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Revision as of 21:09, 20 May 2012
|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: Mechanism of action
- 1.5 Abnormal Function: Diseases associated with Wnt/β-catenin signalling
- 1.6 Treatment:
- 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 which features heavily in development, regeneration, stem cell regulation and cellular processes such as proliferation and migration.
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.
1973: A mutant of Drosophila melanogaster which had no wings was characterised as Wingless (Wg) 
1982: Roel Nusse and Harold Varmus found that mouse mammary tumour virus (MMTV) stimulated tumour formation when the Int1 (integration 1) gene was activated.
1991: The nomenclature of Wingless and Int-1 was combined to form the mnemonic, Wnt.
1994: Mutations in Dishevelled affected phenotype and gene expression, indicating its crucial role in the Wnt signalling pathway
1996: β-catenin levels were reported to be three times higher in tumour tissues than in normal specimens, indicating the link between β-catenin accumulation and colorectal cancers
1997: Huber et al investigates the three dimensional structure of β-catenin
1999: Casein kinase 1 (CK1) was found to regulate β-catenin function 
2003: The tyrosine kinase receptor Derailed in Drosophila (RYK in mammals) was identified as an alternative Wnt receptor 
2007: Dishevelled was found to polymerize at the plasma membrane and to recruit axin upon Wnt stimulation 
Key players in Wnt/β-catenin signalling
|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||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||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||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||Primes b-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|
|APC (APC)||150px||A large 312 kDa protein||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|
|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: 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 
- β-catenin also 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”)
- No extracellular "Wnt signal"
- β-catenin targeted for proteosomal degradation by its incorporation into a “destruction complex” comprising:
- APC and axin assemble into a structural scaffold, allowing the phosphorylation of the N-terminal of β-catenin by the coordinated action of CK1 and GSK-3β
- β-catenin is recognised by E3 ligase β-Trcp, ubiquinated and subsequently degraded by the 26S proteosome 
- Level of β-catenin in the cytosol is low, and therefore does not reach the nucleus at a level sufficient enough to affect transcription
- No transcription of Wnt target genes
In the presence of Wnt (“on state”)
- Extracellular “Wnt signal” binds to a cell surface G-protein coupled receptor of the “Frizzled” (FRZ) family
- Low-density lipoprotein receptor-related protein (LRP) family functions as a transmembrane co-receptor for FRZ
- Activation (phosphorylation and poly-ubiquination) of proteins of the “Dishevelled” (Dsh) family
- With the complex now interrupted, β-catenin resists ubiquination and reaches the nucleus in increased levels where it functions as a transcriptional enhancer alongside TCF
- TCF promotes binding of RNA polymerase to DNA template strand
- Induction of the downstream transcription of Wnt target genes, for example:
- In a tumour cell, the Wnt siganalling pathway is activated abberantly either:
- Innappropriately 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 
Role of Wnt/β-catenin signalling in embryonic development
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.
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.
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).||
For a comprehensive list of human diseases associated with mutations of the Wnt signalling components, please refer to Table 1 in the following article: Wnt/β-catenin signaling: components, mechanisms, and diseases
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 success of NSAIDs was measured, indicating a "40-50% reduction in mortality due to CRC in indicidulas taking NSAIDs". Furthermore, studies are being conducted into the use of NSAIDs to inhibit coclooxygenase-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 .
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 FRZ/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.
Dissecting Molecular Differences between Wnt Coreceptors LRP5 and LRP6
The current understanding of the Wnt signalling pathway is that the LRP5/6 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 LRP6 co-receptors and the individual processes they are responsible for. By investigating the molecular basis for these differences, it has been demonstrated that the LRP6 coreceptor plays a predominant role in embryogenesis and also exhibits a stronger signalling activity in vivo than the LRP5 isotype.
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 inflammation; 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 LRP6 co-receptor, which blocks initiation of the pathway by the Wnt ligand by preventing activation of the FRZ 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 LRP6, 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.
- Article links: Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling | Dissecting Molecular Differences between Wnt Coreceptors LRP5 and LRP6 | Identification of a Novel Inhibitor of the Canonical Wnt Pathway | Inhibition of GSK3 by Wnt signalling – two contrasting models
- What is the mode of translocation of β-catenin into the nucleus upon dissociating with axin?
- Elucidate the mechanism for the hyperphosphorylation of ‘‘‘Dsh’’’ by Wnt/’‘‘Fz /LRP-5/6 binding concurrent to, but mutually exclusive from, axin destabilisation
- The biochemical events connecting the components of the canonical Wnt pathway
- Production of purified Wnt proteins to assist with research
- Production of effective Wnt antibodies
- Structure of the Wnt1 protein
- Complete understanding of the routing and the coincident posttranslational modifications of Wnt proteins in the secreting cell
- The rules that dictate the movement of Wnt proteins between cells
- The biochemistry of the activities of the destruction complex
Taken from Wnt signaling: a common theme in animal development - the research outlined will be explored in the current literature
- How do the ‘‘‘Fz’’’ receptors work?
- What is ‘‘‘Dsh’’’ doing to transduce the signal?
- What is the relationship between ‘‘‘APC’’’ and Wnt signaling?
- How does Arm/β-catenin get into the nucleus?
List of Abbreviations Used
- APC: adenomatous polyposis coli
- CAMKII: Ca2+/calmodulin-dependent protein kinase II
- CK-I: casein kinase I
- CRD: cysteine rich domain
- Dsh: Dishevelled
- Fz: Frizzled
- GSK-3β: glycogen synthase kinase-3β
- HMG: high mobility group
- LEF: lymphoid enhancer-binding factor
- LRP: lipoprotein receptor–related protein
- Nkd: naked cuticle
- 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
- 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
- Homeotic gene: genes involved in embryonic development, specifically controlled the anterior-posterior axis
- Histone deacetylation: the process by which histone deacetylase removes acetyl groups on the histones to increase affinity and condense DNA, thus preventing transcription
- 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
- 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
- Pluripotency: the ability of stem cells to differentiate into ectoderm, mesoderm and endoderm
- 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
- Proteasome: a large intracellular particle which degrades proteins
- Repressor: a protein which binds to the operator and prevents transcription
- Self renewal: the capability to undergo numerous cell divisions and maintain undifferentiation
- Sequester: to isolate or separate
- Scaffolding protein: proteins which regulate signal transduction by organising and assembling signalling complexes
- 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