2012 Group 5 Project
|2012 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7 | Group 8 | Group 9|
- 1 Wnt/β-catenin Signalling Pathway
- 1.1 Introduction
- 1.2 History
- 1.3 Key players in Wnt/β-catenin signalling
- 1.4 Normal function: Roles of Wnt/β-catenin signalling
- 1.5 Normal function: Mechanism of action
- 1.6 Abnormal Function: Diseases associated with Wnt/β-catenin signalling
- 1.7 Current research
- 1.8 Future directions
- 1.9 Glossary
- 1.10 External links
- 1.11 Reference List
Wnt/β-catenin Signalling Pathway
2012 marks the 30th anniversary of the identification of Wnt-1, the first component of the Wnt signaling pathway to be discovered. Since this breakthrough, researchers from around the globe have extensively studied and pieced together the multiple components that form the Wnt signaling pathway as it is known and accepted today.
The classical pathway of Wnt signalling that is best understood in current research is the Wnt/β-catenin signalling pathway. This signal transduction pathway is evolutionarily conserved and features heavily in development, regeneration, stem cell regulation and cellular processes such as proliferation and migration.
The term 'Wnt' was coined to reflect the homolog genes: Wingless in Drosophila and Int-1 in mammals. Wnt proteins encompass a network of secreted glycolipoproteins, forming the basis of this highly conserved pathway. The various elements, agonists and antagonists of this pathway will be discussed, with key emphasis on the structure and function.
Furthermore, mutations in components of this pathway have been associated with cancers, developmental defects and other diseases which will be discussed in further detail.
It is important to note that the Wnt/β-catenin signalling pathway is becoming increasingly complex with the addition of new molecules being discovered in recent years. Areas of current research and future directions will also be outlined.
Key players in Wnt/β-catenin signalling
This table provides an introduction to the various proteins involved in what would be a complex interaction in the Wnt/β-catenin signalling pathway. It is aimed to simplify the following mechanisms of action by elucidating the characteristics of each protein. For each protein listed, the gene from which it is derived is given in brackets, followed by a column providing an illustration to help the reader better picture the respective protein. The general function and antagonists are also outlined for reference.
|Wnt (WNT)||250px||Evolutionarily conserved protein across species. Most Wnt proteins figure in the region of 40 kDa, each possessing a characteristic 23 or 24 cysteine residues highly conserved in their spacing. This suggests that the formation of disulfide bonds plays a significant role in determining tertiary protein structure||Initiates Wnt signalling when it binds as a ligand to the Fz receptor with LRP and destabilises the β-catenin degradation complex, dephosphorylating β-catenin and subsequently enabling its migration and accumulation in the cell nucleus for gene transcription ||
|Fz(FZD)||150px||Fz houses a CRD on the N-terminus, seven transmembrane domains, and a PDZ domain on the C-terminus||Binds the Wnt ligand at the extracellular N-terminus for the initiation of the Wnt/β-catenin signalling pathway||
|LRP-5/6 (LRP)||250px||A protein that spans multiple domains. The extracellular component is comprised of four EGF-like repeats on the N-terminus||Co-receptor to the Wnt ligand in its interaction with the Fz receptor in signal transduction. Causes translocation and association of axin to its intracellular tail, thus destabilising the β-catenin binding activity of axin|
|Dsh (DVL)||250px||A phosphoprotein  approximately 670 amino acids long . Possesses a DIX domain, which is thought to facilitate interaction with Axin .||Prevents phosphorylatory activity of GSK-3β upon hyperphosphorylation by the Wnt/LRP-5/6 complex binding to the Fz receptor, thus preventing β-catenin ubiquitination and degradation, to promote translocation into the nucleus.||
|Axin (AXIN)||250px||A 95.635 kDa protein. Possesses a DIX domain, which is thought to facilitate interaction with Dsh .||In the absence of Wnt signalling, it constitutes the β-catenin ubiquitination complex along with APC and GSK-3β and its role as a scaffolding protein enhances GSK-3β phosphorylation of β-catenin|
|GSK-3β(GSK3β)||250px||46 kDa kinase . Possesses a surface channel on the hydrophobic C-terminal helical domain that binds a 19 amino acid residue on Axin .||Constitutes the β-catenin ubiquitination complex along with axin and APC; its primary role in the Wnt/β-catenin signalling pathway is inhibition of β-catenin nuclear translocation through phosphorylation of three amino acids at the N-terminus after priming by CK-I, for subsequent ubiquitination and degradation by the proteasome. This activity is enhanced by GSK-3β phosphorylation of axin and APC which appears to promote phosphorylation of β-catenin by GSK-3β in the complex.|
|Diversin (ANKRD6)||776 amino acid with multiple ankyrin repeat domains .||Primes β-catenin for degradation by recruiting CK-I to phosphorylate serine 45 on the N-terminus before subsequent phosphorylation of threonine 41, serine 37 and serine 33 by GSK-3β and degradation by E3 ubiquitin ligase β-TrCP||There are no known inhibitors of Diversin in the published literature.|
|APC (APC)||150px||A large 312 kDa protein . Possesses a 25 amino acid SAMP repeat that is sufficient for full Axin binding ||Possesses a numerous and varied set of roles ranging from cell migration and adhesion, cell cycle regulation and chromosome stability. In the Wnt/β-catenin signalling pathway, APC constitutes the β-catenin ubiquitination complex along with axin and GSK-3β. It binds to axin via SAMP elements, and to β-catenin via three 15-amino acid repeats and seven 20-amino acid repeats||
|β-catenin (CTNNB1)||The N-terminus accommodates a critical sequence of thirteen armadillo repeats that competitively bind E-cadherin, LEF-1 and APC. The C-terminus houses a glycine-rich transactivation domain||Involved in mediating both morphogenesis and maintenance of tissue integrity in the endothelium, bound to a-catenin that subsequently binds to the actin cytoskeleton. Gene expression is mediated in conjunction with TCF /LEF, upon translocation from the cytosol into the nucleus||The axin/APC/GSK-β complex phosphorylates β-catenin and marks it for ubiquitination and degradation by the proteasome|
|TCF /LEF (TCF7/LEF)||The N-terminus constitutes the β-catenin interaction domain; TCF /LEF also possesses a HMG box DNA-binding domain||Mediates DNA binding when in complex with β-catenin; the N-terminus of TCF /LEF associates with the β-catenin C-terminus|
Normal function: Roles of Wnt/β-catenin signalling
The Wnt/β-catenin signaling pathway has been implicated as an important pathway in human fetal development. Through immunohistochemical staining, Eberhart and Argani (2001) localised nuclear beta catenin in fetal lung, placenta, kidney, cartilage, capillaries, adrenal glands and skin. This indicates that Wnt signaling regulates the development of specific set of organs and tissues. For example, Wnt genes such as Wnt4 regulate the conversion of mesenchyme to epithelial cells in kidney morphogenesis.
In addition, Wnt/β-catenin signaling is also involved in maintaining the pluripotency of human embryonic stem cells (hESCs). Wnt3a promotes the reprogramming of somatic cells to pluripotency in conjunction with the classical transcription factors, Oct4, Sox2 and Nanog. For example, Oct4 has the effect of repressing Wnt/β-catenin signaling in self renewing hESCs and is depressed during hESC differentiation. Hence, this suggests that Wnt/β-catenin signaling is involved in differentiation rather than self renewal.
Adult tissue self renewal
Wnt/β-catenin signalling features in a great variety of processes during embryogenesis however the interesting concept to note about Wnt/β-catenin signalling is that it is heavily involved in adult tissue self renewal as well . For example, in the gut, Wnt signaling is essential for the establishment of the progenitor compartment in an intestinal crypt . This is further supported by Kuhnert et al (2004) who noted that the inhibition of Wnt signalling results in the loss of crypts. This means Wnt provides the mitotic stimulus for the progenitor cells in the crypts to proliferate.
Wnt/β-catenin signalling is also involved in the self renewal of bone tissue where several mouse models established the importance of Wnt signaling in bone homeostasis. For instance, Babij et al (2003) observed how mice with gain of function mutations in LRP-5 (in osteoblasts) results in increased bone density and osteoclast activation. In contrast, Kato et al (2002) noted how a loss of function in LRP-5 lead to decreased bone mass and faulty osteoblast maturation. Hence, Wnt signalling plays an essential role in maintaining the maturation and proliferation of osteoblasts.
Other tissues involved in self renewal via Wnt signaling include hair follicles and the haematopoietic system.
Muller et al (2002) investigated how Wnt/beta catenin signalling is involved in the stimulation of epithelial cell motility. They discovered that when β-catenin was activated, it had the ability to induce epithelial migration in cell culture. Furthermore, the addition of growth factors enhanced the transcriptional activity of β-catenin and induced more migration than beta catenin alone. This indicates evidence of synergism between Wnt signalling pathways and growth factors, which is particularly relevant in cancers. As a result, the cooperation between Wnt signaling and growth factors are likely stimulators of tumour progression and possibly the capacity to migrate and metastasise.
Comparison with non-canonical Wnt signalling
Wnt/β-catenin signalling is commonly referred to as the canonical Wnt signalling pathway, and its function is primarily centered around the differentiation and proliferation of cells in adults and embryos. In contrast, the non-canonical Wnt signalling pathways include the Planar Cell Polarity (PCP) pathway and Wnt/Calcium pathways, and are involved in regulating cell polarity, neural migration and myogenesis. Despite their differences in intracellular effectors and function, these pathways have been known to cross talk with each other, for example during axis formation in Xenopus
Normal function: Mechanism of action
β-catenin commonly exists as a subunit making up the cadherin protein complex, where it links E-cadherin to α-catenin. Cadherin proteins play an integral role in the formation of adhesion junctions between cells , and β-catenin in particular acts to help anchor the actin cytoskeleton within the cell. The β-catenin gene can be said to function as an oncogene given that the promotion of transcription of Wnt target genes by β-catenin has been shown to be involved in the development of an array of cancers including basal cell carcinoma, colorectal cancer and breast cancer.
In the absence of Wnt (“off state”)
1. No extracellular "Wnt signal"
2. β-catenin targeted for proteosomal degradation by its incorporation into a “destruction complex” comprising:
- Intracellular Axin, GSK-3β (glycogen synthase kinase β) and APC (Adenomatous polyposis coli – coded for by the APC tumour suppressor gene)
5. Level of β-catenin in the cytosol is low, and therefore does not reach the nucleus at a level sufficient enough to affect transcription
6. No transcription of Wnt target genes
- TCF/LEF proteins act as transcriptional repressors, binding to proteins of the TLE /Groucho family 
- HDAC represses transcription via chromatin remodelling in the form of histone deacetylation
In the presence of Wnt (“on state”)
1. Extracellular “Wnt signal” binds to a cell surface G-protein coupled receptor of the “Frizzled” (FZ) family
- Recruitment of axin to the membrane where it is degraded upon initiation of the Wnt signalling cascade
- Dissociation of β-catenin into the cytoplasm without degradation by the “destruction complex" 
3. Activation (phosphorylation and poly-ubiquination) of proteins of the “Dishevelled” (Dsh) family
- Implicated in the inactivation of the “destruction complex” by the recruitment of axin and GSK-3β away from the complex 
- TCF promotes binding of RNA polymerase to DNA template strand
6. Induction of the downstream transcription of Wnt target genes, for example:
- oncogenes such as c-Myc and cyclin D1 involved in cell cycle control 
- MMP-7 and uPA, associated with metastasis and the invasion of cancer cells 
In a tumour cell, the Wnt signalling pathway is activated aberrantly either:
- Inappropriately by an extracellular Wnt signal
- In the absence of an extracellular Wnt signal
- Usually results from a mutation in one of the subunits that comprise the “destruction complex”, resulting in an inability to degrade cytosolic β-catenin 
- Mutant APC proteins characteristic of those associated with cancer, have been shown to upregulate transcription of Wnt target genes due to an inability to regulate levels of cytosolic and nuclear β-catenin 
Abnormal Function: Diseases associated with Wnt/β-catenin signalling
As described above, the Wnt/β-catenin signalling pathway plays a critical role in development, stem cell regulation and cellular processes such as proliferation and migration. Thus mutations in components of this pathway have been associated with cancers, hereditary disorders, developmental defects and other diseases. In addition, overexpression or underexpression of Wnt responsive genes have also been implicated in cardiovascular disorders such as hypertrophy and myocardial infarction, bone diseases and skin disorders.
For a comprehensive list of the range of diseases caused by mutations in Wnt-β-catenin components, please refer to Table 1 in Bryan T MacDonald, Keiko Tamai, Xi He Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev. Cell: 2009, 17(1);9-26
Wnt/β-catenin Pathway and the Onset of Cancer
The high number of repressor genes involved in the Wnt/β-catenin pathway indicates that it is imperative for this pathway to be tightly regulated.  These repressor genes include APC, Axin 1 and Axin 2. Given the pathways involvements in the regulation of stem cell choice to proliferate or self renew, there is a strong correlation between mutations in these genes and the onset of cancer. The table below describes in further detail some mutations of the Wnt signalling pathway and associated cancers.
Mutations of the Wnt signalling pathway and associated cancers
|Gene||Normal function||Mutation||Effects of mutation||Associated cancers |
|β-catenin||Primary Wnt effector. In the nucleus, β-catenin functions as a cofactor for TCF transcription factors which specify a subset of genes, which are responsible for determining cell fate and regulation of proliferation. ||GOF mutation||Any mutations that inhibit its destruction motif would cause constitutively active β-catenin signalling, leading to excessive stem cell renewal and proliferation thus predisposing the cells to the formation of tumours. ||
|APC||Facilitates β-catenin degradation; acts as a tumour suppressor||LOF mutation||Mutational inactivation of APC inhibits the degradation of β-catenin, leading to the over-stabilisation and accumulation of β-catenin in the nucleus of the cell.  (See β-catenin above).
|Axin 1 & Axin 2||Serve as scaffolding components for the β-catenin degradation complex. Acts as a tumour suppressor.||LOF mutation||Mutational inactivation of Axin 1 or Axin 2 inhibits the degradation of β-catenin, leading to the over-stabilisation and accumulation of β-catenin in the nucleus of the cell. (See β-catenin above).||
Current Research on Inhibitors to Treat or Prevent Proliferation of Cancer
There is a large focus on finding more effective means of treating and preventing cancer. Current researchers are looking at inhibiting various components of the Wnt/β-catenin pathway to prevent the proliferation of cancer. Such treatments include:
- Small molecule inhibitors can be used to block the interaction between β-catenin and TCF, thus preventing constitutive transcriptional activities that lead to the proliferation of cancer. 
- Non-steroidal anti-inflammatory drugs (NSAIDs) function by interfering with β-catenin/TFC-dependent transcription, and have proven promising for the treatment and prevention of colorectal cancers.  Examples of NSAIDs include exisulind, sulindac and aspirin. The use of NSAIDs was proven effective with a "40-50% reduction in mortality due to CRC in individuals taking NSAIDs". Furthermore, studies are being conducted into the use of NSAIDs to inhibit cyclooxygenase-2 (COX-2) as an effective method of treatment and prevention of cancers. 
- Frizzled-related proteins can be used as natural antagonists to manage the Wnt pathway.
- A “recombinant adenovirus (Ad-CBR) that constitutively expresses the β-catenin binding domain of APC”  was developed, enabling APC to maintain its function of β-catenin degradation.
- Monoclonal antibodies are being used against Wnt proteins by inducing apoptosis in cancer cells .
Other Areas of Current Research
There are a multitude of research groups investigating an array of components making up the Wnt signalling pathway. Four articles most relevant in the context of this wiki page are listed below.
Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling
De Lau et al (2011) demonstrated that the stem cell markers Lgr5 and Lgr4 are often co-expressed in Wnt-driven cellular proliferation, where they associate with the FZ/LRP Wnt receptor complex. This is of particular importance as the deletion of both the Lgr5 and Lgr4 genes results in decreased proliferation of cells in Wnt-driven compartments (e.g. intestinal crypts in a mouse model), a result similar to the mechanism of the Wnt signalling pathway in its “OFF” state.
The current understanding of the Wnt signalling pathway is that the LRP-5/6 co-receptor plays a critical role in the transduction of signals concerning processes such as skeletal remodelling, osteoporosis pathogenesis and cancer formation. MacDonald et al (2011), expand on the canonical understanding of the pathway by differentiating between the LRP-5 and LRP-6 co-receptors and the individual processes they are responsible for. By investigating the molecular basis for these differences, it has been demonstrated that the LRP-6 coreceptor plays a predominant role in embryogenesis and also exhibits a stronger signalling activity in vivo than the LRP-5 isotype (as depicted in Figure 7).
Identification of a novel inhibitor of the canonical Wnt pathway
Because of the vital role Wnt signalling plays in processes such as inflammation, angiogenesis and fibrosis; the identification of a novel inhibitor of the pathway could provide a viable therapeutic intervention for diseases characterised by excessive activation of these processes. One such inhibitor showing promise is pigment epithelium-derived factor (PEDF), which has been demonstrated to attenuate Wnt signalling induced by conditions such as retinal ischaemia. This is believed to be a result of a physical interaction between PEDF and the LRP-6 co-receptor, which blocks initiation of the pathway by preventing activation of the FZ receptor.
Inhibition of GSK3 by Wnt signalling - two contrasting models
We have discussed the role of GSK-3β in the phosphorylation of β-catenin and subsequent ubiquitylation and proteosomal degradation of the protein (“off” state), although the mechanism by which Wnt activation inhibits these events (“on state”) is unclear. This is an important step because by blocking the phosphorylation of β-catenin, the protein is able to accumulate in the cytoplasm and carry out its biological function by promoting the transcription of Wnt target genes. Metcalfe and Bienz (2011) investigated two models proposing contrasting mechanisms by which GSK-3β is inhibited by Wnt activation. The first model proposed that Wnt induces the phosphorylation of LRP-6, which consequently inhibits the catalytic activity of GSK-3β, thereby preventing phosphorylation of β-catenin. The second model suggested that Wnt signalling induces the uptake of GSK-3β in vesicles called multivesicular bodies (MVBs), and therefore prevents any interaction between the enzyme and its substrate, β-catenin. The study also tested whether either of the models have any inclination to predominate in an acute vs. chronic setting of Wnt signalling.
Although the Wnt/β-catenin signalling pathway has been well-established, there are many aspects that add complexity to this signal transduction pathway. The journal articles below are examples of areas of Wnt signalling that could potentially be investigated in the future.
The Role of Canonical WNT Signaling Pathway in Oral Carcinogenesis: A Comprehensive Review. 
This review article highlights the importance of the Wnt/β-catenin pathway to the pathogenesis of oral carcinogenesis. What is still poorly understood in the quest for a cure is the role, if any, of Wnt/β-catenin signalling in apoptosis, a crucial mode of combatting abnormal cell proliferation. Although much has been established in the mechanism of action of the canonical pathway, this topic of research is also relevant to developing treatments for many other diseases that are impacted by abnormalities in the apoptotic function of cells, and hence the continued interest in such research.
Dermal β-catenin activity in response to epidermal Wnt ligands is required for fibroblast proliferation and hair follicle initiation. 
Chen et al. have demonstrated the necessity of secreted Wnt ligands in not only fibroblast precursors, but also for the proliferation of fibroblasts, leading to thickening of the dermis, as well as hair follicles. However, the mechanism of the involvement of the secreted Wnt ligands in dermal fbroblast proliferation is yet to be defined, still resting on the determination of which broadly expressed Wnt ligands are required for dermal β-catenin activity, as well as what role (if any) dermal cell density plays in hair follicle formation. Elucidating such interactions pose implications for addressing diseases such as congenital human focal dermal hypoplasia and skin fibrosis.
List of Abbreviations Used
- APC: adenomatous polyposis coli
- CAMKII: Ca2+/calmodulin-dependent protein kinase II
- CK-I: casein kinase I
- CRD: cysteine rich domain
- CRM1: exportin-1 protein
- DIX: dishevelled and axin domain
- Dsh: Dishevelled
- Fz: Frizzled
- GOF: gain of function mutation
- GSK-3β: glycogen synthase kinase-3β
- HMG: high mobility group
- LEF: lymphoid enhancer-binding factor
- LMB: leptomycin B
- LOF: loss of function mutation
- LRP: lipoprotein receptor–related protein
- MVB: multi-vesicular body
- NES: leucine-rich nuclear export signal protein
- Nkd: naked cuticle
- NSAIDs: non-steroidal anti-inflammatory drugs
- PEDF: pigment epithelium-derived factor
- PKC: protein kinase C
- SAMP: Ser-Ala-Met-Pro repeated amino acid segment
- Stbm: strabismus
- TCF: T cell factor
List of Terminology
- Agenesis: the inability to form an organ during embryogenesis due to the lack of primordial tissues
- Agonist: a molecule which binds to a receptor to produce a physiological response
- Antagonist: a molecule which inhibits the physiological function of another substance
- Apoptosis: programmed cell death
- C-terminus: the end of the polypeptide chain which features a free carboxyl group (-COOH)
- Canonical: standard and well accepted
- Caveolin: integral membrane proteins involved in receptor mediated endocytosis
- Destruction motif: short sequences which provide stability of other proteins when deleted or mutated
- Differentiation: the process by which cells become mature and specialised in structure and function
- Disulfide bonds: a covalent bond between sulfur atoms
- Gain of function mutation: mutation produces a protein which has an enhanced or abnormal function
- Glycolipoprotein: a protein with attached lipid and carbohydrate groups
- Histone deacetylation: the process by which histone deacetylase removes acetyl groups on the histones to increase affinity and condense DNA, thus preventing transcription
- Homeotic gene: genes involved in embryonic development, specifically controlled the anterior-posterior axis
- Homolog: gene which is related to another gene by common descent
- Immunohistochemistry: detecting antigens on a tissue using labelled antibodies
- Immunoprecipitation: the process of isolating a protein antigen using an antibody that binds to it to form an insoluble complex
- Lethal mutation: a mutation which can result in the death of the organism
- Ligand: a molecule which binds to another entity
- Loss of function mutation: mutation produces a protein which has decreased or no function
- Metastasis: distant non adjacent spread of a tumour
- Mnemonic: a tool which assists memory
- Monoclonal antibody: an antibody engineered from a single clone of cells
- Morphogenesis: the differentiation and growth of tissues and organs during embryo development
- N-terminus: the start of the polypeptide chain which features a free amine group (-NH2)
- Oncogene: genes which transform normal cells into cancerous cells
- PDZ domain: a common structural domain of 80-90 amino acids of signalling proteins, whose name is derived from the first three letters of the first three proteins to be discovered to share the domain: post synaptic density protein, Drosophila disc large tumor suppressor, zonula occludens-1 protein
- Pluripotency: the ability of stem cells to differentiate into ectoderm, mesoderm and endoderm
- Proteasome: a large intracellular particle which degrades proteins
- Repressor: a protein which binds to the operator and prevents transcription
- Scaffolding protein: proteins which regulate signal transduction by organising and assembling signalling complexes
- Self renewal: the capability to undergo numerous cell divisions and maintain undifferentiation
- Sequester: to isolate or separate
- siRNA: small interfering RNA which is involved in RNA interference
- Synergism: the joint action of two substances which increases their effectiveness
- Tumour suppressor gene: produces proteins which control cell growth to prevent uncontrolled cell proliferation
- Ubiquitylation: the process of adding ubiquitin
- Hubrecht Institute: Wnt signaling and cancer
- Children's Hospital Boston: Wnt signaling in development
- University of Pennsylvania: Wnt in developmental biology
- Stanford School of Medicine: Stem Cells, Wnt signaling and Tissue Repair
- University of Nebraska: Convergence of Wnt and PKA signaling
- Centre for Cancer Research: The Role of Wnts in Vertebrate Development and Cancer
- Sharma RP Wingless, a new mutant in D.melanogaster. Drosoph. Inf. Serv: 1973, 50;134
- Cell Signalling Technology. (2012). Axin 1 (human). http://www.phosphosite.org/proteinAction.do?id=4768
- National Center for Biotechnology Information, U.S. National Library of Medicine. (2002). Glycogen synthase kinase-3 beta http://www.ncbi.nlm.nih.gov/protein/P49841.2
- National Center for Biotechnology Information, U.S. National Library of Medicine. Adenomatous Polyposis Coli (Xenopus laevis). http://www.ncbi.nlm.nih.gov/protein/AAB41671.1
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