Wnt signaling pathway Edit Wikipedia article

Figure of the three main pathways of Wnt signaling in biological signal transduction.

The Wnt signaling pathway is a network of proteins that passes signals from receptors on the surface of the cell through the cytoplasm and ultimately to the cell's nucleus where the signaling cascade leads to the expression of target genes. It controls cell-cell communication in the embryo and adult (ie, cell proliferation and differentiation during development and healing). The Wnt signalling pathway is an example of paracrine signalling.^[1][2]

It was identified first for its role in cancer development, and separately in creating normal patterns of embryonic development.^[3] Its role in embryonic patterning was discovered when genetic mutations in critical players in this pathway produced abnormal fruit fly embryos. Later research found that the two genes responsible for this type of breast cancer in the mouse and the abnormal patterning in the fruit fly were in the same family and part of the Wnt signaling pathway.^[3]

The Wnt signaling pathway is evolutionarily conserved, and functions across species ranging from the fruit fly to humans. It is also required for adult tissue maintenance in bone, heart, muscle, and elsewhere. Mutations in this pathway in adults contribute to degenerative diseases and cancers.^[3]

[edit] Discovery

The discovery of Wnt signaling is due largely to several lines of research that led to a better understanding of oncogenic retroviruses. Specifically, a shift in focus to the cancer-causing properties of these viruses led to the discovery of specific cancer-causing genes known as proto-oncogenes. This discovery encouraged the research of Roel Nusse and Harold Vamus in 1982, where they infected mice with the oncogenic retrovirus MMTV (mouse mammary tumor virus) to see which mouse genes would produce breast tumors upon mutation. Using a method known as proviral tagging, they were able to use this research to identify a new mouse proto-oncogene that they named int1 (integration 1).^[3]

However, due to other major mutation discoveries in human tumors at that time, int1 was mostly overshadowed. Its importance was not fully appreciated until researchers noted that it had a high degree of conservation across several species, including humans and Drosophila. Its presence in Drosophila melanogaster was particularly important because in 1987, researchers were able to determine that the int1 gene in Drosophila was actually the already characterized Drosophila gene known as Wingless (Wg). This meant that the mammalian int1 was the homologue of Wg.^[3]

The uncovering of this shared homology was an exciting discovery due to the fact that previous research by Christiane Nüsslein-Volhard and Eric Wieschaus, which would later win the 1995 Nobel Prize in Physiology or Medicine, had already elucidated the function of Wg as a segment polarity gene involved in embryonic development.^[4] Research in int1 and its signaling pathways increased because abnormalities in embryonic development mechanisms have long been implicated in cancer. Consequently, this increase in research would lead to the discovery of further genes related to int1. Since all those genes had not been activated via proviral integration, it quickly became clear that the int gene nomenclature was no longer adequate. Thus, the int/Wingless family was renamed the Wnt family and int1 became Wnt1. The name Wnt was chosen because it is a portmanteau of int and Wg and stands for Wingless-related integration site.^[3]

[edit] Wnt signaling proteins

Crystal protein structure of Wnt8 and the cysteine-rich domain of Frizzled 8.

The Wnt proteins are a diverse family of secreted lipid-modified signaling glycoproteins that are 350-400 amino acids in length.^[5] The type of lipid modification that occurs on these proteins is palmitoylation of cysteines in a conserved pattern of 23-24 cysteine residues.^[1] In Wnt signaling, these proteins act as ligands to activate the different Wnt pathways.^[6]

These proteins are also highly conserved across species.^[3] They can be found in mouse, human, Xenopus, Zebrafish, Drosophila, and many others.^[7]

[edit] Mechanism

[edit] Foundation of Wnt signaling

Wnt signaling begins when one of the Wnt proteins binds the N-terminal extra-cellular cysteine-rich domain of a Frizzled (Fz) family receptor.^[8] These receptors span the plasma membrane seven times and constitute a distinct family of G-protein coupled receptors (GPCRs).^[9] However, to facilitate Wnt signaling, co-receptors may also be required alongside the interaction between the Wnt protein and Fz receptor. Examples include lipoprotein receptor-related protein (LRP)-5/6, receptor tyrosine kinase (Ryk), and ROR2.^[6] Upon activation of the receptor and co-receptors, a signal is sent to the phosphoprotein Dishevelled (Dsh), which is located in the cytoplasm. This signal is transmitted via a direct interaction between Fz and Dsh. Dsh are present in all organisms and they all share the following highly conserved protein domains: an amino-terminal DIX domain, a central PDZ domain, and a carboxy-terminal DEP domain. These different domains are important because after Dsh, the Wnt signal can branch off into several different pathways and each pathway interacts with a different combination of the three domains.^[10]

[edit] Canonical and noncanonical pathways

The three best characterized Wnt signaling pathways are the canonical Wnt pathway, the noncanonical Planar Cell Polarity pathway, and the noncanonical Wnt/Ca2+ pathway. As their names suggest, these pathways belong to one of two categories: canonical or noncanonical. The difference between these two categories is the presence or absence of β-catenin. The canonical Wnt pathway involves β-catenin, while the noncanonical pathways operate independently of it.^[8]

Diagram of the canonical Wnt pathway without Wnt.

Diagram of the canonical Wnt pathway with Wnt.

Diagram of the noncanonical Wnt/calcium pathway.

[edit] The canonical Wnt pathway

The canonical Wnt pathway (or Wnt/β-catenin pathway) is the Wnt pathway that causes an accumulation of β-catenin in the cytoplasm and its eventual translocation into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.^[8] Without Wnt signaling, the β-catenin would not accumulate in the cytoplasm since a destruction complex would normally degrade it. This destruction complex includes the following proteins: Axin, adenomatosis polyposis coli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).^[8] It degrades β-catenin by targeting it for ubiquitination, which subsequently sends it to the proteasome to be digested.^[8] However, as soon as Wnt binds Fz and LRP-5/6, the destruction complex function becomes disrupted. This is due to Wnt causing the translocation of both a negative regulator of Axin and the destruction complex to the plasma membrane.^[6] This negative regulator becomes localized to the cytoplasmic tail of LRP-5/6. Phosphorylation by other proteins in the destruction complex subsequently binds Axin to this tail as well. Axin becomes de-phosphorylated and its stability and levels are decreased.^[6] Dsh then becomes activated via phosphorylation and its DIX and PDZ domains inhibit the GSK3 activity of the destruction complex. This allows β-catenin to accumulate and localize to the nucleus and subsequently induce a cellular response via gene transduction alongside the TCF/LEF transcription factors.^[6]

[edit] The noncanonical Planar Cell Polarity pathway

Diagram of the noncanonical Wnt Planar Cell Polarity pathway.

The noncanonical Planar Cell Polarity (PCP) pathway is one of the two Wnt pathways that does not involve β-catenin. It does not use LRP-5/6 as its co-receptor and is thought to use NRH1, Ryk, PTK7, or ROR2.^[6] As in the canonical Wnt pathway, the PCP pathway is activated via the binding of Wnt to Fz and its co-receptor. The receptor then recruits Dsh, which uses its PDZ and DEP domains to form a complex with Dishevelled-associated activator of morphogenesis 1 (DAAM1).^[6] Daam1 then activates the small G-protein Rho through a guanine exchange factor. Rho activates Rho-associated kinase (ROCK), which is one of the major regulators of the cytoskeleton.^[6] 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 and gastrulation.^[6]

[edit] The noncanonical Wnt/calcium pathway

The noncanonical Wnt/calcium pathway is the other Wnt pathway that does not stimulate accumulation of β-catenin. Its role is to help regulate calcium release from the endoplasmic reticulum (ER) in order to control intracellular calcium levels. Like other Wnt pathways, upon ligand binding, the activated Fz receptor directly interacts with Dsh and activates specific Dsh-protein domains. The domains involved in Wnt/calcium signaling are the PDZ and DEP domains.^[6] However, unlike other Wnt pathways, the Fz receptor also directly interfaces with a trimeric G-protein. This co-stimulation of Dsh and the G-protein can lead to the activation of either PLC or cGMP-specific PDE. If PLC is activated, the plasma membrane component PIP2 is cleaved into DAG and IP3. When IP3 binds its receptor on the ER, calcium is released. Increased concentrations of calcium and DAG can activate Cdc42 through PKC. Cdc42 is an important regulator of cell adhesion, migration, and tissue separation. Increased calcium also activates calcineurin and CaMKII. Calcineurin induces activation of the transcription factor NFAT, which regulates ventral patterning.^[6] CamKII activates TAK1 and NLK kinase, which can interfere with TCF/ß-Catenin signaling in the canonical Wnt pathway.^[11] However, if PDE is activated, calcium release from the ER is inhibited. PDE mediates this through the inhibition of PKG, which subsequently causes the inhibition of calcium release.^[6]

[edit] Other pathways

Along with the three pathways, Wnt signaling also regulates a number of other signaling pathways that have not been as extensively elucidated. One such pathway includes the interaction between Wnt and GSK3. During cell growth, Wnt can inhibit GSK3 in order to activate mTOR in the absence of β-catenin.^[12] However, Wnt can also serve as a negative regulator of mTOR via activation of the tumor suppressor TSC2, which is upregulated via Dsh and GSK3 interaction. During myogenesis, Wnt has been shown to use PKA and CREB to activate the genes MyoD and Myf5. Wnt has also been seen to act in conjunction with Ryk and Src to allow for regulation of neuron repulsion during axonal guidance. Wnt regulates gastrulation when CK1 serves as an inhibitor of Rap1-GTPase in order to modulate the cytoskeleton during gastrulation. Further regulation of gastrulation is achieved when Wnt uses ROR2 along with the CDC42 and JNK pathway to regulate the expression of PAPC. Dsh can also interact with aPKC, Par3, Par6, and LGl in order to control cell polarity and microtubule cytoskeleton development. While these pathways overlap with components associated with PCP and Wnt/Calcium signaling, they are considered distinct pathways because they produce entirely different responses.^[6]

[edit] Regulation

In order to insure proper functioning, Wnt signaling is constantly regulated at several points along its signaling pathways. For instance, as previously mentioned, Wnt proteins are palmitoylated. The protein porcupine has been shown to be involved in this palmitoylation process, which means that it helps regulate when the Wnt ligand is secreted by determining when it is fully formed. Secretion of Wnt protein is further controlled with proteins such as wntless and evenness interrupted and complexes such as the retromer complex. Upon secretion, the ligand can also be prevented from reaching its receptor through the binding of certain proteins such as the stabilizers Dally and glypican 3, which inhibit diffusion. At the Fz receptor, the binding of proteins other than Wnt can antagonize signaling. Specific antagonists include Dickkopf (Dkk), Wnt inhibitory factor 1 (WIF-1)^[13][14], secreted Frizzled-related proteins (SFRP), Cerebrus, Frzb, Wise, and SOST. All of these constitute inhibitors of Wnt signaling; however, other molecules have been shown to act as activators as well. For example, Norrin and R-Spondin2 have been shown to activate Wnt signaling in the absence of Wnt ligand. Interactions with between signaling pathway has also been seen to regulate the different Wnt pathways. As previously mentioned, this has been seen in the case of the Wnt/calcium pathway, which inhibits the TCF/β-catenin signaling of the canonical Wnt pathway.^[6]

[edit] Wnt-induced cell responses

[edit] Embryonic development

[edit] Neural tube patterning

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, which is the dorsal most region of the neural tube. While evidence that Wnt and Shh 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 Shh via the canonical Wnt pathway.^[15][16][17]

The balance of Wnt and Shh morphogenetic signaling gradients pattern the developing neural tube. Wnt/B-catenin pathway components emanate from the top dorsal region and activate Gli3, which suppresses Shh coming from the bottom most ventral portion of the neural tube and notochord.

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 Shh response in neural tube explants in chicks.^[16] Chick electroporation assays have shown that ectopic activation of the Wnt/β-catenin pathway components results in an expansion of the dorsal genes Pax7 and Pax6 into the 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 Olig2 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.^[15] 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.^[15]

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.^[15] 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.^[15][17]

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 since cross-talk exists 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.^[17]

[edit] 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.^[18] 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.^[18][19]

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.^[20]

[edit] 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.^[21] 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.^[22]

[edit] Stem cell differentiation

Traditionally, it is assumed that Wnt proteins can act as Stem Cell Growth Factors, promoting the maintenance and proliferation of stem cells.^[23]

However, a recent study 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.^[24]

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.^[25] 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.^[25] TCF3, a transcription factor regulated by Wnt signaling, has been shown to repress nanog, a gene required for stem cell pluripotency and self-renewal.^[26] Over expression of another gene associated with pluripotency, OCT4 leads to increased beta-catenin activity, suggesting Wnt involvement.^[27]

Studies of embryoid bodies 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.^[28] 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.^[29] 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.^[30] 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.^[25]

[edit] 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^[31] It seems that Wnt signaling might be part of the means by which experience regulates synapse numbers and hippocampal network structure.^[31]

[edit] Clinical implications

[edit] Cancer

Ever since its initial discovery, Wnt signaling has had an association with cancer. When Wnt1 was discovered, it was first identified as a proto-oncogene in a mouse model for breast cancer. Furthermore, the fact that Wnt1 is a homolog of Wg shows that it is involved in embryonic development, which often calls for rapid cell division and migration. Thus, misregulation of these processes can cause unwanted cell growth and movement, which can lead to tumor development.^[3]

Activity of the canonical Wnt pathway has been directly measured in the development of benign and malignant breast tumors. Its presence is indicated with elevated levels of β-catenin in the nucleus and/or cytoplasm, which can be detected with immunohistochemical staining and Western blotting. Furthermore, increased β-catenin expression has been strongly correlated with poor prognosis in breast cancer patients. This accumulation may be due to several factors such as mutations in β-catenin, deficiencies in the β-catenin destruction complex, overexpression of Wnt ligands, loss of inhibitors, and/or decreased activity of regulatory pathways (such as the Wnt/calcium pathway).^[32][33] Breast tumors have also been seen to metastasize due to Wnt involvement in the epithelial-mesenchymal transition (EMT). Research looking at metastasis of basal-like breast cancer to the lungs has shown that repression of Wnt/β-catenin signaling can prevent EMT, which can inhibit metastasis.^[34]

Wnt signaling has also been implicated in the development of more than just breast-type cancers. Changes in CTNNB1 expression, which is the gene that encodes β-catenin, has been seen in not just breast cancer, but also colorectal cancer, melanoma, prostate cancer, lung cancer, and several other cancer types. Increased expression of Wnt ligand-proteins such as Wnt 1, Wnt2, and Wnt7A have been observed in the development of glioblastoma, oesophageal cancer, and ovarian cancer respectively. Other proteins known to cause multiple types of cancer in the absence of proper functioning include ROR1, ROR2, SFRP4, Wnt5A, WIF1, and those of the TCF/LEF family.^[35]

[edit] Type II diabetes

[edit] See also

• Signal transduction
• Morphogenesis
• Developmental biology
• Embryogenesis
• Cancer
• Catenin
• GSK-3
• Frzb
• Wingless localisation element 3 (WLE3)
• Baldness treatments
• GPR177

[edit] External links

• Mouse Wnt proteins from Signaling Gateway Molecule Pages^[36]
• Mouse Frizzled proteins from Signaling Gateway Molecule Pages
• The Wnt Homepage from Nusse Lab, Stanford
• Wnt pathways, their relationship, disease, and therapies by healthvalue.net
• Drosophila Wnt pathway from KEGG
• mouse Wnt pathway from KEGG
• humanα Wnt pathway from KEGG
• Homo sapiens (human) Wnt pathway from KEGG
• video of grey hair reversal
• Netpath - A curated resource of signal transduction pathways in humans
• Wnt Proteins at the US National Library of Medicine Medical Subject Headings (MeSH)
• Wnt game

[edit] References