid activation of ERK by TGF-β has been observed in
epithelial cells, breast cancer cells, airway smooth muscle cells and fibroblasts [97,98].
Autophosphorylation or Src-dependent phosphorylation of TβR-II allows the docking of Shc
to the membrane which in turn recruits Grb2/Sos complex leading to Ras/MEK1/ERK
cascade activation. Another mechanism is the tyrosine phosphorylation of TβR-I and
activation of ShcA recruiting Grb2/Sos complex to the membrane and leading to the Ras-
MAPK cascade activation [97].
In addition to ERK, TGF-β can also activate the p38 and JNK pathway in various cell types
[97]. The activation of p38 and JNK is mediated by a complex TNF receptor-associated
factor6 (TRAF6)-TAK1 cascade where it constitutes a key signaling event in TGF-β-induced
apoptosis [99,100]. TAK1, first identified as a mitogen-activated kinase kinase kinase
(MAP3K7) activated by TGF-β, is a critical regulator in inflammatory, immune and stress
response signaling. TAK1 constitutes an integral part of IL1, TLR and TNF signaling,
activating NFκB and MAPK pathways [101,102]. In TGF-β signaling, TAK1 interacts with
TβR-I and is required for JNK and p38 activation. Mechanistically, TGF-β signaling activates
the recruitment and subsequent Lysine 63-linked polyubiquitination of TRAF6.
Polyubiquitinated TRAF6 interacts with and recruits TAK1 to the TβRI complex.
Subsequently, TRAF6 ubiquitinates TAK1 at Lysine 34 triggering its activation which, in
turn, leads to p38 and JNK activation [100] (Figure 2B).
1.3.2 TGF-β in airway remodeling
TGF-β exerts extensive immunomodulatory and profibrotic effects on the constituents of
airways contributing to airway remodeling in asthma [103]. The key TGF-β-regulated
features of airway remodeling are discussed below.
1.3.2.1 Epithelial Shedding
Damage and loss of epithelial integrity is observed in airway remodeling. Repeated allergen
exposures and ensuing chronic inflammation in asthmatic airways may keep epithelium
under duress. The presence of TGF-β in such circumstances can initiate p38 signaling
leading to apoptosis and epithelial damage [104].
In addition to direct effects, TGF-β can also potentiate apoptosis by other pathways, for
instance, TNF-related apoptosis-inducing ligand (TRAIL). TRAIL is expressed by many
structural and inflammatory cells in asthmatics such as fibroblasts, epithelial, endothelial
cells, eosinophils and macrophages [105]. It is a pro-apoptotic protein and can induce both
the extrinsic and intrinsic cell death pathways in cells expressing its receptors- TRAIL-RI
and TRAIL-RII. TGF-β induces expression of TRAIL in epithelial cells and TRAIL can induce
TGF-β expression suggesting a vicious apoptotic cycle in epithelial cells leading to extensive
damage and airway remodeling [105].
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1.3.2.2 Subepithelial Fibrosis
Enhanced ECM deposition under the airway epithelium is present in airway remodeling.
TGF-β is a highly potent inducer of ECM proteins, mainly from cells of mesenchymal lineage
which also crowd the subepithelial space in asthmatics. TGF-β induces proliferation and
activation of fibroblasts [106,107] and increases their survival by anti-apoptotic mechanisms
[108-110]. In addition, TGF-β promotes their differentiation into more active
myofibroblasts. TGF-β can induce pulmonary fibroblast activation and myofibroblast
differentiation by several mechanisms. For instance, it can limit the antagonistic cAMP
response element-binding protein (CREB) phosphorylation via glycogen synthase kinase
(GSK)-3 [111] or activate β-catenin signaling [107] leading to these effects. TGF-β can also
induce the release of growth factors such as fibroblast growth factor-2 (FGF-2) and
connective tissue growth factor (CTGF) [86], both of which can induce mesenchymal cell
proliferation and the release of angiogenic mediators. In addition, CTGF can also induce
ECM production, cell adhesion and migration [86]. Together with fibroblasts,
myofibroblasts produce and deposit ECM proteins such as collagen, fibronectin and
proteoglycans leading to the subepithelial fibrosis.
1.3.2.3 Airway smooth muscle cell remodeling
One of the most important features of ASM remodeling attributed to TGF-β is ECM
production. TGF-β can induce expression of various ECM components like fibronectin,
perlecan, collagen (I, II, III, IV, V), versican, elastin, CTGF and laminin (α1, β1, β2 and γ2)
by ASM cells. It can also promote expression of MMPs and TIMPs (20, 21) by ASM cells
thereby influencing ECM turnover. Altered composition of ECM modulates the mechanical
properties of ASM bundle influencing the stiffness as well as transfer of force between the
ASM bundle and surrounding tissues [46].
Additionally, TGF-β promotes ASM hyperplasia and survival by direct or indirect
mechanisms. TGF-β induces activation of MAPKs ERK, p38 and JNK in ASM cells leading
to cell proliferation [98] whereas it promotes cell survival via a p38/PI3K signaling axis
[110]. ECM proteins induced by TGF-β such as fibronectin, collagen and CTGF also have
promitogenic and prosurvival effects on ASM cells [46]. The molecular mechanisms behind
these effects are still not clearly understood but widely believed to be mediated via integrins.
Indeed, a study has shown that administration of an integrin-binding peptide RGDS, which
inhibits fibronectin, collagen and laminins binding to integrins, prevented allergen-induced
ASM hyperplasia [112,113].
Another feature of ASM remodeling in increased expression of contractile apparatus in
asthmatic ASM cells. TGF-β is a potent inducer of contractile proteins such as α-smooth
muscle actin, sm-MHC, calponin and SM22 in ASM cells [114]. These smooth muscle cell
(SMC) -specific genes essentially contain CArG box DNA elements [CC(A/T)6GG] in their
promoters which serve as binding sites for serum response factor (SRF)- a ubiquitously
expressed transcription factor [115]. SRF can activate both the proliferative and contractile
gene programs via CArG boxes. Interestingly, SRF selectively associates with SMC-specific
gene promoters in SMCs whereas it activates growth-related genes in both the SMCs and
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non-SMCs [116]. The specificity to SRF function is conferred by sets of specific
transcriptional coregulators in a tissue- and stimulus-specific manner. The myocardin family
of transcription factors, which includes myocardin and myocardin-related transcription
factors –A and –B (MRTF-A and MRTF-B) is a principle binding partner of SRF for
regulation of SMC-specific gene expression program. In smooth muscle cells, myocardin
partners with SRF and binds to the CArG box elements regulating SMC-specific genes. On
the other hand, presence of serum or a growth factor such as PDGF promotes association of
SRF with ternary complex factor family members such as Elk-1, SAP-1 and SAP-2, thereby
displacing myocardin from SRF and attenuating SMC-specific gene expression [117]. While
expression of myocardin is restricted to cardiac- and smooth muscle-specific lineages,
MRTFs are more ubiquitously expressed. MRTFs possess an N-terminal RPEL domain
which can interact with monomeric globular actin (G-actin) resulting in their retention in
the cytoplasm. At the same time, the association with G-actin also makes MRTFs sensitive
to the cellular actin dynamics. Myocardin, on the other hand, has poor interaction with G-
actin, and thus, remains predominantly nuclear and insensitive to actin dynamics [118,119].
Induction of actin polymerization by various stimuli such as TGF-β and mechanical stress
depletes the monomeric G-actin pool by its progressive incorporation into the filamentous
actin (F-actin) stress fibers in a process known as actin treadmilling. This releases MRTFs
from G-actin which subsequently translocate to the nucleus and partner with SRF
concluding a transcriptional complex that binds to the CArG box elements and initiate
smooth muscle-specific gene transcription [118,119]. Interestingly, mere nuclear
translocation of MRTFs from cytosol is not sufficient for transcriptional activation as they
can be efficiently sequestered by the nuclear G-actin pool. Thus, a substantial depletion of
both the cytosolic and nuclear G-actin pool is required for complete MRTF activity [118,119].
RhoA plays a critical regulatory role in this actin-MRTF axis, mainly as an upstream activator
and regulator of actin treadmilling.
TGF-β has been shown to induce differentiation of fibroblasts into myofibroblasts in the
MRTF-dependent manner [120,121]. In pulmonary fibroblasts, TGF-β induces
myofibroblastic differentiation by activation and nuclear translocation of SRF. Similarly,
TGF-β induces RhoA-dependent SRF-MRTF activation and binding to the CArG element
and subsequent contractile gene expression in renal epithelial cells during EMT [122,123].
In addition, SMAD3 and the canonical WNT signaling effector β-catenin, both downstream
effectors of TGF-β in ASM cells, can also modulate MRTF transcriptional activity via direct
and indirect mechanisms [123-125].
While a direct evidence of TGF-β-induced RhoA-SRF-MRTF axis activation in ASM cells is
still awaited, considering the evidence discussed above, it is plausible that TGF-β regulates
contractile gene expression program in ASM cells via MRTFs thereby promoting airway
remodeling.
1.3.2.4 Vasculature
TGF-β is a potent inducer of proangiogenic vascular endothelial growth factor (VEGF) via
GSK-3β and SMAD signaling [126]. Exaggerated VEGF abundance is present in the lung
tissue, BAL fluid and induced sputum of asthma patients [127-129]. TGF-β also possesses
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23 | P a g e
direct angiogenic properties. While VEGF opposes endothelial cell apoptosis, it mediates
TGF-β-induced apoptosis. Interestingly, the angiogenic properties of TGF-β are dependent
on VEGF-mediated apoptosis as inhibition of VEGF blocks TGF-β induced apoptosis and
angiogenesis [130]. In addition, TGF-β also augments production of plasminogen activator
inhibitor-1 (PAI-1), a vascular remodeling factor, via SMAD and WNT/β-catenin pathway
[131-133]. These studies shed light on a possible link between TGF-β and bronchial vascular
remodeling in asthma, direct evidence, however, is still unclear.
1.4 WNT signaling
WNT signaling is a key pathway involved in various aspects of embryonic morphogenesis,
maintenance of adult tissue homeostasis, repair and regeneration and stem cell renewal
[134-138]. WNT is a broad multicomponent signaling pathway and is highly conserved
among species, with varied number of WNT members. Investigating the WNT signaling
pathway in the context of airway remodeling in asthma could be of importance as 1] loss of
various components of WNT signaling leads to abnormalities in lung development including
complete agenesis, underscoring its importance in lung biology; and 2] aberrant activation
of WNT signaling has been associated with a myriad of human pathologies including cancer,
inflammatory disorders and fibrosis [139]. Of note, fibrosis and inflammation are key
components of asthma pathophysiology.
The term WNT is derived from a combination of two homologues genes integrase 1 (int1)
and wingless (wg) [137]. Int1 gene was first identified for its activation by integration of
mouse mammary tumor virus DNA and involvement in the development of virally-induced
breast tumors in mice. Wg, which was identified for its role in the development of wing tissue
in Drosophila and regulation of larval segment polarity, was later found to be a homologue
of Int1 [137]. The WNT signaling family has grown multifold since then both in the number
of its members and complexity.
In humans, the WNT family is comprised of 19 WNT ligands, 10 Frizzled (FZD) receptors,
low-density lipoprotein receptor-related protein (LRP) 5/6 coreceptors, several non-frizzled
receptors such as RYK, ROR2, PTK7 along with intracellular mediators, several extracellular
and intracellular antagonists and a range of modulators [139]. These WNT ligands can
function through signaling mechanisms broadly categorized on the basis of the requirement
of an intracellular mediator-β-catenin. The β-catenin-dependent WNT signaling pathway is
termed as canonical WNT signaling whereas all the WNT ligands activated signaling
cascades functioning independent of β-catenin are collectively described as the non-
canonical WNT signaling.
1.4.1 WNT ligands
WNT ligands are secreted, cysteine-rich, lipid modified and heavily glycosylated proteins
which act as autocrine and paracrine signaling cues and elicit myriad of cellular responses.
Structurally, WNT ligands are composed of ~350 amino acids and contain a signal sequence
for secretion along with a highly conserved distribution of 22 cysteine residues [140]. Before
secretion, WNT ligands undergo posttranslational modifications in the endoplasmic
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reticulum by oligosaccharyltransferase complex (OST) and Porcupine, which adds
oligosaccharide moieties via N-glycosylation to the WNT peptide. WNT ligands are, then,
secreted by a poorly understood mechanism involving Wntless/Evenness-interrupted
(Wls/Evi), into the extracellular space where they remain localized in the vicinity of the cell
surface, adhered to various ECM proteins such as perlecan, syndecan, glypican and biglycan
[139,140]. The significance of extensive modifications on WNT ligands is not completely
understood but may have a role in their secretion and function. For instance, N-linked
glycosylation at Asparagine 87 and 29, Serine 209- and Cysteine 77-linked palmitoylation of
murine WNT-3A are required for its secretion and palmitoylation at Cysteine 77 is also
crucial for its signaling activity [141,142]. Importantly, these modifications could also be a
major contributor to the hydrophobicity and poor solubility of WNT ligands which, in turn,
govern the limited diffusion of WNT ligands observed in the aqueous extracellular space.
Poor diffusion allows the formation of a concentration gradient of WNT ligands with highest
density near the secretory cell surface and may define their autocrine and paracrine nature
of signaling activity. However, WNT ligands have also been suggested to signal long-range
assisted by carrier proteins (flotillin-2, lipoprotein particles) or conformational changes
(multimerization of WNT ligands) that allows shielding of the hydrophobic motifs and
increases their solubility and diffusion [139,140].
1.4.2 WNT receptors
WNT ligands signal through membrane-bound receptors, most common are the seven-pass
transmembrane FZD receptors and the single-pass LRP coreceptors. FZD receptors contain
an extracellular N-terminal cysteine-rich domain (CRD), a seven-pass transmembrane
domain and a short cytosolic C-terminal tail. WNT ligands bind to FZD receptors via their
CRD whereas cytosolic domain which is composed of PDZ-binding domain is required for
the intracellular signaling where it facilitates interaction of various signaling mediators such
as DVL and heterotrimeric G-proteins in a poorly understood process [143]. Due to the
presence of the seven transmembrane domain, FZD receptors are listed as a novel family of
G-protein coupled receptor (GPCR) by the International Union of Pharmacology as class
FZD [144]. While the association of G-proteins with FZDs have been addressed in many
studies using biochemical approached, a direct evidence for the contribution of WNT-
induced association of FZD-G-proteins in the WNT signaling and its physiological relevance
remains unclear [145].
LRP5 and LRP6 are highly homologous single-pass transmembrane proteins basically
involved in receptor-mediated endocytosis of lipoproteins and protein ligands [146]. LRPs
are composed of ~1600 amino acid with an N-terminal signal peptide, four tandem β-
propeller (bp) domains each connected by an epidermal growth factor (EGF)-like domain
followed by three low-density lipoprotein-type A repeats, a transmembrane domain and a C-
terminal intracellular signaling domain [147]. The β-propeller domains, bp1 and bp3,
provide the binding sites for WNT ligands and other LRP interaction partners which are also
suggested to have some degree of specificity. For instance, WNT-1, -2 and -6 bind to bp1
domain, WNT-3 and -3A bind to bp3 whereas Dickkopf-1 (DKK-1) binds to both. In WNT
signaling, they function as coreceptors and form a membrane complex with FZD receptors
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25 | P a g e
and WNT ligands [147]. Of note, LRP5 and 6 are required for β-catenin-dependent canonical
WNT signaling [148].
1.4.3 Alternative ligands and receptors
In addition, Noggin and R-spondins also participate in WNT signaling as secreted ligands
and can signal through FZD receptors alone or in combination with leucine-rich repeat-
containing G-protein coupled receptor 4/5 (LGR4/5) receptors leading to WNT pathway
activation [149].
Ryk is a mammalian ortholog of Drosophila derailed and functions as an alternative receptor
in WNT signaling. It is a receptor tyrosine kinase type I receptor and has been shown to play
key roles in axonal guidance and pattern formation [150]. It contains an extracellular WNT
inhibitory factor (WIF) domain but lacks the CRD domain, a characteristic of WNT
receptors. Ryk can bind to WNT-1, -3A and -5A and can also function as coreceptor by its
association with FZD receptors such as FZD8 [150-152]. Ryk can participate in both the
canonical and noncanonical WNT signaling pathways depending on the receptor- and cell-
context. Similarly, other receptor tyrosine kinase type I receptors- ROR1 [153], ROR2 [154]
and PTK7 [155-157] also function as alternative WNT receptors.
1.4.4 WNT modulators and mediators
Several extracellular and intracellular modulators of WNT signaling have been described
that play key roles in fine tuning and regulation of WNT signaling.
1.4.4.1 Secreted Frizzled related proteins
Secreted Frizzled related proteins (sFRPs) are secreted glycoproteins and contain an N-
terminal CRD domain resembling the WNT-binding CRD of FZD receptors and a C-terminal
containing a hydrophilic heparin-binding region. sFRPs can bind to WNT ligands preventing
their interaction with the FZD receptors and/or bind directly to FZD receptors preventing
the assembly of an active complex thereby antagonizing the downstream WNT signaling
[139]. Contrary to their antagonistic function, sFRP1 and 2 have recently been shown to
positively regulate canonical WNT/β-catenin signaling and assist in WNT ligands gradient
formation [158]. sFRPs, thus, modulate WNT signaling positively and negatively in a
context-dependent manner.
1.4.4.2 WNT Inhibitory Factor 1
WNT inhibitory factor 1 (WIF1) is an evolutionary conserved protein that can bind to WNT
ligands and antagonize WNT signaling. Considered as a member of sFRP class of proteins, it
lacks CRD domain but contains a highly conserved N-terminal WIF domain (WD) as found
in Ryk receptors, five EGF-like repeats as found in LRPs and a hydrophilic C-terminal
domain. WIF1 has been shown to bind to WNT-3A, -4, -5A, -7A, -9B and -11 via its WIF
domain with varying affinities (WNT-5A>WNT-9B>WNT-11>WNT-4>WNT-7A>WNT-3A)
[159]. The crystal structure of the WIF domain of WIF1 shows a binding pocket for the acyl
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groups present on WNT ligands providing an interaction surface. In addition, the EGF-like
domains, particularly, EGFII-V are suggested to constitute a heparan sulfate proteoglycan
(HSPG)-binding site [160]. Thus, WIF1 binds to WNT ligands via its WIF domain and tethers
this WIF1-WNT complex in the extracellular matrix by its interaction with HSPG-glypican
via EGF-like domains [161]. Glypicans can have modulatory effects on WNT signaling [162].
Interestingly, formation of this WNT ligand-WIF1-glypican complex is required for complete
WNT antagonizing activity of WIF1 [161]. Thus, WIF1 can antagonize WNT signaling by
interfering with the formation of a functional WNT/FZD complex. A role of additional
mechanisms of WIF1, however, cannot be ruled out. Interstingly, owing to the crucial role of
WIF1 in WNT signaling regulation, suppression of WIF1 is often associated with
malignancies [163].
1.4.4.3 Dickkopf
Dickkopf (DKK) proteins are another class of extracellular WNT modulators associated with
regulation of β-catenin-dependent canonical WNT signaling. Four members of the DKK
family are currently known – DKK-1-4, with DKK-1, -3 and -4 having antagonistic function
whereas DKK-2 can be inhibitory or stimulatory depending on cellular context [139]. DKK
proteins do not interact with WNT ligands but inhibit WNT signaling by their interaction
with LRP5/6 coreceptors interfering with the formation of a WNT-FZD-LRP5/6 complex
[139]. Kremen1 and Kremen2 are type-I transmembrane proteins and are recently identified
as high-affinity receptors for DKK-1. DKK-1 interacts with LRP6 and Kremen2 forming a
ternary complex that leads to the endocytosis and membrane depletion of LRP6, thereby,
inhibiting canonical WNT signaling [164].
1.4.4.4 β-catenin
β-Catenin is remarkable in its ability to perform dual cellular functions. As a membrane-
bound protein, it constitutes a key component of adherens junctions where it interacts with
the cadherins and connects them to the cytoskeleton [165]. In addition to its role in adherens
junctions, β-catenin is the central mediator of canonical WNT signaling where it functions
as the transcriptional co-activator for WNT-responsive genes [165].
The ability of β-catenin to perform dual functions is conferred by its structural composition
[165]. β-Catenin consists of an N-terminal domain, a central region of twelve Armadillo
repeats followed by a C-terminal domain and a helix located between the last Armadillo
repeat and a C-terminal domain. β-Catenin binds to the adherens junction protein- cadherin
via its central domain, a region also shared by some critical interaction partners involved in
its transcriptional co-activator function such as adenomatous polyposis coli (APC) and T-cell
factor (TCF)/ lymphoid enhancer factor (LEF) [139,165]. Thus, the mutually exclusive
binding of β-catenin interaction partners involved in the adhesion and transcription
functions confers the versatility in β-catenin functions.
The membrane-bound pool represents the predominant cellular fraction of β-catenin where
it is present as part of the adherens junctions. Adherens junctions are cell-cell adhesion
complexes that contribute to the polarity and integrity of epithelium [166]. Cadherins, the
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27 | P a g e
core component of adherens junctions, are single-pass transmembrane glycoproteins which
interact with cadherins of the adjacent cells by a Ca2+-dependent homophilic association
[166]. The intracellular cytoplasmic tail of cadherins assembles as complex comprised of the
catenins- p120-catenin and β-catenin. β-Catenin links the cadherins to the α-catenin, which
in turn, links with the actin cytoskeleton. In addition, β-catenin also protects cadherins from
proteasomal degradation, probably by masking a PEST sequence, as disassembly of the
adherens junction complex leads to the degradation of E-cadherin [165,167-170].
β-Catenin can be released from the cadherin complex by various mechanisms and can
contribute to its transcriptional pool. Cleavage of cadherins by metalloprotease ADAM10 has
been shown to disrupt cell junctions and release β-catenin which translocates to the nucleus
and targets gene transcription [171,172]. Thus, loss of cadherins under various physiological
or pathological processes [173-176] may increase the transcriptional pool of β-catenin.
Precise regulation of the membrane-bound pool of β-catenin is still unclear and is a matter
of intense investigation. However, a range of phosphomodifications of β-catenin have been
described that modulate its affinity for the cadherin complex and regulate its structural and
signaling functions. For instance, phosphorylation at Serine 684, 686 and 692 by casein
kinase (CK) 2 and GSK-3β tremendously increases β-catenin-cadherin interaction whereas
phosphorylation at Tyrosine 142 by Fyn, Fer or c-Met attenuates α-catenin-β-catenin
interaction thereby impairing its structural function [165]. Phosphorylation of β-catenin by
JNK at various points such as Serine 31, 191, 605 and Threonine 41 also negatively regulates
cadherin-β-catenin interaction and promotes its nuclear translocation [165,177]. Similarly,
phosphorylation of β-catenin at Serine 552 by EGF receptor-activated AKT kinase leads to
its dissociation from the cadherin complex and translocation to the nucleus and
augmentation of its transcriptional activity [178]. Tyrosine 654 phosphorylation of β-catenin
by EGF receptor or c-Src also impairs β-catenin binding to cadherins. An additional
phosphorylation at Serine 675 by protein kinase A (PKA) is suggested to be required for
complete augmentation of transcriptional function of Tyrosine 654-phosphorylated β-
catenin, presumably by recruiting various co-activators such as CREB-binding protein
(CREBBP or CBP) and TATA-binding protein (TBP) [165]. Thus, various mechanisms exist
that promote the transcriptional role of β-catenin at the expense of its structural function,
however, it might not be a universal scenario and β-catenin released from adherens junctions
could also participate in functions other than transcription.
While the β-catenin released from the membrane contributes to the free cytosolic pool of β-
catenin, it is believed to be predominantly maintained by the newly synthesized nascent β-
catenin. The cytosolic abundance of this pool is tightly controlled by a multiprotein
destruction complex which is an integral part of canonical WNT signaling. The multiprotein
destruction complex is comprised of scaffold proteins Axin and APC and kinases GSK-3β
and CK1α. Under the steady state conditions, free β-catenin is captured by Axin and APC and
phosphorylated by CK1α at Serine 45, priming it for subsequent phosphorylation by GSK-3β
at Threonine 41, Serine 37 and Serine 33. The phosphorylated β-catenin is ubiquitinated by
E3 ligase Jade1 or more predominantly, by E3 ubiquitin ligase complex Skp-Cullin-F-box
protein/β-transducin repeat-containing protein (SCFβ-TRCP) and degraded by 26S
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28 | P a g e
proteasome [139,165]. Thus, free β-catenin is constantly degraded thereby maintaining its
low cytosolic levels.
The presence of canonical WNT ligands leads to the inhibition of destruction complex by
poorly understood mechanisms. This allows β-catenin to evade the phosphorylation and
subsequent degradation thereby increasing the cytosolic levels of β-catenin. The free
cytosolic β-catenin, then, translocates to the nucleus. Inside the nucleus, β-catenin binds to
the TCF/LEF and activates target gene transcription.
In addition to WNTs, several other growth factors can also stabilize β-catenin and activate
β-catenin-dependent processes. GSK-3 mediated phosphorylation is a key event in β-catenin
degradation. GSK-3, encoded by two isforms-α and β, is a constitutively active kinase and is
predominantly regulated by an inactivating phosphorylation at Serine 21 at GSK-3α and
Serine 9 for GSK-3β [179]. Canonical WNT signaling, however, may not utilize
phosphoinactivation of GSK-3 for β-catenin stabilization [180,181] but engages alternative
strategies such as changing the compartmentalization of GSK-3 [182] or its dissociation from
the destruction complex to separate GSK-3 activity on β-catenin [183,184], thus, rescuing β-
catenin. Several growth factors, on the other hand, can inactivate GSK-3 by inhibitory
phosphorylation and, in turn, stabilize β-catenin. For instance, PDGF and fetal bovine serum
(FBS) stabilize β-catenin and promote its nuclear localization via GSK-3β inactivation in
airway smooth muscle cells [185]. Similarly, protein kinase C (PKC) mediates inactivation of
GSK-3β in bronchial epithelial cells and activates β-catenin signaling in an in vitro model of
tissue injury [186]. A study has proposed an interesting mechanism for non-WNT growth
factor-mediated rescue of β-catenin from the destruction complex. PDGF stimulation of
colon cancer cell lines led to activation of c-Abl which phosphorylated a RNA helicase p68 at
Tyrosine 593. The phosphorylated p68 blocked GSK-3β mediated phosphorylation of β-
catenin by displacing it from the Axin complex and activated the TCF/LEF-dependent gene
transcription [187]. Although GSK-3 can target a plethora of cellular proteins, it is suggested
to modify β-catenin only in the destruction complex in association with Axin and APC. It is,
thus, tempting to speculate that non-WNT growth factors may inactivate destruction
complex-associated GSK-3 along with other cellular pools of GSK-3 leading to β-catenin
rescue.
1.4.5 Canonical WNT signaling
Stabilization of cytosolic β-catenin is the key process in canonical WNT signaling, although
the sequence of events is unclear. In a widely-accepted model of canonical WNT signaling
(Figure 3), binding of WNT ligands to the FZD receptor and LRP5/6 co-receptor leads to the
formation of a heteromeric ternary membrane complex. This leads to the polymerization of
dishevelled (DVL) proteins and their recruitment to the ternary complex. DVL, in turn,
recruits Axin and GSK-3β to the receptor complex. GSK-3β and CK1γ both phosphorylate
the cytosolic tail of LRP coreceptors in their Pro-Pro-Ser-Pro repeats resulting in a multifold
increase in the affinity of Axin for LRP. Subsequently, Axin is recruited to the phosphorylated
LRP cytosolic tail leading to the depletion of free cellular pool of Axin. Axin, being present
in limited amounts, is a rate limiting factor for β-catenin phosphorylation by destruction
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29 | P a g e
Figure 3. Canonical WNT signaling pathway. In the absence of WNT ligands, a destruction
complex comprising axin, GSK-3β, CK1 and APC captures cytosolic β-catenin and phosphorylates it
sequentially via CK1 and GSK-3β activity. The phosphorylated β-catenin is degraded by the ubiquitin-
proteasome system. Activation of the FZD and LRP5/6 receptor complex by extracellular WNT
ligands leads to sequestration of the β-catenin destruction complex to the membrane receptor
complex, primarily mediated by the scaffold protein DVL. This prevents phosphorylation of β-catenin
by GSK-3β and CK1 and subsequent proteasomal degradation culminating into the accumulation of
cytosolic β-catenin. Unphosphorylated and stabilized β-catenin translocates to the nucleus and
activates β-catenin-dependent gene transcription. APC, Adenomatous polyposis coli; CK1, Casein
kinase 1; DVL, dishevelled; GSK-3β, Glycogen synthase kinase-3β; FZD, Frizzled; LEF, Lymphoid
enhancer factor; LRP5/6, Lipoprotein receptor-related protein 5/6; TCF, T-cell factor. (Schematic is
taken from Chapter 2)
complex and can modulate rapid assembly and disassembly of the complex. This
sequestration of Axin at the membrane by LRPs leads to the disassembly of the destruction
complex leading to an increase in the non-phosphorylated cytosolic pool of β-catenin [188].
While membrane sequestration of Axin and GSK-3 is a prevelant model, several alternative
mechanisms of WNT-dependent β-catenin activation have also been reported. A study has
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suggested that WNT ligands stimulation leads to LRP5/6 and Axin mediated sequestration
of GSK-3 into multivesicular bodies. This change in GSK-3 compartmentalization effectively
segregates β-catenin from GSK-3 leading to its accumulation [182]. In another study,
authors have shown that DVL can bind directly to the Axin, leading to disruption of its
interaction with GSK-3 which may lead to the disassembly of destruction complex [183,189].
Alternatively, GSK-3 interacting proteins such as GSK-3-binding protein (GBP)/frequently
rearranged in advanced T-cell lymphomas 1 (FRAT-1), may compete with GSK-3 for its Axin-
binding domain leading to the dissociation of GSK-3 from Axin and destruction complex.
This process is facilitated by DVL which can also bind to GBP and FRAT-1 [139,184,190,191].
Another recent model proposed WNT ligand-induced monoubiquitination of GSK-3β
leading to its association with β-TRCP which renders both the GSK-3β and β-TRCP
unavailable to interact with and ubiquitinate β-catenin [192].
Furthermore, a recent study has shown that WNT ligand stimulation doesn’t lead to
disassembly of the destruction complex or recruitment of individual Axin and GSK-3 to the
membrane, nor does it lead to inhibition or any alteration in the activity of destruction
complex constituents [193]. The authors have shown that β-catenin is not only
phosphorylated but also ubiquitinated by β-TRCP inside the destruction complex. WNT
ligand stimulation leads to dissociation of β-TRCP from the destruction complex and
recruitment of the entire destruction complex to the WNT-FZD-LRP ternary complex at the
membrane. In the absence of β-TRCP, the destruction complex-associated β-catenin cannot
be ubiquitinated and hence can’t be removed leading to saturation of the destruction
complex by phospho-β-catenin [193]. As such, no nascent β-catenin interacts with the
destruction complex leading to accumulation of β-catenin in the cytosol and its nuclear
translocation [193].
Interestingly, Yes-associated protein/transcriptional coactivator with PDZ-binding motif
(YAP/TAZ) have been shown to mediate association of β-TRCP with the destruction complex
[194]. YAP and TAZ are transcriptional cofactors whose nuclear shuttling is primarily
believed to be controlled by cell density sensing Hippo signaling [195] and mechanical stress
sensing pathways [196]. In a novel mechanism, YAP and TAZ have been shown to be an
integral part of the canonical WNT signaling. YAP and TAZ associate with Axin and reside
in the destruction complex in the absence of WNT ligands [194] where they recruit β-TRCP
to the destruction complex which degrades phosphorylated β-catenin. In the presence of
WNT ligands, the destruction complex is recruited to the phosphorylated intracellular
domain of LRP5/6 via Axin. As both LRP5/6 and YAP/TAZ compete for the same domain of
Axin, YAP/TAZ dissociate from the destruction complex which also dislodges β-TRCP from
the destruction complex [194]. Thus, impaired destruction complex-β-TRCP association
stabilizes cytosolic β-catenin.
Once stabilized, β-catenin can readily translocate to the nucleus by an unidentified
mechanism as it lacks nuclear localization sequence (NLS) and also seem to be independent
of importin-β and Ran-GTPase, the common nuclear transporters [165]. Inside the nucleus,
β-catenin partners with various primary and auxiliary proteins including transcription
factors, histone modification proteins and chromatin remodeling complexes, to relay the
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effects of its inducers [165,197]. The most common binding partners for β-catenin in
canonical WNT signaling are TCF/LEF transcription factors. In the absence of WNT
signaling, TCF/LEF occupy the WNT responsive elements (WREs) in association with
transducin-like enhancer of split (TLE)/Groucho where it suppresses the gene transcription.
Binding of β-catenin displaces TLE/Groucho from TCF/LEF allowing the transcriptional
activation of WNT responsive genes [165]. While TCF/LEF occupy central Armadillo repeats
of β-catenin, it recruits several cofactors via its N-terminal and C-terminal domains such as
BCL9, Parafibromin, MED12, Histone acetyltransferases (eg. CBP/p300), chromatin
remodeling complexes (eg. BRG1) and histone methyltransferases (eg. MLL complexes,
COMPASS) that modulate the gene transcription [197].
1.4.6 Noncanonical WNT signaling
In addition to the β-catenin dependent canonical WNT signaling, WNT ligands can activate
multiple signaling cascades broadly classified as noncanonical WNT signaling [198,199]
(Figure 4). Noncanonical WNT signaling is essentially independent of both β-catenin and
LRP5/6 coreceptors but utilizes FZD and alternative receptors. Additionally, a different set
of intracellular mediators such as Ca2+-dependent factors, MAPKs and small GTPases are
employed to relay cellular effects of noncanonical WNT ligands. The major cellular effects of
noncanonical WNT signaling are transcriptional activation of target genes, reorganization of
cytoskeleton and cell movement.
1.4.6.1 WNT/planar cell polarity (WNT/PCP)
Cell polarity is an important feature of living organisms whether unicellular or multicellular
[201]. It arises by an asymmetrical distribution and organization of cell contents such as cell
membrane, intracellular organelles and cytoskeleton. Cell polarity is involved in almost all
aspects of eukaryotic life from cell movement and migration to asymmetric cell divisions and
organization of a well-structured metazoan body. For instance, polarization of cells is
absolutely required in gastrulation to generate germ layers, in the development of tissues
such as neurons and epithelium or polarized structures such as limbs and in determining the
proximal-distal (P-D) and anterio-posterio (A-P) axes of the body. In adult life, cell polarity
is also critical for the directional migration of motile cells such as fibroblasts and immune
cells, required for repair and regeneration and is intrinsic to the maintenance of tissue
architecture and integrity. In malignant diseases, cells modulate their polarity to migrate
during metastasis. One of the major underlying mechanisms of cell polarity is directional
organization of the cytoskeleton which can be affected by a multitude of factors [201].
Generation and maintenance of cell polarity is a tightly regulated process and is associated
with the noncanonical WNT signaling.
Most of the WNT/PCP pathway is characterized in Drosophila with the identification of
various homologues proteins in vertebrates. The core WNT/PCP pathway is comprised of
FZD, Van Gogh (Vangl1 and 2 in vertebrates), flamingo, prickle and DVL [198]. Binding of
noncanonical WNT ligands to FZD receptors may activate the heterotrimeric G-proteins
which, in turn, leads to the recruitment and phosphoactivation of DVL [198,199].
Alternatively, noncanonical WNT ligands such as WNT-5A can bind to ROR2 and Vangl2
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leading to the formation of a ternary signaling complex- WNT-5A-ROR2-Vangl2 recruiting
DVL via Vangl2 [202]. Activated DVL, in turn, can engage multiple pathways leading to actin
cytoskeleton reorganization and/or transcriptional responses. DVL associates with the DVL-
associated activator of morphogenesis 1 (Daam1) and RhoA and subsequently activates
RhoA [203]. Activated RhoA, in turn, leads to the activation of its downstream kinase- Rho-
associated coiled-coil containing protein kinase 1 (ROCK1) which regulates actin
cytoskeleton remodeling. Additionally, activated Daam1 interacts with profilin, an actin
binding protein, and initiates actin polymerization and cytoskeletal remodeling [204].
In another pathway, interaction of DVL with Rac1 leads to the activation of JNK kinase [205].
It has also been suggested that DVL can directly interact with JNK via its DEP domain and
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33 | P a g e
activate it independently of Rac1 [206]. The requirement of small GTPases in DVL mediated
JNK activation seems to be cell- and stimulus-dependent. JNK, in turn, may regulate actin
remodeling [207] or induces activation of multiple downstream signaling cascades including
activation of a transcription factor- c-Jun [208]. In addition, RhoA can also activate JNK
signaling in the WNT/PCP pathway as demonstrated in Xenopus convergent extension
movements, the key morphogenetic movements wherein tissue narrows down along one
direction and elongates in the perpendicular direction thereby shaping the body axis [209].
The noncanonical WNT ligand WNT-11 stabilizes XRNF185, a Xenopus homologue of
human RING finger protein RNF185 and induces its interaction with DVL [210]. This, in
turn, facilitates XRNF185 interaction with paxillin [210]. Paxillin is a focal adhesion complex
protein and participates in actin cytoskeleton remodeling and cell motility. XRNF185-
paxillin interaction results in increased paxillin ubiquitination and subsequent degradation
leading to enhanced paxillin turnover and actin cytoskeleton remodeling [210].
1.4.6.2 WNT/Ca2+ pathway
Ca2+-dependent signaling is another key noncanonical WNT pathway. Binding of WNT
ligands leads to the recruitment of DVL to the FZD receptor, presumably via G-proteins,
which in turn, may activate phospholipase C (PLC) leading to generation of diacylglycerol
(DAG) and inositol trisphosphate (IP3) by hydrolysis of phosphatidylinositol 4,5-
bisphosphate (PIP2). IP3 leads to the release of Ca2+ from intracellular stores and rise in Ca2+
concentrations whereas DAG activates PKC [211].
Figure 4. Noncanonical WNT signaling pathways. (A) WNT/planar cell polarity (PCP)
pathway. Binding of WNT ligand to Fz receptor leads to the activation of DVL and its subsequent
recruitment to the receptor. Additionally, AP2 and βarr2 may be recruited leading to the receptor
internalization. DVL activates multiple pathways leading to the cytoskeletal remodeling and
transcriptional regulation as shown in the figure. FZD-induced membrane recruitment of DVL is
regulated by various kinases such as PAR1, PKCδ and CK1ε. In addition, Glypican, Syndican, PAPC,
RTK7, DVL-binding proteins such as Div and Inv also regulate WNT/PCP signaling. (B) WNT/Ca2+
pathway. Binding of WNT ligand to Fz receptor leads the activation of DVL which promotes
generation of DAG and IP3. IP3 induces intracellular Ca2+. DVL also activates PDE6 leading to decline
in intracellular cGMP levels and subsequent inhibition of PKG activating Ca2+ release. Ca2+ activates
CaMKII-TAK1-NLK cascade inhibiting β-catenin-TCF signaling. DAG and Ca2+ also activate PKC
which activates Cdc42 and regulates actin cytoskeletal remodeling. Additionally, Ca2+ activates the
phosphatase CAN leading to activation and nuclear translocation of NFAT. DVL, dishevelled; Fz,
Frizzled; βarr2, β-arrestin 2; AP2, adaptor protein complex 2; Daam1, DVL-associated activator of
morphogenesis; ROCK, Rho-associated coiled-coil containing protein kinase; MRLC, myosin
regulatory light chain; PXN, paxillin; XRNF185, Xenopus ring finger protein 185; CapZIP, CapZ
interacting protein; Dub, duboraya; PAPC, paraxial protocadherin; RTK7, receptor tyrosine kinase
7; TK, tyrosine kinase; PAR1, partitioning-defective 1; PKCδ, protein kinase Cδ; CK1ε, casein kinase
1ε; Inv, inversin; Div, diversin; PLC, phospholipase C; PIP2, Phosphatidylinositol 4,5-bisphosphate;
IP3, inositol trisphosphate; DAG, diacylglycerol; PKC, protein kinase C; Cdc42, cell division cycle 42
protein; PDE6, phosphodiesterase 6; PKG, protein kinase G; CaMKII, Ca2+/calmodulin-dependent
protein kinase 2; TAK1, TGF-β-activated kinase 1; NLK, nemo-like kinase; β-cat, β-catenin; TCF, T-
cell factor; CAN, calcineurin; NF-AT, nuclear factor of activated T cells; G, G protein αβγ subunits
(Schematics are adapted from Semenov et al (2007) [200])
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34 | P a g e
In addition, WNT ligands activate cGMP phosphodiesterase 6 (PDE6) which leads to the
depletion of cGMP and inhibition of protein kinase G (PKG) [212-214]. PKG is a negative
regulator of Ca2+ mobilization. Thus, inhibition of PKG leads to a rise in intracellular Ca2+
concentrations. Alternatively, heterotrimeric G-proteins can activate PDE6 in a DVL-
independent manner via p38 MAPK stimulation [215]. While these studies mention a role
for G-proteins in WNT/Ca2+ signaling, direct evidence supporting the physiological
relevance of WNT-induced FZD-G-protein interaction is still awaited [145].
High intracellular Ca2+ activates protein kinase C (PKC), calmodulin-dependent kinase II
(CaMKII) and calcineurin-NFAT signaling. Activation of PKC by Ca2+ and/or DAG leads to
actin cytoskeleton remodeling, probably, via small GTPases [216-218]. Further, increased
Ca2+ leads to activation of calmodulin which in turn activates a protein phosphatase
calcineurin. Activated calcineurin dephosphorylates NFAT transcription factors which
translocate to the nucleus and activate gene transcription [219].
In addition, calmodulin also activates CaMKII which can antagonize the canonical WNT
signaling pathway. CaMKII activates TAK1 which, in turn, stimulates nemo-like kinase
(NLK) activation. NLK phosphorylates TCF/LEF transcription factor which prevents their
interaction with β-catenin thereby inhibiting canonical WNT signaling pathway [220,221].
1.4.6.3 Other noncanonical WNT signaling pathways
In addition to the WNT/PCP and WNT/Ca2+ pathways, several other signaling cascades are
activated by WNT ligands in a β-catenin independent manner. For instance, WNT ligands,
in particular WNT-5A, can regulate the cell polarity complex in developing neurons. WNT-
5A leads to association of DVL with members of polarity complex- partitioning-defective
(PAR) 3, PAR6 and atypical PKC (aPKC) which inhibits the activity of PAR1 leading to
inhibition of microtubule organization thereby regulating axonal guidance [222].
Noncanonical WNT signaling activates CREB in various systems. WNT-5A binding to FZD3
leads to rise in cAMP concentrations which, in turn, activate protein kinase A (PKA) [223].
PKA directly phosphorylates and activates CREB. In addition, it also phosphorylates a
dopamine and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), an antimigratory
protein [223]. Activated DARPP-32 inhibits PP1, a protein phosphatase, and potentiates
WNT-5A-PKA mediates CREB phosphorylation. Also, DARPP-32 suppresses Cdc42
inhibiting filopodia formation [223]. During mammalian myogenesis, WNT ligands WNT-1
and -7A activate G-protein dependent increase in cAMP levels leading to PKA activation in
mouse presomitic mesoderm. PKA, in turn, phosphorylates CREB leading to activation of
myogenic transcriptional factors such as Pax3, MyoD and Myf5 [224]. In addition, WNT
ligands can activate p38, JNK, ERK and NFkB signaling in β-catenin independent manner
expanding the array of noncanonical WNT signaling pathways [225].
General introduction
35 | P a g e
1.5 WNT-5A: a noncanonical WNT ligand(adapted from chapter 7)
1.5.1 WNT-5A
WNT-5A is one of the most studied WNT ligands associated predominantly with the
noncanonical WNT signaling [226]. WNT-5A is highly conserved among species and plays
key roles in embryonic development and post-natal homeostatic processes. Homozygous
WNT-5A knock-out mice show perinatal lethality, primarily due to respiratory failure, and
present extensive developmental abnormalities. It is involved in lung [227], heart [228] and
mammary gland morphogenesis [229] and regulates stem cell renewal [230,231] and tissue
regeneration [232]. In addition, it has also been associated with a myriad of pathological
conditions such as cancer, fibrosis, inflammation and neurodegeneration.
1.5.2 WNT-5A gene
WNT-5A cDNA was first isolated from mice fetal tissues [233] followed by isolation and
sequencing from human cells [234]. The human WNT-5A gene is located on chromosome
3p14-p21. The WNT-5A gene generates two very identical transcripts by utilization of
alternative transcription start sites. The corresponding upstream sequences are termed as
promoter A and B [235] and their products as WNT-5A-L and WNT-5A-S, respectively [236].
Both the promoters have comparable transcriptional potential; their activity, however, is
highly context-dependent. WNT-5A promoter A has been suggested to be more active in
human and murine fibroblasts compared to promoter B [237]. Both the isoforms have
similar biochemical properties such as stability, hydrophobicity and signaling activity [236].
While the significance of individual WNT-5A isoforms is not completely understood,, a
recent study showed that they might have different functions [236]. When ectopically
expressed, WNT-5A-L inhibited proliferation of various cancer cells lines whereas WNT-5A-
S lead to stimulation of growth [236].
Figure 5. Human WNT-5A promoter. Schematic representation of various
transcription factor binding sites on WNT-5A promoter A as derived from in silico analysis.
1.5.3 WNT-5A transcription
WNT-5A is a transcriptional target of an array of cytokines and growth factors. CUTL1 [238],
STAT3 [239], TBX1 [240] and NFκB [241,242] have been reported as transcription factors
for WNT-5A in various cell types. TGF-β has been shown to induce WNT-5A expression in
pancreatic cancer cells [238]. Similarly, pro-inflammatory cytokines-IL-1β [242], TNF-α
[241] , LPS/IFNγ [243], IL-6 family members- leukemia inhibitory factor and cardiotropin-
CU
TL
1
Sp
3/S
p1
CU
TL
1/S
RY
Pa
x-2
/c-F
os
CU
TL
1
E2
F-1
/Sp
1
CU
TL
1
E2
F-1
Sp
1
c-E
ts-1
Sp1
Pax-
2c-M
yb
c-E
ts-1
Sp1
TC
F-4
E
Pax-
2
E2
F-1
c-M
yb
Pax-
2
TC
F-4
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-1448 -1257 -837 -571 -179
Chapter 1
36 | P a g e
1 [244], high extracellular Ca2+ concentration [245] all augment whereas amino acid
limitation [246] represses WNT-5A expression in various cell types. A schematic
representation of WNT-5A promoter A is presented in Figure 5 showing the predicted
transcription factor binding sites (Figure 5).
Numerous AU-rich motifs are present in the 3’-untranslated region of WNT-5A mRNA which
is about ~2.5-fold longer than the coding region in humans and evolutionary conserved
among species [247]. AU-rich element binding proteins (ARE-binding proteins) associate
with the AREs and tightly regulate their stability by posttranscriptional mechanisms. HuR,
A
B
WNT-5A-L MKKSIGILSPGVALGMAGSAMSSKFFLVALAIFFSFAQVVIEANSWWSLGMNNPVQMSEV
WNT-5A-S ---------------MAGSAMSSKFFLVALAIFFSFAQVVIEANSWWSLGMNNPVQMSEV
WNT-5A-L YIIGAQPLCSQLAGLSQGQKKLCHLYQDHMQYIGEGAKTGIKECQYQFRHRRWNCSTVDN
WNT-5A-S YIIGAQPLCSQLAGLSQGQKKLCHLYQDHMQYIGEGAKTGIKECQYQFRHRRWNCSTVDN
WNT-5A-L TSVFGRVMQIGSRETAFTYAVSAAGVVNAMSRACREGELSTCGCSRAARPKDLPRDWLWG
WNT-5A-S TSVFGRVMQIGSRETAFTYAVSAAGVVNAMSRACREGELSTCGCSRAARPKDLPRDWLWG
WNT-5A-L GCGDNIDYGYRFAKEFVDARERERIHAKGSYESARILMNLHNNEAGRRTVYNLADVACKC
WNT-5A-S GCGDNIDYGYRFAKEFVDARERERIHAKGSYESARILMNLHNNEAGRRTVYNLADVACKC
WNT-5A-L HGVSGSCSLKTCWLQLADFRKVGDALKEKYDSAAAMRLNSRGKLVQVNSRFNSPTTQDLV
WNT-5A-S HGVSGSCSLKTCWLQLADFRKVGDALKEKYDSAAAMRLNSRGKLVQVNSRFNSPTTQDLV
WNT-5A-L YIDPSPDYCVRNESTGSLGTQGRLCNKTSEGMDGCELMCCGRGYDQFKTVQTERCHCKFH
WNT-5A-S YIDPSPDYCVRNESTGSLGTQGRLCNKTSEGMDGCELMCCGRGYDQFKTVQTERCHCKFH
WNT-5A-L WCCYVKCKKCTEIVDQFVCK
WNT-5A-S WCCYVKCKKCTEIVDQFVCK
104 114
120
312 326
380
365
60
45
105
180
165
240
225
300
285
360
345
3801
C104
N114 N120 N312 N326
43 44
General introduction
37 | P a g e
a member of the embryonic lethal abnormal vision (ELAV) -like family of ARE-binding
proteins, binds to the 3’-UTR AREs in WNT-5A mRNA and suppresses its translation [247].
1.5.4 WNT-5A protein
WNT-5A-L and WNT-5A-S, composed of 380 and 365 amino acids respectively, are heavily
glycosylated and lipid modified proteins. Each isoform consists of an N-terminal
hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus
sequence and a highly conserved distribution of 22 cysteine residues [234] (Figure 6). The
cleavage of N-terminal signal sequence is predicted to generate mature proteins containing
either 343 or 338 amino acids [236]. However, N-terminal sequencing of mature WNT-5A
isoforms revealed that WNT-5A-L is cleaved after 43rd amino acid whereas WNT-5A-S has
much longer signal sequence with cleavage after 46th amino acid, generating 337 and 319
amino acid containing mature proteins, respectively (Figure) [236]. Interestingly, mouse
WNT-5A which is ~99% homologues to the human WNT-5A generates same mature protein
as human WNT-5A-S [234,248]. Asparagine 114, 120, 312 and 326 have been identified as
the N-linked glycosylation sites whereas a palmitoylation has been identified at Cysteine 104.
The palmitoylation of WNT-5A is necessary for its binding to FZD5 and signaling activity but
not required for its secretion [249,250]. In contrast, glycosylation of WNT-5A is required for
its secretion but dispensable for its signaling activity [249].
1.5.5 WNT-5A: receptors and signaling
WNT-5A binding to receptors activates various β-catenin-independent noncanonical WNT
signaling cascades, however, it can also activate canonical WNT signaling depending on the
cell and receptor context.
WNT-5A can signal through multiple receptors and according to current understanding
FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, RYK, ROR2 and CD146 may function as
WNT-5A receptors [245,248,251-261].
WNT-5A has been shown to bind to FZD2 inducing intracellular Ca2+ release and PKC
activation in Xenopus [262] and zebrafish embryos [263] and WNT-5A-FZD2-induced Ca2+
spikes in neurons are also implicated in traumatic brain injury [264]. WNT-5A binds to
FZD2 in a ROR1- or ROR2-dependent manner and recruits DVL and β-arrestin to FZD2
leading to the clathrin-mediated internalization of FZD2 [251]. Internalization of FZD2 is
Figure 6. Human WNT-5A protein. (A) A comparative analysis of amino acid sequences of WNT-
5A-L and WNT-5A-S isoforms. Grey highlighted area represents N-terminal signal sequence in
respective protein. Bold arrows mark the N-terminal of mature protein of respective isoform post-
signal sequence cleavage. The amino acids marked in red-bold represent posttranslational
modification sites on protein backbone. Number represents the respective position of the amino acid
from the first N-terminal amino acid. The protein sequences are taken from NCBI; NP_003383.2
(WNT-5A-L) and NP_001243034.1 (WNT-5A-S). (B) Diagrammatic representation of WNT-5A-L
protein. N-terminal signal sequence is represented by blank box. ( ) represents palmitoylation and
( ) represents N-linked glycosylation on the protein backbone. The respective amino acids locations
are marked above the modification sites.
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38 | P a g e
essential for WNT-5A-induced Rac activation [251]. WNT-5A also induces clathrin-mediated
internalization of FZD4 [265] in a PKC- and β-arrestin-dependent process and that of ROR2
in a PKC-dependent manner [258]. Binding of WNT-5A to FZD5 [249] also leads to its
internalization but the functional relevance is unknown. Internalization of receptors is
considered as a critical step in WNT signaling and a reflection of active signaling. Although
the exact mechanisms underlying the functional significance of receptor internalization are
not clear, it is believed to facilitate intracellular signaling activation by recruitment of
scaffolding proteins such as β-arrestin and may also facilitate the termination of signaling
and receptor recycling [266].
WNT-5A binding to FZD7 activates pro-survival PI3K/AKT cascade in human melanoma
cells which can account for the resistance of these cells to BRAF inhibitors [259]. Similarly,
WNT-5A can activate the PI3K/AKT cascade via FZD3 in human dermal fibroblasts and
promotes integrin-mediated adhesion of these cells [252]. In contrast, WNT-5A-activated
PI3K/AKT signaling induces migration in human osteosarcoma cells [267]. Similarly, WNT-
5A induces migration in gastric cancer cells by activating the PI3K/AKT pathway which
phosphorylates and inactivates GSK-3β and activates RhoA leading to cytoskeleton
remodeling [268]. Indeed, cytoskeletal reorganization and cell migration are major cellular
effects of WNT-5A signaling.
WNT-5A-FZD6 interaction is suggested to regulate cell fate in hair-follicles [261] whereas
WNT-5A-FZD5 signaling plays critical role in tuberculosis immunology regulating the
immune responses by antigen presenting cells and activated T cells in response to
mycobacterium infection [253].
WNT-5A binding to an adhesion molecule CD146 leads to the recruitment of DVL2 to the
complex and activation of downstream noncanonical JNK signaling cascade [256]. CD146
has been linked to cell migration via RhoA-dependent cytoskeletal rearrangements [269]. In
line with that, WNT-5A-CD146 axis regulates polarity and migration of cells [256].
ROR2 is a key receptor for WNT-5A-induced effects during development as demonstrated
by remarkable phenotypic resemblance between the ROR2 knock-out and WNT-5A knock-
out mice [270]. Multiple mechanisms have been suggested to explain the close functional
relationship between WNT-5A and ROR2. WNT-5A interacts with ROR2 and Vangl2 to form
a ternary complex leading to the CK1δ-induced phosphorylation of Vangl2 which serves to
relay the gradient effects of WNT-5A thereby regulating WNT-5A-induced planar cell
polarity and embryonic morphogenesis [271]. WNT-5A associates with FZD7 in the presence
of ROR2 to form a complex required for DVL polymerization and activation of Rac-
dependent noncanonical WNT signaling [260]. WNT-5A activates ERK1/2 in intestinal
epithelial cells via ROR2 [272], whereas it activates JNK-mediated c-Jun transcriptional
activity to induce production of receptor activator of nuclear factor-κB (RANK), a regulator
of osteoclast differentiation and activation, in osteoclast precursor cells via ROR2 [273].
WNT-5A activates intracellular Ca2+ release to fine tune neuronal growth by axonal
outgrowth and repulsion. WNT-5A signals via Ryk leading to Ca2+ release from stores
through IP3 receptors as well as Ca2+ influx through transient receptor potential (TRP)
General introduction
39 | P a g e
channels inducing axonal outgrowth. On the other hand, simultaneous association of WNT-
5A with Ryk and FZD releases Ca2+ from TRP channels without involvement of IP3 receptors
and induces axonal repulsion [274]. WNT-5A also forms a ternary complex with Ryk and
Vangl2 to relay the WNT/PCP effects [275] whereas WNT-5A-Ryk signaling is required for
inhibition of reactive oxygen species (ROS) production and maintenance of hematopoietic
stem cell quiescence [151]. WNT-5A engages ROR2 to activate JNK signaling and regulates
convergent extension movements [226] and human dental papilla cell migration [276]
whereas it induces assembly of DVL-aPKC and polarity complex (PAR3 and PAR6) to
regulate neuronal differentiation and polarity [222,277].
Noncanonical WNT ligands counteract WNT/β-catenin signaling by preventing β-catenin
and TCF/LEF interaction. Interestingly, WNT-5A can inhibit or activate WNT/β-catenin
signaling depending on the receptor- and cell-context. Indeed, a study has shown that WNT-
5A can both activate and inhibit canonical WNT signaling during mouse embryonic
development [278]. The WNT-5A-activated CaMKII-TAK1-NLK1 cascade has been
implicated in WNT/β-catenin suppression [220]. In addition, WNT-5A inhibits WNT-3A-
induced β-catenin signaling via ROR2 and CD146 [248,256]. In hematopoietic stem cells,
WNT-5A inhibits β-catenin signaling, probably via suppression of ROS production [151].
Similarly, WNT-5A inhibits β-catenin signaling by promoting degradation of β-catenin
through an alternative E3 ubiquitin ligase complex comprised of siah2-APC-Ebi [279].
Purified WNT-5A, on the other hand, can activate β-catenin-dependent transcription in the
presence of FZD4 and LRP5 [248]. Similarly, osteoblast-lineage cells from WNT-5A knock-
out mice show reduced WNT/β-catenin signaling and WNT-5A pre-treatment potentiated
the WNT/β-catenin signaling in bone marrow stromal cells via upregulation of LRP5 and
LRP6 expression [280].
1.5.6 WNT-5A: functions
1.5.6.1 Embryogenesis:
WNT-5A has been identified for its key involvement in defining the body outgrowths in
addition to many other specific features. WNT-5A expression is most abundant during early
embryonic developmental stages between 10-14 day post conception [233,281]. Importantly,
homozygous WNT-5A knock-out mouse embryos show perinatal lethality underlining its
vital role in embryogenesis. During development, regions undergoing extensive outgrowth
like limbs, tail and facial structures exhibit prominent WNT-5A expression where it is
present in a graded fashion with highest abundance at the tips of these structures and lowest
in the proximal areas [233,281]. WNT-5A knockout leads to severe malformations in the
outgrowth structures, a shortened A-P axis and severely compromised P-D axis. These
malformations could be traced back to the underlying axial skeleton which exhibited a
shortened vertebral column due to smaller vertebrae size and absence of caudal vertebrae
[281]. The phenotype apparently originates from the critical role of WNT-5A as a mitogen
required for the proliferation of the mesodermal progenitors early in embryonic
development. The mesodermal stem cells which arise early in development can continue to
develop in the primitive streak even in the absence of WNT-5A but lack the ability to divide
and give rise to the progeny [281]. Impaired self-renewal capacity leads to progressive
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40 | P a g e
depletion of the stock of these stem cells resulting in insufficient numbers of cells to develop
the distal skeleton and leading to the absence of related structures [281].
Similar to WNT-5A knock-out mice, WNT-5A transgenic mice show perinatal lethality when
WNT-5A is induced early in development exhibiting severe deformities resembling the
WNT-5A knock-out phenotype [282]. Overexpression of WNT-5A induced malformations of
limbs, tail and facial structures. Underdeveloped limb skeletal elements, reduced number of
tail vertebrae and shortened upper and lower jaw bones constituted the mutant phenotype.
Interestingly, overexpression of WNT-5A in later embryonic stages and in adult animals was
well-tolerated with no visible phenotype [282]. This study highlights a critical window
during embryonic development when WNT-5A activity is most required [282].
Further studies have looked into the organ-specific developmental roles of WNT-5A and
have identified a crucial role for distal morphogenesis of internal organs. For instance, WNT-
5A knock-out mice fail to develop the genital tubercle [281] and have intestinal deformities
[283]. Prominent WNT-5A expression is observed in the gut mesenchyme during intestinal
morphogenesis which persists throughout the development of the small intestine [281,284].
In line with that, WNT-5A knock-out mice show severe malformations in the small intestine
with drastically reduced length and the presence of a secondary cavity. In addition, the
mutants present an imperforated anus [283]. Interestingly, overexpression of WNT-5A
during embryonic development also leads to gut malformations resembling the WNT-5A
knock-out phenotype. Specifically, WNT-5A overexpression caused shortening of the small
and large intestine, caecum and stomach and also presents anal imperforation [282]. Of
note, both the loss and overexpression of WNT-5A doesn’t interfere with the intestinal
differentiation or cell fate decisions. The underlying mechanisms that lead to the
malformations observed in WNT-5A transgenic mice are not clear yet. However, the
observation that overexpression of WNT-5A leads to the downregulation of ROR2 in
intestine [282] could reveal the reason behind the similarities in both the WNT-5A
overexpressed and WNT-5A knock-out phenotypes. ROR2 is a receptor for WNT-5A and
ROR2 knock-out mice show a phenotype resembling that of WNT-5A knock-out [270].
Therefore, increased expression of WNT-5A which leads to the downregulation of ROR2
could present a similar phenotype as ROR2 knock-out. Although the downstream WNT-5A
signaling after overexpression remained intact, it is tempting to speculate that ROR2-
dependent WNT-5A signaling is crucial for the embryonic development and that the loss of
ROR2 in WNT-5A transgenic mice underlies the similarity with the WNT-5A knock-out
phenotype.
Lungs are complex organs with extensive branching, a large number of different types of
specialized cells and distinct P-D polarity. WNT-5A, as a major determinant of P-D polarity,
is prominently expressed in the embryonic lungs [227,233] where it is localized in both the
mesenchymal and epithelial compartments. WNT5A signaling is most enhanced at the tip
and around the branching epithelium [227]. In later stages, WNT-5A is predominantly
localized to the lung epithelium and attains a typical P-D gradient with most expression in
the distal branching epithelium and almost no presence in the proximal regions [227].
Analysis of lungs obtained from WNT-5A knock-out mice revealed extensive developmental
General introduction
41 | P a g e
malformations. The trachea was truncated with reduced number of cartilages [227]. The
branching morphogenesis of WNT-5A knock-out lungs was compromised as revealed by
increased number and overexpanded terminal airways. Also, the intersaccular walls were
thick and hypercellular indicating failed maturation of lungs in WNT-5A knock-out embryos.
Further analysis revealed that loss of WNT-5A didn’t interfere with cell differentiation but
led to hyperproliferation resulting in intersaccular septum thickening and disrupted
vasculature [227]. Interestingly, WNT-5A knock-out lungs presented increased expression
of sonic hedgehog/patched (SHH/PTC), FGF and bone morphogenetic protein 4 (BMP4)
indicating the molecular mechanisms involved in the observed WNT-5A knock-out
phenotype [227]. Notably, lungs of WNT-5A knock-out mice show resemblance with the
FGF-10 knock-out [285], SHH knock-out [286,287], SHH transgenic [288] and BMP4
transgenic [289] lung phenotype, which underlines the interactive network of WNT-5A,
FGF-10, SHH/PTC and BMP4 in lung development. Lung-specific WNT-5A transgenic
expression also disrupts lung morphogenesis as demonstrated by dilated terminal airways,
loss of branching and smaller size of the lungs [290]. Interestingly, supporting a role for
WNT-5A in regulating other signaling cascades, WNT-5A overexpression repressed
SHH/PTC expression and distribution in the lung epithelium whereas it augmented FGF-10
abundance in the mesenchyme [290]. While FGF-10 expression is increased, WNT-5A
overexpression severely impairs the ability of epithelium to respond to FGF-10 [290]. Thus,
WNT-5A fine-tunes the developmental signaling underlying the epithelial-mesenchyme
communication which is required for proper lung morphogenesis [290].
1.5.6.2 Migration
Cell migration requires acquisition of new asymmetry and polarity along with reorganization
of the cytoskeleton and breaking and/ or reprocessing cell-cell and cell-substrate adhesions.
As such, the WNT/PCP and WNT/Ca2+ pathways have been linked with migration of cells.
WNT-5A-activated noncanonical WNT signaling pathways have been associated with the
convergent extension movements. Several studies have elucidated the significance and
molecular mechanisms of WNT-5A-induced cell migration. For instance, a study has
identified the WNT-5A-ROR2 axis in regulating cell motility. WNT-5A interacts with ROR2
and induces its association with filamin A, an actin binding protein, which, in turn, leads to
formation of filopodia [291]. Filopodia are actin based structures projecting at the leading
edge of migrating cells and are important in formation of focal adhesions attaching to the
substrate and facilitating directional cell movement [292]. WNT-5A-induced ROR2-Filamin
A association activates aPKC which in turn activates JNK. Activated JNK may mediate cell
migration by microtubule organizing center (MTOC) reorientation and actin remodeling via
phosphorylation and activation of CapZ-interacting protein (CapZIP) [293]. In addition,
JNK can also phosphorylate paxillin regulating focal adhesion complexes [294,295] and
modulating cell motility in response to WNT-5A. In another mechanism, WNT-5A induces
cell migration via Daple (DVL-associating protein with a high frequency of leucine residues)-
mediated Rac activation [296]. Daple interacts with DVL in response to WNT-5A and
facilitates its interaction with aPKC consequently inducing Rac activation. This leads to
cytoskeletal reorganization promoting lamellipodia formation and cell migration [296]. In
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addition to aPKC, WNT-5A can also employ Rab35 to activate Rac in a DVL-dependent
manner and induce cell migration [297].
Besides noncanonical WNT signaling, WNT-5A can also activate β-catenin-dependent
signaling to promote cell migration. In melanoma cells, WNT-5A activates small GTPase
ADP-ribosylation factor 6 (ARF6) via FZD4-LRP6 binding. ARF6 released membrane-
bound β-catenin from N-cadherin increasing its cytosolic abundance and triggering β-
catenin-dependent transcriptional program that induces invasion and metastasis [257].
1.5.6.3 Stem cell differentiation and regeneration
Owing to its property of regulating cell polarity, cell movement and cell proliferation along
with antagonistic effects on WNT/β-catenin signaling, WNT-5A may play a critical role in
modulating cell fate determination and differentiation of stem cells.
Hematopoietic stem cells exhibit a shift from canonical to noncanonical WNT signaling with
ageing where high levels of WNT-5A are present in the aged cells [231]. Interestingly,
treatment of young hematopoietic stem cells with WNT-5A induced age-related changes
such as ageing-associated stem-cell apolarity, reduced regenerative capacity and an ageing-
like myeloid–lymphoid differentiation shift via activation of small Rho GTPase Cdc42 [231].
On the other hand, reduction of WNT-5A expression in aged hematopoietic stem cells leads
to their functional rejuvenation [231].
Similarly, WNT-5A is also critical in mesenchymal stem cell (MSC) biology. MSCs can
differentiate into multiple cell types such as adipocytes and osteocytes. The presence of
WNT-5A in human bone marrow MSCs inhibits adipogenesis and promotes
osteoblastogenesis by inhibition of peroxisome proliferator-activated receptors γ (PPARγ)
activation via a CaMKII-TAK1-TAK1-binding protein2 (TAB2)-NLK signaling axis and
simultaneous induction of runt-related transcription factor (RUNX) expression [298].
In line with its role in morphogenesis and stem cell differentiation, WNT-5A has recently
been shown to be involved in tissue repair and regeneration after injury. A study
demonstrated robust induction of WNT-5A-positive mesenchymal cells following an
intestinal injury which are specifically localized in the wound bed [232]. The presence of
WNT-5A provided a demarcation of the regenerating proliferative area via potentiation of
TGF-β signaling. This allowed a fine-tuning of regeneration and proper wound healing [232].
Increased amount of WNT-5A is observed in lung tissue from mouse model of acute
respiratory distress syndrome (ARDS) which could be the repair response of damaged lungs
to resolve the injury [299]. Indeed, WNT-5A can promote the survival of bone marrow-
derived MSCs following an oxidative-stress injury and can induce their differentiation into
the type II alveolar epithelial cells (ATII) via activation of JNK and PKC signaling [299].
1.6 WNT signaling in pulmonary diseases(adapted from Boorsma et al (2014) [300])
Aberrant WNT signaling has been linked to a myriad of pathological conditions including
fibroproliferative, malignant and inflammatory disorders [137]. A growing body of literature
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43 | P a g e
implicates WNT signaling in pulmonary fibrosis but the underlying mechanisms are not well
understood. Increased expression of WNT signaling pathway genes (WNT-1, WNT-7B,
WNT-10B, FZD2, FZD3, β-catenin and LEF1) have been detected in lung biopsies from
idiopathic pulmonary fibrosis (IPF) patients [301] whereas elevated expression of WNT-3A,
LRP5 and LRP6 have been reported in peripheral blood monocytes isolated from IPF
patients and has been linked with disease progression [302]. Immunohistochemistry
analysis revealed an increased activation of WNT/β-catenin signaling in the bronchial and
alveolar epithelium of IPF lung sections [301] whereas increases immunoreactivity to WNT-
7B is observed in the IPF lung tissues which was localized in the regions of active fibrotic
changes [303]. Similarly, in lung biopsies from IPF/UIP patients there was an increase in
nuclear β-catenin, a hallmark of active β-catenin signaling, associated with the regions of
proliferative bronchiolar lesions and fibroblast foci [304]. Interestingly, increased
expression of matrilysin/MMP7 [304], a target of WNT/ β-catenin signaling, overlapped
with the regions of high nuclear β-catenin [305]. Genetic ablation of matrilysin/MMP7
confers protection against bleomycin-induced lung injury [306] supporting an important but
detrimental functional role of WNT/β-catenin signaling in pulmonary fibrosis. Increased
nuclear β-catenin is also observed in fibroblasts from different fibrotic conditions including
IPF and systemic sclerosis pulmonary fibrosis patients [307,308]. Whilst these studies
identify a detrimental role for WNT/β-catenin signaling in pulmonary fibrosis, alveolar
epithelial cell-specific genetic ablation of β-catenin augmented cell death and impaired the
repair response post-lung injury suggesting a protective role for β-catenin [309] as
accelerated apoptosis of alveolar epithelial cells contributes to the progression of pulmonary
fibrosis [310]. This observation by Tanjore et al (2013) highlights an important role for
WNT/β-catenin signaling in alveolar cells where it regulates the repair process after lung
injury [309]. Perhaps, the strong increase in WNT/β-catenin signaling could signal a repair
response of the lung in response to an insult. β-Catenin, thus, may serve different roles in
different structural compartments of the lung, The differential roles could be attributed to
the downstream interaction partners which associate with β-catenin concluding the
transcriptional complex and activating target gene transcription. Indeed, preventing β-
catenin-CBP interaction rescues mice from bleomycin-induced lung injury [311] whereas
inhibition of β-catenin-p300 interaction worsens lung epithelial repair during inflammation
[312] underlining a detrimental role for β-catenin-CBP and a protective role for β-catenin-
p300 interaction. These observations reveal that fine-tuning of β-catenin-mediated
responses by its interaction partners can channel β-catenin signaling into a positive or
negative outcome.
In addition to β-catenin, other components of WNT signaling are also implicated in
pulmonary fibrosis. Global knockout of LRP5, the canonical WNT signaling coreceptor,
protects against pulmonary fibrosis by decreased β-catenin signaling which, in turn, reduced
the expression of TGF-β [302].
The profibrotic cytokine, TGF-β, also suppresses the expression of a WNT antagonist- DKK-
1, thereby facilitating increased WNT/β-catenin signaling suggesting a causal link between
altered WNT signaling and development of fibrosis [307]. WNT-5A, a non-canonical WNT
ligand, is also highly upregulated in fibroblasts isolated from IPF patients and regulates cell
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proliferation, survival and expression of fibronectin [313]. Furthermore, in a murine model
of mechanical ventilation-induced pulmonary fibrosis, WNT-5A expression is increased
considerably and contributes to lung injury and fibrosis [314]. As mentioned in the earlier
sections, disrupted ECM homeostasis is an important denominator of fibrosis. Increasing
evidence suggests a potential regulatory role of WNT signaling in ECM expression and
deposition. TGFβ and WNT signaling can crosstalk at multiple levels playing a crucial role
in fibrotic disorders [96]. Interestingly, TGF-β exerts a wide modulatory effect on WNT
ligand and receptor expression in airway smooth muscle cells and β-catenin is required for
TGFβ-induced ECM expression in airway smooth muscle cells [315].
Interactions between the WNT signaling pathway and the ECM are not unidirectional, as the
ECM can also influence WNT signaling. For instance, mechanical stretch, which is partly
defined by the composition and extent of the ECM, can lower the expression of the WNT
antagonist- DKK-1 in a dose-dependent manner, thereby activating WNT/β-catenin
signalling [316]. Although the phenomenon is not confirmed in lungs, decreased abundance
of DKK-1 in the fibrotic lungs could be explained by progressive stiffening of the disease
afflicted organ.
Altered expression of microRNAs (miRNAs) has been associated with pulmonary fibrosis in
clinical and experimental studies [317,318]. miRNAs are small noncoding RNA molecules
of ~22 nucleotides which can repress expression of protein-coding genes by blocking the
translation and/or promoting the degradation of specific target mRNAs [319]. WNT
signaling and miRNAs can cross-regulate each other at multiple levels. Interestingly,
abnormal expression of some of the miRNAs in pulmonary fibrosis can also be linked to
WNT signaling modulation [318]. For instance, let-7d expression localizes to alveolar
epithelium and is significantly downregulated in lung explants from IPF patients, an effect
which is linked to TGF-β. Inhibition of let-7d in animal model induces features of severe lung
fibrosis [320]. Interestingly, WNT/β-catenin signaling suppresses expression of let-7 family
miRNAs [321]. miRNA-21 expression is increased in the lungs and serum from IPF patient
[322,323] and can be induced by WNT/β-catenin signaling [324]. High levels of miRNA-21
can also be linked to enhanced WNT pathway activation as miRNA-21 targets and suppresses
DKK-2 expression, a WNT antagonist, thereby promoting WNT/β-catenin signaling [325].
WNT-1 inducible signaling pathway protein 1 (WISP1) or CCN4, a target of WNT/β-catenin
signaling pathway, is a cysteine rich, secreted matricellular protein of CCN family and
regulates cell proliferation, survival and differentiation [326,327]. WISP1 has been linked to
lung fibrosis in both experimental and human IPF. Considerable upregulation of WISP1 is
found in the lung biopsies from IPF patients where its expression is localized to the
hyperplastic, proliferating ATII cells in close proximity to epithelial lesions and fibroblast
foci [328]. Similarly, marked augmentation in WISP1 expression, both at the gene and
protein level, is also observed in the lungs obtained from mice subjected to bleomycin-
induced lung fibrosis [328]. Interestingly, WISP1 expression in bleomycin-treated lungs is
also primarily localized to the ATII cells [328]. In line with its prohypertrophic role,
treatment with exogenous recombinant WISP1 led to a strong proliferative effect on the
primary ATII and lung epithelial cell line-A549 cells [328]. Interestingly, recombinant
WISP1 induced myofibroblast differentiation and ECM expression in mouse and human lung
General introduction
45 | P a g e
fibroblasts [328] whereas orthotracheal administration of neutralizing anti-WISP1
antibodies attenuated bleomycin-induced lung fibrosis and partially restored normal lung
function [328]. These observations underline a critical role for WISP1 in WNT/β-catenin-
induced pulmonary fibrosis.
Based on the insights from current literature, WNT signaling homeostasis clearly emerges
as a major determinant of healthy and diseased conditions. Our understanding about the
mechanisms and direct consequences of WNT signaling activation in fibrotic pulmonary
disorders such as airway remodeling is poor. Further studies are warranted to understand
the role of WNT signaling in airway remodeling and maintenance of its homeostasis in order
to achieve our ultimate goal of utilizing WNT signaling modulation for treating fibrosis and
other ailments involving deregulation of this developmental pathway.
1.6.1 WNT signaling: therapeutic potential
WNT signaling and its target genes are linked to proliferation, survival, matrix protein
expression, inflammatory responses, stemness and differentiation. With the ever evolving
understanding about its role in disease, WNT signaling is fast emerging as a promising
therapeutic target. Many small molecule inhibitors and modulators are available and many
more are under development for targeting WNT signaling pathway at different levels in the
cascade [139]. As discussed in previous sections, β-catenin partners with various proteins to
channel the effects of its activators, many strategies target the specific interaction of β-
catenin with its downstream partners. For instance, small molecules like PKF115-584 are
available to prevent β-catenin/TCF/LEF transcriptional assembly whereas ICG-001 and IQ-
1 can inhibit association of β-catenin with transcriptional coactivators -CBP and p300,
respectively [311,312]. Both CK1α and Axin facilitate β-catenin degradation. Pyrvinium, a
small molecule inhibitor, can promote β-catenin degradation by activating CK1α whereas
XAV939, an inhibitor of axin destabilizing kinase- tankyrase, does the same by promoting
Axin stability. In addition, small molecule inhibitors also exist for targeting DVL and WNT
ligand secretion and availability of recombinant WNT antagonists like DKK and sFRPs
further add to the resources [139].
1.7 TGF-β and WNT signaling crosstalk: potential implications in airway
remodeling (adapted from Yeganeh et al (2013) [96])
Both TGF-β and WNT signaling regulate a myriad of processes including development, cell-
fate determination, and cellular differentiation and regeneration across the phyla from
Drosophila to mammals [136,329]. Studies have implicated extensive cross-talk between
TGF-β and WNT signaling pathways at various levels in their multi-component signaling
system, from early development to post-natal tissue homeostasis (Figure 7). For instance, in
Xenopus, both TGF-β and WNT signaling play important role in establishing the Spemann’s
organizer mediated by association of SMAD4 with β-catenin in a LEF1/TCF-dependent
manner [330]. Another study demonstrated that SMAD3 physically interacts with high
mobility group (HMG) domain of LEF1 inducing transcriptional activation of twin and
contributing to the patterning of embryo [331]. Other studies have also shown the TGF-β-
dependent and independent association of SMAD proteins with β-catenin and LEF1/TCF in
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46 | P a g e
human and other mammalian cell systems [126,332-334]. Furthermore, TGF-β and various
growth factors have been shown to activate β-catenin signaling through GSK-3β inactivation
[107,126,315,335]. Observations suggest that TGF-β/WNT pathway cross-talk is highly
context dependent. For instance, β-catenin is not required and doesn’t affect TGF-β-induced
expression of PAI-1 in ASM cells and pulmonary fibroblasts [107,315], whereas in renal
epithelial cells PAI-1 is a target of β-catenin-dependent WNT signaling [132]. Also, it is
interesting to note that TGF-β-activated β-catenin doesn’t drive expression of canonical
WNT target genes [107] but contributes to the activation of specific TGF-β target genes,
probably those with LEF1/TCF- and / or SMAD-binding sites in their promoters. These
differential effects of TGF-β/β-catenin axis can be attributed to the association of β-catenin
with various nuclear partners in a stimuli- and cell-dependent manner.
Fig. 7. TGF-β-WNT signaling crosstalk in airway smooth muscle cells. β-Catenin is the key
effector of canonical WNT signaling wherein WNT ligand-induced GSK3 inhibition leads to protection
of β-catenin from proteasomal degradation. TGF-β also prevents degradation of β-catenin by
inactivating GSK3, which in combination with ERK1/2 mediated transcriptional nupregulation of β-
catenin, leads to the cytosolic accumulation of this transcriptional co-activator. Subsequently, non-
phosphorylated transcriptionally active β-catenin contributes to the TGF-β-induced cellular effects in
ASM cells. In addition, TGF-β may also induce expression of WNT ligands, however, their role in TGF-
β signaling is not known (Taken from Yeganeh et al (2013) [96]).
ASM cells show remarkable phenotypic plasticity and accumulating evidences suggest a
central role for TGF-β-WNT pathway cross-talk in regulating various processes governing
structural and functional features of ASM cells. It has been shown that TGF-β increases
abundance of β-catenin in ASM cells by transcriptional up-regulation [185,315]. TGF-β can
also activate β-catenin signaling via inactivation of GSK-3β and rescuing it from proteasomal
degradation leading to subsequent increase in active β-catenin in ASM cells [315]. Besides
General introduction
47 | P a g e
inactivating GSK-3β, TGF-β signaling can protect β-catenin from degradation using SMAD3
[336]; however, this observation is not yet confirmed in ASM cells. Components of TGF-β
and WNT signaling have been shown to be mutual targets of each other. For instance, WNT
signaling can induce expression of TGF-β [337] whereas TGF-β can modulate expression of
β-catenin in ASM cells [185]. The contribution of autocrine WNT production and signaling
to TGF-β-induced β-catenin signaling is not yet established in ASM. Nonetheless, β-catenin
signaling can regulate various aspects of ASM plasticity and contributes actively to TGF-β
responses in ASM cells [338]. Interestingly, stabilization of β-catenin is both required and
sufficient to induce ECM protein production in ASM cells [315]. This finding is important as
deregulated ECM homeostasis is linked to airway remodeling in chronic lung diseases [46].
Accordingly, a study in smooth muscle cells linked TGF-β-induced inactivation of GSK-3β to
increased cell size and expression of contractile proteins [339]. Indeed, hypertrophy and
hypercontractility of ASM cells are features of airway remodeling [46]. Deng et al (2008) did
not show any evidence concerning activation or involvement of β-catenin [339]; however it
has been shown that β-catenin contributes to smooth muscle cell contractility. As
demonstrated by Jansen et al (2010), β-catenin is required for force generation in bovine
tracheal smooth muscle (BTSM) strips [340]. This effect is attributed to the β-catenin
mediated stabilization of cell-cell adhesions [340]. A change in the expression of contractile
proteins was not observed in this study, but other studies have implicated β-catenin-
dependent contractile protein expression in fibroblasts [107,337]. In addition to contributing
to force generation, β-catenin has also been shown to regulate proliferation of ASM cells.
Nunes et al (2008) showed that growth factors induce inactivation of GSK-3β and
subsequent increase in nuclear β-catenin levels, an effect which was required for the increase
in DNA synthesis as cell proliferation was attenuated by β-catenin siRNA [341].
Further studies are therefore required for delineating the cross-regulatory mechanisms by
which TGF-β and WNT pathways regulate ASM phenotype and functions. Also, the
functional role of autocrine WNT secretion in response to TGF-β needs to be investigated
further. A role for TGF-β-WNT signaling networks in hypercontractility, hypertrophy and
hyperplasia of ASM cells along with their contribution to enhanced and altered deposition
of ECM proteins emerges strongly. Hence, a crucial role of TGF-β-WNT cross-talk in airway
remodeling can be envisaged.
1.8 Scope of the thesis
As discussed above, WNT signaling is a major player in lung morphogenesis and has been
linked to several respiratory diseases with a large body of literature supporting the role of
canonical WNT signaling mediator-β-catenin in various fibrotic disorders including those
afflicting lungs. A study from our group has also reported a novel role for β-catenin in TGF-
β-induced ECM production by airway smooth muscle [315]. Additional work from our lab
has revealed the importance of β-catenin signaling in fibroblasts [107]. Nonetheless, the role
of noncanonical WNT signaling is not known in airway remodeling. The primary aim of this
thesis is to investigate the role of noncanonical WNT signaling in the initiation of cellular
responses associated with airway remodeling. To this aim, in vitro experiments are
performed using cultured ASM cell lines and human primary bronchial smooth muscle cell
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48 | P a g e
to identify the role of noncanonical WNT signaling in various aspects of airway remodeling.
Underlying molecular mechanisms are dissected by employing pharmacological inhibitors
and gene-specific knock-down strategies.
Chapter 2 provides a review of current understanding of the functional significance of β-
catenin in airway remodeling in asthma and its therapeutic potential. A detailed account is
provided about the contribution of β-catenin in various aspects of airway remodeling and
the current strategies which can possibly target β-catenin signaling to counter airway
remodeling.
Chapter 3 identifies WNT-5A, a noncanonical WNT ligand, as a novel player in airway
remodeling. A comprehensive analysis of WNT signaling pathway alteration by TGF-β is
performed in ASM cells. siRNA-mediated gene silencing was used to probe the specific roles
of WNT-5A and for identification of its receptors. WNT-5A-activated downstream signaling
was identified using recombinant protein antagonists and pharmacological inhibitors for
JNK and Ca2+ signaling.
In Chapter 4, we assessed the role of noncanonical WNT ligands in TGF-β-induced
expression of α-SMA- a key contractile protein of ASM cells. We identified a role of actin
remodeling by observing the globular and filamentous actin fractions using specific binding
dyes, specific pharmacological inhibitors such as latrunculin A and Y27632, and probed for
the possible transcription mechanism linked to actin remodeling.
Chapter 5 describes the molecular mechanisms involved in TGF-β-induced WNT-5A
expression and identifies Sp1 as a novel transcription factor for WNT-5A. We have dissected
the signaling cascade employed by TGF-β in WNT-5A transcriptional upregulation using
pharmacological inhibitors and siRNA mediated gene silencing. Furthermore, we did in
silico analysis of WNT-5A promoter to predict candidate transcription factors and performed
chromatin immunoprecipitation (ChIP) to identify the transcription factor.
The WNT signaling family is complex with large number of members with varied functions.
Many of them are implicated in pulmonary diseases as discussed previously. Chapter 6
provides insights from a mouse model of chronic airway inflammation into modulation of
WNT signaling in the lungs of an animal model of asthma. We performed a comprehensive
gene expression analysis of WNT signaling family in whole lung extracts to identify novel
candidates and mechanisms involved in asthma.
Chapter 7 reviews the current knowledge about WNT-5A signaling, functions and roles in
various pathologies including pulmonary diseases.
Finally, in chapter 8, we summarize our results and discuss our findings providing a
broader perspective and provide future perspectives.
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