Wnt signaling pathway

The Wnt signaling pathway is a network of proteins best known for their roles in embryogenesis and cancer, but also involved in normal physiological processes in adult animals.

Discovery
The first Wnt gene was discovered by Roeland Nusse and Harold Varmus in 1982 when they observed activation of Int1 (integration 1) in the breast tumors of mice infected with mouse mammary tumor virus (MMTV), in which Int was identified as a vertebrate gene near several integration sites of MMTV. The origin of the name Wnt comes from a hybrid of Int and Wg (wingless) in Drosophila, which is the best characterized Wnt gene. Although the wingless gene was originally identified as a recessive mutation affecting wing and haltere development in Drosophila melanogaster, wingless has been important towards understanding the Wnt signaling system in other organisms, and its function as a segment polarity gene was discovered by Christiane Nüsslein-Volhard and Eric Wieschaus through their work on Drosophila mutants with phenotypes related to mutations in the Wg signaling pathway. As a result of their work, they later went on to win the Nobel Prize in Physiology and Medicine in 1995. It was not until 1987 that researchers found that Wg was the homologue to mammalian Int1 due to a common evolutionary origin evidenced by similar amino acid sequences of their encoded proteins.

Mutations of the wingless gene in the fruit fly were found in wingless flies, while tumors caused by MMTV were found to have copies of the virus integrated into the genome forcing overproduction of one of several Wnt genes. The ensuing effort to understand how similar genes produce such different effects has revealed that Wnt proteins are a major class of secreted morphogenic ligands of profound importance in establishing the pattern of development in the bodies of all multicellular organisms studied.

Members
The following is a list of human genes that encode WNT signaling proteins:
 * WNT1
 * WNT2, WNT2B
 * WNT3, WNT3A
 * WNT4
 * WNT5A, WNT5B
 * WNT6
 * WNT7A, WNT7B
 * WNT8A, WNT8B
 * WNT9A, WNT9B
 * WNT10A
 * WNT10B, WNT11
 * WNT16

Wnt signaling proteins
The Wnt proteins are a group of secreted lipid-modified (palmitoylation) signaling proteins of 350-400 amino acids in length. Following the signal sequence, they carry a conserved pattern of 23-24 cysteine residues, on which palmitoylation occurs on a cysteine residue. These proteins activate various pathways in the cell that can be categorized into the canonical and noncanonical Wnt pathways. Through these signaling pathways, Wnt proteins play a variety of important roles in embryonic development, cell differentiation, and cell polarity generation.

Mechanism
The Wnt pathway involves a large number of proteins that can regulate the production of Wnt signaling molecules, their interactions with receptors on target cells and the physiological responses of target cells that result from the exposure of cells to the extracellular Wnt ligands. Although the presence and strength of any given effect depends on the Wnt ligand, cell type, and organism, some components of the signaling pathway are remarkably conserved in a wide variety of organisms, from Caenorhabditis elegans to humans. Protein homology suggests that several distinct Wnt ligands were present in the common ancestor of all bilaterian life, and certain aspects of Wnt signaling are present in sponges and even in slime molds.

The canonical Wnt pathway describes a series of events that occur when Wnt proteins bind to cell-surface receptors of the Frizzled family, causing the receptors to activate Dishevelled family proteins and ultimately resulting in a change in the amount of β-catenin that reaches the nucleus (Figure 2). Dishevelled (DSH) is a key component of a membrane-associated Wnt receptor complex (Figure 2), which, when activated by Wnt binding, inhibits a second complex of proteins that includes axin, GSK-3, and the protein APC (Figure 1). The axin/GSK-3/APC complex normally promotes the proteolytic degradation of the β-catenin intracellular signaling molecule. After this "β-catenin destruction complex" is inhibited, a pool of cytoplasmic β-catenin stabilizes, and some β-catenin, is able to enter the nucleus and interact with TCF/LEF family transcription factors to promote specific gene expression (interaction 2, Figure 2).

Cell surface Frizzled (FRZ) proteins usually interact with a transmembrane protein called LRP (Figure 2). LRP binds Frizzled, Wnt and axin and may stabilize a Wnt/Frizzled/LRP/Dishevelled/axin complex at the cell surface ("receptor complex" in Figure 2).

In vertebrates, several secreted proteins have been described that can modulate Wnt signaling by either binding to Wnts or binding to a Wnt receptor protein. For example, Sclerostin (not shown in a figure) can bind to LRP and inhibit Wnt signaling.

The part of the pathway linking the cell surface Wnt-activated Wnt receptor complex to the prevention of β-catenin degradation is still under investigation. There is evidence that trimeric G proteins (G in Figure 2) can function downstream from Frizzled. It has been suggested that Wnt-activated G proteins participate in the disassembly of the axin/GSK3 complex.

Several protein kinases and protein phosphatases have been associated with the ability of the cell surface Wnt-activated Wnt receptor complex to bind axin and disassemble the axin/GSK3 complex. Phosphorylation of the cytoplasmic domain of LRP by CK1 and GSK3 can regulate axin binding to LRP (interaction 1 in Figure 2). The protein kinase activity of GSK3 appears to be important for both the formation of the membrane-associated Wnt/FRZ/LRP/DSH/Axin complex and the function of the Axin/APC/GSK3/β-catenin complex. Phosphorylation of β-catenin by GSK3 leads to the destruction of β-catenin (Figure 1).

Feedback regulation of canonical Wnt signaling occurs via a variety of mechanisms, including induction or repression of Wnt signaling components themselves, including AXIN2 and Naked cuticle.

Ligands that act on Wnt signaling

 * 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine as an agonist of Wnt signaling.


 * The signaling molecule Cerberus inhibits Wnt, thus repressing the inhibition of β-Catenin on SoxB1 family members. This enables the specification of Neuroepithelium in Drosophila neural induction.

Wnt-induced cell responses
Several important effects of the canonical Wnt pathway include:


 * Cancers. Alterations of Wnts, APC, axin, and TCFs are all associated with carcinogenesis.


 * Body axis specification. Ectopic placement of Wnt in Xenopus eggs during early gastrulation gives rise to a secondary body axis and head, while inhibition of Wnt signaling results in a lack of dorsal structures in the frog embryo. Wnt is extensively involved in the formation of dorsal structures and the nervous system in early frog development and is found in high concentrations in the region known as Spemann's Organizer.


 * Morphogenic signaling. Wnts produced from specific sites, such as the edge of the developing fly wing or the dorsal region of the neural tube of the developing vertebrate, are distributed throughout adjacent tissues in a gradient fashion.  The Wnt pathway becomes activated to different degrees in cells of these tissues depending on how close they are to the production site, leading to subtle but crucial differences in the level of genes regulated by the Wnt pathway.

Wnt and patterning the neural tube
In vertebrates, dorso-ventral patterning of the developing neural tube is achieved by the counteracting activities of morphogenetic signaling gradients set up by Sonic Hedgehog (Shh) in the ventral floor plate and notochord, and the canonical Wnt/β-catenin pathway acting in the roof plate, the dorsal most region of the neural tube. While evidence that Wnt and Sonic hedgehog are direct antagonists of one another remains to be seen, the role of Wnt in patterning the neural tube is thought to work in an indirectly inhibitory manner towards Sonic Hedgehog via the canonical Wnt pathway. Studies in early neural tube development have shown that the Wnt/β-catenin pathway is largely responsible for regulating Shh expression in the dorsal region of the neural tube. Addition of the GSK3 inhibitor LiCl, which stabilizes β-catenin by preventing its destruction, has been shown to attenuate Sonic Hedgehog response in neural tube explants in chicks. Chick electroporation assays have shown that ectopic activation of the Wnt/β-catenin pathway components results in an expansion of dorsal genes, Pax7 and Pax6, into more ventral regions of the neural tube. These regions become dorsalized due to the ectopic presence of the active Wnt components, which are thought to inhibit the expression of genes such as Oligo2 and Nkx2.2 that are normally found there. Furthermore, misexpression of activated canonical Wnt pathway components β-catenin and TCF-lef results in complete dorsalization of the neural tube. Inhibition of Wnt signaling in the neural tube by introduction of a dominant-negative inactive form of TCF, the transcription factor activated by the Wnt/β-catenin pathway, results in shift of ventral genes into more dorsal regions of the neural tube.

Wnt signaling in the dorsal region of the neural tube also controls the expression of a transcription factor Gli3, one of the main inhibitors of the Shh/Gli pathway. It is by signaling through the canonical Wnt/β-catenin pathway that Wnt is able to activate and control the expression of the Gli3 transcription factor to repress transcriptional activity of Shh/Gli in the dorsal region of the neural tube and elicit dorsal cell fates.

In addition to Wnt and Shh signaling, studies have shown that Bone Morphogenetic Proteins (Bmp) are also necessary for Shh regulation in the dorsal neural tube, and, because of cross-talk between the Bmp and Wnt pathways, it was thought that Bmps were regulating the activities of Wnt. Recent studies have shown that Wnt may not be mediated by Bmps, but rather that Bmps may be mediated by Wnts. However, The interactions of the Wnt and Bmp pathways remain unclear and further research needs to be done to identify exactly how Bmps and Wnts work together to elicit dorsal cell fates in the developing neural tube.

Non-canonical Wnt signaling
There are many non-canonical pathways, but the two best-studied pathways are the Planar Cell Polarity (PCP) and Wnt/Calcium Pathways. The most distinctive differences between the canonical and non-canonical pathways include the specific ligands activating each pathway, ß-Catenin, LRP5/6 co-receptor, and Dsh-DEP domain independence, and the ability of the non-canonical pathway to inhibit the canonical pathway. Ligands that activate the non-canonical pathways are Wnt4, Wnt5a, and Wnt11.

In the PCP pathway, ligand binding to the receptor recruits Dishevelled (Dsh), which forms a complex with Daam1. Daam1 then activates the small G-protein Rho through guanine exchange factor. Rho activates ROCK ( Rho-associated kinase), which is one of the major regulators of the cytoskeleton. Dsh also forms a complex with rac1 and mediates profilin binding to actin. Rac1 activates JNK and can also lead to actin polymerization. Profilin binding to actin can result in restructuring of the cytoskeleton.

In the Wnt/Calcium pathway, Wnt5a and Frizzled regulate intracellular calcium levels. Ligand binding causes the coupled G-protein to activate PLC, leading to the generation of DAG and IP3. When IP3 binds to its receptor on the ER, intracellular calcium concentration increases. Ligand binding also activates cGMP-specific phosphodiesterase (PDE), which depletes cGMP and further increases calcium concentration. Increased concentrations of calcium and DAG can activate Cdc42 (cell division control protein 42) through PKC. Cdc42 is an important regulator of cell adhesion, migration, and tissue separation. Increased calcium also activates calcineurin and CamKII (calcium/calmodulin-dependent kinase). Calcineurin induces activation of transcription factor NFAT, which regulates ventral patterning. CamKII activates TAK1 and NLK kinase, which can interfere with TCF/ß-Catenin signaling in the canonical pathway.

Planar cell polarity
An example of the control of planar cell polarity in insects like Drosophila is determining which direction the tiny hairs on the wings of a fly are aligned. Planar cell polarity is distinct from and perpendicular to apical/basal polarity. The signaling pathway that is involved in planar cell polarity includes frizzled and dishevelled but not the axin complex proteins. The non-classical cadherins Fat, Dachsous, and Flamingo appear to modulate frizzled function. Other proteins including prickle, strabismus, rhoA, and rho-kinase act downstream of frizzled and dishevelled to regulate the cytoskeleton and planar cell polarity.

Some of the proteins involved in planar cell patterning of the Drosophila wing are used in vertebrates during regulation of cell movements during events such as gastrulation. A common feature of both hair patterning in Drosophila and cell movements such as vertebrate gastrulation is control of actin filaments by G proteins such as Rho and Rac.

Axon guidance
Wnt has some diverse roles in axon guidance. For example, the Wnt receptor Ryk is required for Wnt mediated axon guidance on the contralateral side of the corpus callosum. Another example is in the growing spinal cord commissural neurons: after their extending axons cross the midplate of the spinal cord, they are guided by a Wnt gradient, which is active through the Frizzled receptors in this case.

Stem cells
Traditionally, it is assumed that Wnt proteins can act as Stem Cell Growth Factors, promoting the maintenance and proliferation of stem cells.

However, a recent study conducted by the Stanford University School of Medicine revealed that Wnt appears to block proper communication, with the Wnt signaling pathway having a negative effect on stem cell function. Thus, in the case of muscle tissue, the misdirected stem cells, instead of generating new muscle cells (myoblasts), differentiated into scar-tissue-producing cells called fibroblasts. The stem cells failed to respond to instructions, actually creating wrong cell types.

Understanding the mechanisms by which pluripotency, self-renewal, and subsequent differentiation are controlled in embryonic stem cells is crucial to utilizing them therapeutically. In addition, control of Wnt signaling may allow for minimizing the use of animal products, which can introduce unwanted pathogens, in stem cell cultures. Wnt signaling was first identified as a potential component to differentiation because of its established role in development. Recent research has supported this hypothesis. There are data to suggest that Wnt signaling induces differentiation of pluripotent stem cells into mesoderm and endoderm progenitor cells.

There are several pieces of evidence to suggest that Wnt signaling is important in stem cell differentiation. TCF3, a transcription factor regulated by Wnt signaling, has been shown to repress nanog, a gene required for stem cell pluripotency and self-renewal. Over expression of another gene associated with pluripotency, OCT4 leads to increased beta-catenin activity, suggesting Wnt involvement.

Studies of embryoid bodies (see embryoid body) have led to new insights regarding the role of Wnt signaling in human embryonic stem cells. Researchers at Stanford School of Medicine observed that embryoid bodies spontaneously begin gastrulation. They determined that gastrulation in embryoid bodies mimics the in vivo process in human embryos; in vivo gastrulation has been previously linked to the Wnt pathway. Formation of the primitive streak in particular was associated with localized Wnt activation in the embryoid bodies. Once the Wnt pathway is activated, it is self-reinforcing. It is unclear, however, what induces the initial Wnt signaling that begins gastrulation.

Research published in the Journal of Biological Chemistry has suggested that activation of the Wnt pathway in mouse embryonic stem cells induces differentiation into multipotent mesoderm and endoderm cells. This study showed that upon inducing Wnt signaling in mono-layer embryonic stem cell cultures, the cells express high levels of markers associated with mesoderm development, particularly T-brachyury and Flk-1. The cells also expressed high levels of Foxa2, Lhx1, and AFP, which are associated with endoderm development. The progenitor cells created via Wnt activation seemed to have particularly high potential to differentiate into bone and cartilage. The researchers suggested that beta-catenin plays an important role in skeletal development. They demonstrated that the progenitor cells could also develop into endothelial, cardiac, and vascular smooth muscle lineages.

A publication from the American Society of Hematology extended the previous study to human embryonic stem cells (hESCs) by demonstrating that Wnt signaling can induce hematoendothelial cell development from hESCs. This study showed that Wnt3 leads to mesoderm committed cells with hematopoietic potential. Overexpression of Wnt1 led to faster, more efficient hematoendothelial differentiation than Wnt3 overexpression. Wnt1 has also been shown to antagonize neural differentiation; this observation suggests a variety of roles for the Wnt pathway in stem cell activity. In contrast to Wnt3, which is associated with mesoderm and endoderm differentiation, Wnt1 serves the opposite function in neural stem cells. Wnt1 appears to be a major factor in self-renewal of neural stem cells. Wnt stimulation is also associated with regeneration of nervous system cells, which is further evidence of a role in promoting neural stem cell proliferation.

Environmental enrichment
Changes in Wnt signaling mimic in adult mice the effects of environmental enrichment upon synapses in the hippocampus with regard to reversible increase in their numbers, and spine plus synapse densities at large mossy fiber terminals It seems that Wnt signaling might be part of the means by which experience regulates synapse numbers and hippocampal network structure.

Wnt pathway in cancer stem cells
The canonical and non-canonical pathways show that the Wnt pathway is strictly regulated by many different elements. Thus, it can be expected that misregulation of these various components can have drastic effects throughout an organism. One interesting aspect of the Wnt pathway in particular is its involvement in the fate of stem cells and its role as a regulator of stem cell choice to proliferate or self-renew. Given these properties, it is not surprising that a strong correlation between Wnt signaling and the onset of cancer exists. In the normal pathway, APC and Axin prevent β-catenin from traveling to the nucleus by engaging it in the destruction complex. However, an APC deficiency or mutations to β-catenin that prevent its degradation can lead to excessive stem cell renewal and proliferation, predisposing the cells to the formation of tumors. Alteration of Wnt5a, a tumor suppressor gene, could also lead to tumor formation.

One of the potential ways to treat cancer is to affect β-catenin, a central component of the canonical Wnt pathway. Non-steroidal anti-inflammatory drugs (NSAIDs) that interfere β-catening signaling have been shown to be promising for the prevention of colorectal cancer. NSAIDs inhibit prostaglandin production, which interferes with β-catenin/TCF-dependent transcription. Another suggested method of treatment is to use natural antagonists of the Wnt pathway, such as secreted frizzled-related proteins (sFRPs) or Dkk. Furthermore, using small molecules to block the interaction between β-catenin and TCF could stop the proliferation of cancer. Researchers have also developed a recombinant adenovirus (Ad-CBR) that constitutively expresses the β-catenin binding domain of APC. This enables the tumor suppressor activity of APC, thus preventing β-catenin translocation to the nucleus. Scientists are also using monoclonal antibodies against Wnt proteins to induce apoptosis in cancer cells. Currently, there are many investigations underway to target various components of the Wnt pathway as a means of finding more effective treatments for cancer.