User:Kinkreet/Wnt - 2nd. ed.

Wnt (for Wingless and Int-1) signalling is a signalling pathway conserved in many species, and in both vertebrates and invertebrates, which have diverse functions both in development and in homeostasis.

There are three Wnt signalling pathways:
 * 1) Canonical Wnt/β-catenin pathway
 * 2) Non-canonical planar cell polarity (PCP) pathway
 * 3) Wnt/Ca2+ pathway

Functions
There are about 20 distinct Wnt proteins in humans, separated into 12 sub-families. The functions of Wnt is diverse. One Wnt signal can bind to many different receptors and thus able to activate multiple pathways; this is known as pleiotropy.

Development
In embryogenesis, it acts mainly as a morphogen for development. Morphogens are molecules which induces different cellular activity dependent on its concentration. It is important in pattern formation along axis; for example, wild-type Drosophila develops a cuticle (the hard exoskeleton that covers the epidermis) which is segmented, with alternating portions of naked belts and denticles. Mutations in three genes involved in the Wnt pathways - porcupine, dishevelled and armadillo genes (of which their functions will be described later) - lead to the phenotype where the flies produced cuticles consisting of only denticles. Likewise, mutations of the shaggy/zeste-white 3 gene lead to a naked cuticle.

A specific example can be found in Wnt5, where it has been shown to be required for axon guidance in the central nervous system.

Crypt stem cells
The cells that line the inner walls of the gut contain villi, which increases the surface area for efficient absorption. At the base of the villi are crypts, which contains slowly dividing stem cells (>24 hours cycle time) that self-renew as well as producing progenitors (>12 hours cycle time) which migrates upwards that replaces cells which are shed near the tip of the villi. The time it takes for cells to move from the bottom of the crypt to being shed at the top of the villus is about 3-5 days.

The primary signalling pathway involved in crypt formation is Wnt, as the down-regulation of Wnt using Dkk-1 (an inhibitor of Wnt signals) led to the loss of crypts; and the up-regulation of Wnt using R-spondin-1 lead to the hyperproliferation of crypts. Wnt is involved in causing the proliferation of progenitors and thus its absence will lead to terminal differentiation. Furthermore, the down-regulation of TIF4 lead to the absence of the progenitor section of the crypt, although the differentiated cells were unaffected.

At the crypt base, Wnt, hedgehog and maybe other signals induces bone morphogenetic protein 4 (BMP4) to be expressed in the villus core ('upwards' of the crypt base). The BMP4 in turn inhibit Wnt and hedgehog signalling further up the villus, and so the cells will not proliferate, and so preventing them from turning into a crypt. Patients with BMP4 mutations may develop Juvenile Polyposis Syndrome (JPS), characterized by predisposition to hamartomatous polyps (growths, like tumours found in organs as a result of faulty development) in the gastrointestinal (GI) tract. This is due to ectopic crypts forming further up the villus, because BMP4 signal is blocked.

At the crypt base, Wnt keeps gut stem cells dividing and induces Notch expression. Notch, in turn, along with hedgehog, induces differentiation where when one cell expressing a signal would inhibit nearby cells to have the same cell fate. As a result, the villus is lined with absorptive cells, with regularly-spaced secretory cells.

Cells at the crypt base expresses EphB and those up the crypt expresses ephrin. Ephrin and EphB repulse each other, and so this signalling keeps the cells fo the crypt base at the crypt base, and prevent those further up the villus from migrating down. This signal is also controlled by Wnt signalling. Similarly, the position of the panenth cells are also determined by Wnt signalling.

To emphasize, Wnt is involved in the proliferation and fate determination of crypt stem cells, therefore, dysregulation of this pathway can lead to cancer. Mutations in APC or axin, the proteins which exports β-catenin out of the nucleus, leads to a rare condition called familar adenomatous polyposis (FAP). If one of the PAC genes are mutated, it usually leads to the formation of a polyps, or a benign adenoma; this will develop into a malignant adenocarcinoma later on. Mutations in both the APC genes leads to sporadic colorectal cancer. Similar results are also observed where the phosphorylation site (S/T) of β-catenin is mutated, meaning it cannot be degraded and accumulates in the nucleus, inducing transcription.

Bulge stem cells
Bulge stem cells are multipotent epidermal stem cells in the bulge region of hair follicles. They are capable of forming all cells of the hair lineage, as well as sabaceous gland cells and interfollicular epidermis. Very similar to crypt cells, Wnt is essential for follicle formation: the absence of Lef1 reduced the number of hair follicles whereas increasing β-catenin levels increased the number of follicles. The up-regulation of Lef1 allows de novo follicle formation. Here, the gene that TCF binds include multiple keratin genes, which is the primary protein in hair. Wnt signalling is only observed for development along the hair lineage, and thus Wnt signalling is thought to commit bulge stem cells to the hair lineage, in place of sebaceous or epidermal lineages.

Transgenics with constitutively active β-catenin presents pilomatricoma-like lesions, and tamoxifen-induced overexpression of β-catenin lead to another hair follicle tumour called trichofolliculoma.

Haematopoietic stem cells
in vitro, soluble Wnt proteins promoted the proliferation and inhibited differentiation of murine HSCs. Overexpression of β-catenin ensured the self-renewal of murine and human HSCs. Wnt is, like in crypt cells, involved in the fate determination of HSCs. Organisms lacking in Tcf1 or Lef1 lead to a lack of the earliest thymocyte progenitor.

Bone
The generation of bone tissue is a dynamic process in which osteoblasts produce bone matrix, and osteroclasts that remove these tissues by demineralizing the cells as well as breaking down the organic bone matrix (90% collagen). Wnt promotes the production of bone tissues by osteoblasts. Up-regulation of Wnt produced bones with more mass; and the lack of SOST/Sclerostin, an inhibitor of Wnt signals, lead to increased bone mass.

S-Palmitoylation
After translation by ribosomes, immature Wnt peptides enter into the endoplasmic reticulum (ER), where they are S-palmitoylated by acyl transferases - porcupine (in Drosophila) or mom-1 (in worms) - on conserved cysteine residues. This palmitoylation makes the Wnt signal hydrophobic, which is important for its function, as it allows the Wnt signal to better associate with the membrane of its target cells, and thus stabilize the interaction between Wnt and its receptors.

Exocytosis
The Wnt peptide also contains a N-terminal signal peptide, which signals it for secretion. It first must pass through the Golgi and into vesicles which directs them to the plasma membrane, where the vesicle fuses with the membrane and Wnt is released into the extracellular space.

Although its exact role is not clear, a 7-pass transmembrane protein wls/evi (wntless/evenness Interrupted, in Drosophila) or mom-3 (in worms) and hWLS (in humans), localized in the Golgi apparatus, is required for secretion. The retromer, a complex that is known to be required for recycling and trafficking from endosomes back to the trans-Golgi, is also required.

Delivery
Wnt, fitting with its nature as a morphogen, is delivered to the target cell using paracrine signalling. The actual mode of delivery can vary:
 * Diffusion - Because of the hydrophobicity of Wnt signals, they cannot diffuse easily in the extracellular fluids, and thus usually stay on or near membranes. It may be stabilized by lipoprotein particles/molecules, such as heparan sulfate proteoglycans.
 * Intercellular vesicles
 * Cytonemes - Cytonemes are long and thin tubes formed from the plasma membrane that connect different animal cells over long distances. It can carry components of the cytoplasm between cells, such as vesicles and organelles.

Reception
The Wnt signal associates with the Frizzled (Fz) receptor and also with Arrow (in Drosophila) or LRP5/6 (in vertebrates). Fz is a 7-pass transmembrane receptor, whereas LRP5/6 is a single-pass protein. LRP5/6 is transported to the plasma membrane using chaperon proteins Boca (in Drosophila) or Mesd (in mice).

Interactions
Most commonly, the Wnt signal binds to the cysteine-rich domain (CRD) of Frizzled (Fz), a 7-pass transmembrane receptor, and initiate the canonical pathway. However, Wnt can also bind other molecules, which can inhibit or promote Wnt signalling.

To Wnt
Wnt inhibitor factors (WIFs) and soluble Frizzled-related proteins (SFRPs) can both bind extracellularly to Wnt signals, this prevents them from binding to the Frizzled receptor and thus down-regulate Wnt signalling. However, it can also be the case that WIF and SFRP binds and stabilize Wnt signals, and thus up-regulate Wnt signalling.

To Fz/LRP

 * Dickkopf (Dkk) binds to LRP5/6 and crosslinks it to Kremens, a transmembrane molecule. Binding causes the internalization of LRP5/6 and prevent Wnt binding.
 * Wise also bind to LRP5/6

To Wnt
There are two alternative receptors to Fz to which Wnt signals can bind to:
 * Derailed (in Drosophila) or RYK (in vertebrates) - a member of the receptor tyrosine kinase family, which binds to Wnt using its WIF domain. In humans, it can be viewed as a co-receptor to Fz.
 * Ror

To Fz/LRP

 * Norrin, a cysteine-knot protein can also bind to Fz4 and initiate the canonical pathway
 * R-spondins can also activate the canonical pathway

Mechanism
There are two modes in Wnt signalling - on or off - presence or absence of a signal.

Absence
In the absence of a signal, the scaffolding protein axin, the kinases GSK-α/-β and CK1α/-δ/-ε interact in the cytosol to form a degradation complex for β-catenin, the effector of the canonical pathway. APC also associate with the complex, but it is dispensable, as mutants lacking APC can still signal using Wnt, as long the levels of axin was still high. This suggests that axin is essential for the formation of the degradation complex, and APC stabilizes it, aid in the loading/unloading of, or act as a secondary scaffolding protein. Because CK1, Dsh, βTrCP and GSK3 are also involved in other pathways, a low level of axin is essential to ensure the activation of these other pathways do not activate Wnt signalling, because axin is unique to Wnt signalling.

When the degradation complex is formed, β-catenin is phosphorylated on specific serine/threonine residues near its N-terminus, first by CK1α/-δ/-ε in the priming step, then subsequently GSK-α/-β on a different site. The F box/WD repeat protein βTrCP is a component of the E3 ubiquitin ligase complex, and it recognizes the phosphorylated β-catenin and induces it ubiquitination. The ubiquinated β-catenin is then transported to the proteosome for degradation.

In the nucleus, a complex of Groucho/Grg/TLE proteins inhibits the transcription of Wnt target genes. β-catenin act by physically displacing Groucho and initiating transcription. Therefore, if β-catenin is degraded in the cytosol, transcription remains inhibited. APC is also thought to involved in repression.

Presence
The Wnt protein binds to both Fz and LRP5/6 (whether Fz and LRP5/6 interact is not clear) and each receptor then recruits their own intracellular signalling proteins. LRP5/6 recruits CK1-γ and GSK3; CK1-γ is unique amongst the CK1 seen in the degradation complex CK1α/-δ/-ε, because CK1-γ has a palmitoyl group that allows it to tether to the plasma membrane. First GSK3 phosphorylates the cytoplasmic tail of LRP5/6 at a conserved PPP(S/T)P site; this creates a docking site for axin. Caseine kinase 1-γ then also phosphorylates at multiple sites surrounding the PPP(S/T)P site. Note the order of phosphorylation is the reverse order to the phosphorylation of β-catenin in the degradation complex; it is also unclear which phosphorylation reaction (perhaps both) is controlled by Wnt binding. Axin is essential for the formation and continual existence of the degradation complex; when Wnt signal is present, it is pulled to the plasma membrane by the creation of the docking site; this means it would be unavailable to form the degradation complex in the cytosol, and so a Wnt signal leads to the non-degradation of β-catenin.

Fz recruits and phosphorylates Dishevelled (Dvl), although the function of Dishevelled is not clear, it may be involved in recruiting or stabilizing axin binding to the cytoplasmic tail of LRP5/6, as we described.

β-catenin can act in two ways within the cell - as a protein which associates with the cytoplasmic tail of cadherins near the plasma membrane, or as cytoplasmic β-catenin involved in Wnt signalling. The pool of β-catenin associated with cadherins cannot be removed and thus stable, and so it is thought that β-catenin first fill up this pool, before filling in the unstable pool of the Wnt pathway. Whether the two uses of β-catenin are interlinked is unclear.

The build up of β-catenin causes more β-catenin to move into the nucleus, simply by its increased concentration. It may also be that a nuclear localization mechanism exists, but this has been shown to be independent of the nuclear localization signal/importin pathway. β-catenin resembles importin and can bind to the nuclear pore complex itself for import.

β-catenin can then move into the nucleus and displaces Groucho, binding with lymphoid enhancer factor/T cell factor (LEF/TCF) transcription factors to activate transcription of Wnt target genes. The CBD near the N-terminus of TCF binds to β-catenin while the Hmg domain near the C-terminus binds to the minor grooves of the target genes at conserved AGATCAAAGG sequences. Upon binding, TCF binds the DNA by 90°. There are four different TCF, and there are much redundancy.

The N-terminus of β-catenin also binds to Bcl9, which is the linker molecule in the trimeric complex of β-catenin/Bcl9/Pygopus; the trimer ensures retention of β-catenin in the nucleus. The retention of β-catenin is countered by the export by binding with axin or APC.

The C-terminus of β-catenin is bound by other activating factors - CBP and Brg-1 are histone acetylases that acetylate histone to remodel chromatin to the open comformation; homologs of yeast Cdc37 (Parafibromin in humans, and Hyrax in flies) is a component of the PAF complex, and interacts with RNA polymerase II to regulate transcription and initiation and elongation. Together, these three proteins open up chromatin and recruits RNAP to initiate transcription.

The genes that are induced depends largely on the temporal and spatial coordinate of the cell, as well the type of tissue the cell is in. For example, in embryogenesis, it has a morphogenic role, whereas in adults, it has a homeostatic and fate-determining role; hair follicle cells react to Wnt differently to gut crypt stem cells. However, the effect of the Wnt signal depends more on the receiving cell than the Wnt signal itself. As to the actual genes which are induced, it includes both positive (LRP, heparan sulfate, LCF/Lef and Frizzled) and negative (Axin2) proteins of the Wnt pathway, and oncogenes (cMyc and cyclin D). cMyc encodes for transcription factors that induces transcription of cell proliferation genes; cyclin D is involved in regulating the G1/S phase transition in the cell cycle. Mutations in these oncogenes leads to proliferation of cells caused by uncontrolled mitosis, and cause cancer.