β‑catenin pathway: Searching for an activation model

Cellular Signalling 40 (2017) 30–43

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Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig

Review

Complexity of the Wnt/β‑catenin pathway: Searching for an activation model

MARK

Giovane G. Tortelotea,⁎, Renata R. Reisb, Fabio de Almeida Mendesb, Jose Garcia Abreub,⁎ a b

Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

A R T I C L E I N F O

A B S T R A C T

Keywords: Wnt β‑catenin LRP5/6 Axin Development Stem cell Cancer

Wnt signaling refers to a conserved signaling pathway, widely studied due to its roles in cellular communication, cell fate decisions, development and cancer. However, the exact mechanism underlying inhibition of the GSK phosphorylation towards β-catenin and activation of the pathway after biding of Wnt ligand to its cognate receptors at the plasma membrane remains unclear. Wnt target genes are widely spread over several animal phyla. They participate in a plethora of functions during the development of an organism, from axial specification, gastrulation and organogenesis all the way to regeneration and repair in adults. Temporal and spatial oncogenetic re-activation of Wnt signaling almost certainly leads to cancer. Wnt signaling components have been extensively studied as possible targets in anti-cancer therapies. In this review we will discuss one of the most intriguing questions in this field, that is how β–catenin, a major component in this pathway, escapes the destruction complex, gets stabilized in the cytosol and it is translocated to the nucleus where it acts as a cotranscription factor. Four major models have evolved during the past 20 years. We dissected each of them along with current views and future perspectives on this pathway. This review will focus on the molecular mechanisms by which Wnt proteins modulate β-catenin cytoplasmic levels and the relevance of this pathway for the development and cancer.

1. Introduction Wnts are a family of secreted lipid-modified glycoproteins involved with paracrine and autocrine signaling events. They are broadly studied due to their influence on processes such as embryonic development, regeneration, cancer and cellular differentiation [1–5]. The first Wnt gene mapped was found in a Drosophila melanogaster recessive mutation screening. It was characterized by segmentation defects resulting in wingless flies [6]. About a decade later, scientists investigating a model of induced mammary carcinoma in mice, driven by DNA integrations of the mouse mammary tumor virus (MMTV), identified a proviral activation of a putative mammary oncogene (int-1 for integration site 1) on mouse chromosome 15 [7]. Interestingly, analysis of the genetic mapping of the Int-1 into host DNA and the Drosophila melanogaster wingless mutation indicated that wingless and

int-1 were in fact orthologues [7,8]. Thus, the term “Wnt” was coined by the junction of the names Int-1 and wingless (Wingless + Int1 = Wnt1) [9]. Over the years, phylogenetic comparisons reviewed that Wnt genes spread across many different phyla of metazoa [10–12]. It is present and functional from simple multicellular organisms such as sponges (Phylum porifera) [13] and the fresh water polyp, Hydra (Phylum Cnidaria) [10], through to worms like planaria (Phylum Platyhelminthes) [14], insects (Phylum Arthropoda) [1,6], vertebrates, e.g. the amphibian X. leavis [2,15], and mammals including Homo sapiens [1,16]. Vertebrates have an elaborated set of Wnt genes and genes associated with Wnt signaling. Together, they are involved in several biological functions, mentioned above [1,3,16,17]. So far, 19 Wnt ligand genes have been identified in humans and mice [1,3,18]. This pathway,

Abbreviations: APC, adenomatous polyposis coli; BCL-9, B-cell lymphoma 9; BMPs, bone morphogenetic proteins; CARM, coactivator-associated arginine methyltransferase; CBP, CREBbinding protein; CK1α, casein kinase 1α; CRD, Cysteine-Rich Domain; DIX, Dishevelled interaction with axin; DKK, Dickkopf; Dvl, Dishevelled; EGF, epidermal growth factor; EVR2, Exudative Vitreoretinopathy 2 protein; GPCR, G-protein coupled receptor; GSK3-β, glycogen synthase kinase 3 beta; HDAC, histone deacetylases; LDLR, low-density lipoprotein receptor; LGR, Leucine-Rich Repeat Containing G-protein coupled receptors; LRP, low-density lipoprotein receptor-related protein; MMTV, mouse mammary tumor virus; NDP, Norrie Disease Protein; SET1, SET Domain Containing 1; sFRP, secreted Frizzled related protein; TCF/LEF, T-cell factor/lymphoid enhancer factor; TLE, transducing-like enhancer of split; β-TrCP, βtransducin repeat-containing protein; WIF1, Wnt inhibitory factor; Wnt, Int-1 + wingless; WTX, Wilms' tumor protein; WT1, Wilm's tumor protein 1 ⁎ Corresponding authors. E-mail addresses: [email protected] (G.G. Tortelote), [email protected] (J.G. Abreu). http://dx.doi.org/10.1016/j.cellsig.2017.08.008 Received 10 May 2017; Received in revised form 8 August 2017; Accepted 23 August 2017 Available online 26 August 2017 0898-6568/ © 2017 Elsevier Inc. All rights reserved.

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receptor-related protein (LRP) [35] comprises 12 genes in the mammalian genome, however, LRP5 and LRP6 seems to have a stronger and undeniable connection with the canonical Wnt signaling [1,32,38,39], as their orthologue arrow in D. melanogaster [40,41]. A role for LRP/ arrow in Wnt signaling came from genetic studies showing that mutations in arrow phenocopied the wingless phenotype in flies [41]. These observations were the basis for classifying arrow as an orthologue of LRP5/6 and for its epistatic position in the Wnt pathway [40,41]. Investigations involving knockout of either or both LRP5/6 in mice confirmed the LRP requirement for activation of the Wnt pathway [42]. A number of other studies using overexpression, deletion and domainspecific deletion of LRPs helped to elucidate their function in the canonical Wnt signaling [43]. LRPs are type I single-pass transmembrane proteins, with a long extracellular N-terminus that mediates interaction with extracellular ligands and/or antagonists of canonical Wnt signaling, and a short Cterminus that mediates interactions with intracellular proteins. The Ctermini of LRPs also contain target motifs for kinase phosphorylation thought to be important for Wnt signaling [42–44]. Arrow/LRP5/6 has a small variation in length containing 1678, 1615 and 1613 amino acid residues, respectively, with calculated molecular weights of ~200 kDa. Their extracellular region contains 4 epidermal growth factor (EGF) like domains and 3 low-density lipoprotein-related receptor (LDLR) repeats required for ligand binding [35,41]. The intracellular portion contains the proline-rich PPPSP motif, which is the sequence target of glycogen synthase kinase 3 beta (GSK3-β) phosphorylation required for activation of the β-catenin-dependent Wnt pathway [32,38,43].

however, is far more complex than suggested by the number of ligands alone. In the literature, it has been separated into 3 groups: i) the socalled canonical Wnts or β–catenin-dependent (Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8) [1,3], ii) β–catenin-independent [19,20], and iii) the non-canonical Wnt (Wnt5a, Wnt11, Wnt7a). Though some Wnts (e.g. Wnt11) do not fit this classification, because it can activate both canonical and non-canonical pathways [21,22]. The main focus of this review will be the activation mechanisms of the so-called canonical Wnt signaling pathway, recently mentioned as Wnt/LRP6 β‑catenin-mediated transcription pathway [23]. Nevertheless, β‑catenin-independent Wnt pathways should not be seem as totally separate since they may cross-talk by sharing components in several cellular processes [24–26]. Over the years, several disease-causing Wnt mutations have been extensively characterized [27–29]. This information is recorded in “The Wnt homepage” (http://web.stanford.edu/group/nusselab/cgi-bin/ wnt/human_genetic_diseases), therefore it will not be discussed in this review. Here we will present the main players of the Wnt pathway and describe four proposed models of activation of the canonical Wnt signaling: i) a classical biochemical model, in which the destruction complex is partially disrupted and retained in close proximity to the plasma membrane in its inactive form; ii) a cell biology model that describes formation of a multivesicular body that traps the destruction complex inside a double layered structure rendering it incapable of phosphorylate β-catenin thus, incapable to tag it to the proteasomemediated proteolysis pathway; iii) a model in which phosphorylated βcatenin clogs the destruction complex, which in turns, is captured to the plasma membrane and kept in its inactive form; iv) a recently proposed model, which shows the auto-inhibition of the destruction complex driven by Axin conformational changes. All four models describe distinctive mechanisms resulting accumulation of β-catenin in the cytosol, therefore activating the canonical Wnt pathway.

3. Extracellular co-activators of WNT signaling 3.1. Norrin or Norrie Disease Protein (NDP) or X-linked Exudative Vitreoretinopathy 2 protein (EVR2) Norrin or Norrie Disease Protein (NDP) or X-linked Exudative Vitreoretinopathy 2 protein (EVR2) is a cysteine-knot like growth factor protein in humans encoded by the NDP gene [45–47]. Norrin is a extracellular protein that binds and activates Wnt receptors at the plasma membrane [48,49]. Similarities of phenotypes between frizzled4 mouse mutants, NDPs and mouse model for Norrie disease suggested that Norrin protein might activate Wnt signaling [50–52]. Indeed, Norrin binds to Frizzled 4 on its CRD in the extracellular environment, a process that seems to require Wnt co-receptors LRP5/6 [51,53]. Interestingly, Norrin binds, with high affinity, to the CRD of Frizzled 4, but not to Frizzled 8 [50,52], revealing some degree of specificity to the system. In Xenopus embryos, maternal Norrin is necessary to activate Wnt signaling and for the formation of the anterior central nervous system [54]. Norrin can also bind to Lgr4/5 and 6, but only binding to Lgr4 activates Wnt signaling [55]. The complete relationship of Norrin protein and Wnt signaling is not fully understood, but structural analysis had shed light on the binding mechanism and shows how mutation can affect the function of the mature protein [49,50] (Fig. 1A).

2. WNT ligands and WNT receptors Two major classes of receptors have substantial roles in activating Wnt signaling. The primary Wnt receptors are called Frizzled receptors, and belong to a very specific G-protein coupled receptor (GPCR) subfamily [30] (Fig. 1A). 2.1. Frizzled receptors Frizzled receptors have 7 transmembrane-spanning domains, a large N-terminal domain that contains both a signal sequence directing the protein to the plasma membrane, and a Cysteine-Rich Domain (CRD), named after a conserved pattern of 10 cysteine residues found in this part of the mature protein. A large C-terminal domain, that mediates interaction with intracellular proteins, such as Dishevelled (Dvl), Gproteins, Arrestins and a number of intracellular loops that contain phosphorylation sites targeted by intracellular kinases [30–33]. Phylogenetically, there is variation in the number of Frizzled genes; for instance, mammals have 10 different genes encoding Frizzled receptors, whereas D. melanogaster and X. laevis have 4 and 11 orthologues, respectively [30,34]. The name Frizzled was derived from a recessive mutation in D. melanogaster, which gives rise to flies with curly and disorganized bristles and cuticle hair, therefore looking frizzled. Frizzled receptors range in length from 500 to 700 amino acids with molecular weight of ~71 kDa. But differences in sequence and posttranslational modifications lead to variation on SDS-PAGE separation and in molecular weight determination [34–37].

3.2. R-spondins In addition to Wnt ligands, other Non-Wnt molecules are capable of boosting activation of Wnt signaling [56,57]. R-Spondins are a family of secreted proteins that prevent LRP5/6 internalization and potentially enhancing the activation of the canonical Wnt pathway [57]. One early piece of evidence suggesting a link between R-spondin and the Wnt signaling pathway came from an analysis of Wnt-1/3a double knockout mouse that showed less expression of R-spondin on the roof plate during the development of the neural tube, indicating some involvement in dorsal neural tube formation/patterning regulated by Wnts [58]. R-spondins actively participate in the development of vertebrates, functioning extracellularly to regulate receptor-ligand interactions, and synergizing with Wnt ligands during activation of Wnt signaling

2.2. The low-density lipoprotein receptor-related protein (LRP) Wnts have a co-receptor that is required for activation of the βcatenin-dependent Wnt pathway (Fig. 1A). The low-density lipoprotein 31

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Fig. 1. Extracellular regulators of Wnt signaling pathway. Wnt ligands use diverse co-receptors to activate and modulate different downstream signals in Wnt signaling pathway. Wnt ligand bind to Frizzeled and co-receptors LRP5/6 to activate Wnt signaling; Norrin might acts as Wnt ligand, inducing Wnt signaling stimulation and Norrin can also binding to Lgrs4/5 and 6, but only binding to Lgr4 is capable to activate Wnt signaling. However, the relationship between Norrin and Wnt signaling is not completely uncovered (A). After binding of Wnt ligands to Frizzeled receptor and LRPs co-receptors, Wnt signaling is activated and causes the transcription of gene targets [1,2]; Wnt stimulation induces the transcription of targets as Lgr5, ZNRF3 and RNF43 in a feedback loop to add coreceptors in plasma membrane [3,4]; R-spondins are secreted proteins that prevent internalization of Wnt receptors by the binding to Znrf3 and Rnf43, and Lgrs receptors. Thus, Rspondins act as ligands of Lgrs receptors forming a complex that neutralizes Znrf3 and Rnf43, two E3 ubiquitin ligases that remove Wnt receptors from the membrane thought of endocytosis [5,6] (B). Models of Wnt signaling inhibition: Dkks are secreted glycoproteins that inhibit Wnt signaling by recruitment of Kremen transmembrane protein and internalization of LRPs receptors by endocytosis; Binding of Wise/SOST to LRPs receptors blocks the Frizzeled-LRP complex formation and Wnt signaling; (C).

expression. This expression appears to mark the intestinal crypts where the adult stem cells (the cells responsible for the high renewing rate of this tissue) are located [64]. Lgr5 expression pattern have been suggested to mark not only stem cells but also cancer cells (also highly proliferative cells) [64,65]. Homologues of Lgr5 such as Lgr4 also participate of the full activation of Wnt signaling by R-spondins [66]. A recent study taking advantage of a transgenic mice model identified human R-spondin 1 as a potent proliferative agent in intestinal crypts [67]. R-spondins have been associated to potencialization of Wnt/βcatenin pathway through the binding to Lgr4 and Lgr5 leading to LRP6 phosphorylation subsequently [63]. Interestingly, not all cells in the intestinal crypt secrete Wnt ligands. Thus, what would be the source of Wnt to the Lgr + cells? Paneth cells, a specific cell type of located in the crypts, were pointed as the source of Wnt ligand, precisely Wnt3 for Lgr + cells during intestinal organoids

[59,60]. Although strong evidence supports the view that R-spondins are co-activators of the canonical Wnt signaling, in zebrafish, R-spondin 3 (which has three Furin-like repeats instead of two), negatively regulates Wnt signaling during development [61]. The mechanisms by which R-spondins modulate the Wnt signaling pathway are not fully understood, but it seems that R-spondins act as ligands binding to the Leucine-Rich Repeat Containing G-protein couple receptors 4 and 5 (Lgr4 and Lgr5) [62]. Gain- as well as loss-of-function experiments indicated that depletion of Lgr levels did not itself affect Wnt signaling, but it did affect the ability of R-spondin to enhance Wnt signaling, suggesting again that R-spondins are extracellular “enhancers” of canonical Wnt signaling [59,62,63]. The importance of the loop R-spondin/Lgr for Wnt signaling may be better appreciated in vivo. The epithelium of the small intestine is a rapidly renewed tissue in adult mammals with high levels of Lgr5 32

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similar to gain-of-function mutations in LRP5 [81,82]. SOST knockout mice have a high bone mass phenotype, thereby confirming the role of SOST in bone formation and Wnt signaling [83]. Structural analysis of Sclerostin indicated that inhibition of Wnt signaling is based on its ability to bind to LRP5/6, thereby inhibiting activation of the canonical Wnt pathway [84]. Due to Sclerostin's mechanism of action and its effect on bone mass, different groups worldwide have focused on it to develop new therapies for bone-related disorders [85,86].

formation [68,69]. Curiously, in vivo experiments have shown that Wnt3 was dispensible for maintenance of the intestinal stem cells, suggesting that something else in that specific environment may replace this specific Wnt ligand [69]. Following on that, Foxl1-expressing mesenchymal cells that are located in a subepithelial region show expression of Wnt2b, Wnt5a and Rspondin 3 and appeared to be a source of R-spondin 3 for these cells. Ablation of Foxl1-expressing mesenchymal cells, disrupt gut epithelia, due to loss of proliferation and reduced Wnt signaling [70]. Therefore, the renewing of the gut epithelia and maintenance of this stem cell niche appears to be a intrigate event regulated by different cell types and biological factors. The disruption of this regulatory network may lead to unwanted event like deleterious morphological changes and/or cancer. R-spondin 1 is another R-spondin involved with activation of the Wnt signaling. It has been implicated with female sex determination together with Wnt4. Loss-of-function causes female to male sex reversal. Simultaneous ablation of R-spondin 1 and Wnt4 impairs proliferation of cell in the coelomic epithelium, reducing the numbers of progenitors of Sertoli cell (the supporting cells of the testis) in XY mutants gonads [71]. R-spondins appeared also to be involved with regulation of the strength of the Wnt signal. The Lgr5/R-spondin complex act neutralizing Znrf3 and Rnf43, two E3 ubiquiting ligases that remove Wnt receptors from the membrane. Znrf and Rnf43 are Wnt target genes part of a negative feedback loop present in stem cells [72] (Fig. 1B).

4.3. Wnt inhibitory factor (WIF1), secreted Frizzled-Related Protein (sFRP) and Cerberus (Cer or Cer-l)

The Dkk family contains 4 members in vertebrates (Dkk1–4). Dkks are secreted glycoproteins ranging in length from 255 to 350 amino acids with molecular weights of 24–29 kDa for Dkk1,2,4 and an estimated molecular weight of 38 kDa for Dkk3 [75]. Dkks seem to inhibit Wnt by their ability to bind and inactivate LRP5/6 and recruit another single-pass transmembrane protein, Kremen [76,77]. Binding of Dkk brings together LRP5/6 and Kremen, which in turn leads to internalization of the LRP5/6 receptors by endocytosis, thereby shutting down the canonical Wnt pathway [78] (Fig. 1C).

Another group of Wnt antagonists comprises WIF1, sFRP and Cerberus (Fig. 1C). Their mechanism of action is based on binding to Wnt ligands in the extracellular environment, thereby sequestering the Wnt ligand from the Wnt receptors [73,87,88]. The mechanism of inhibition, however, is different for this group, since the target is the Wnt ligand, not its receptors. Wnt inhibitory factor 1 (WIF1), for instance, binds to the lipid-derived appendages of the Wnt molecule, thereby preventing the Wnt ligand from binding to its cognate receptors [88]. Mice lacking WIF1 activity are viable and fertile, but have an increased likelihood of developing osteosarcoma [89], again reinforcing the idea of a dosage control mechanism. WIF1 is highly conserved gene among vertebrates that encodes for a 379 amino acid protein that contains a WIF domain [90]. In the mature protein it forms a hydrophobic pocket that binds to the acyl chains of secreted Wnt proteins [88,91]. Thus the acyl chain of the Wnt protein is necessary not only for correct sorting and function but, interestingly, is involved in dosage/gradient control through inhibition. The secreted Frizzled-Related Protein (sFRP) family comprises 5 known members (sFRP1–5). Expression patterns depend on cell type and different stimuli [92,93]. Unlike WIF1, sFRPs contain a CysteineRich Domain thought to mediate binding not only to Wnts, but to Frizzled receptors [77,87]. Secreted FRP binds to Wnt ligands, preventing the latter from interacting with Frizzled receptors and activating Wnt signaling. Thus the resulting inhibition occurs at the same strata of WIF1, although the mechanism is quite different. Secreted FRP molecule also binds to Frizzled receptors; the resulting complex is probably non-functional. These interactions might be mediated by CRD present on both molecules [77,87,94]. Cerberus is another Wnt antagonist that targets Wnt ligands. Its name is based on its ability to induce formation of ectopic heads in X. laevis embryos [95,96]. Cerberus protein contains a cysteine-knot domain that seems to be important for its function [95,97]. It is also an antagonist of bone morphogenetic proteins (BMPs) and Nodal [97,98]. The mouse orthologue of Cerberus (Cer-l) does not seem to have a high degree of conservation, and it remains debatable whether it indeed is involved in Wnt inhibition in vivo [98,99].

4.2. Sclerostin and Wise

4.4. The metalloprotease Tiki and the deacylase Notum

Sclerostin and Wise are 2 more molecules involved in inhibition of the canonical Wnt pathway (Fig. 1C). Wise was identified in a screen to find factors that can change the anteroposterior identity of neural tissues [79]. It is considered to be an antagonist of Wnt by binding to LRP5/6 receptors, preventing activation of the canonical Wnt pathway [80] However, this view may not always reflect experimental data. For instance, Wise can counteract the posteriorizing effects of Wnt8 when Wise RNA is co-injected with Wnt8 RNA in X. laevis. Conversely, injection of Wise RNA alone in animal caps of X. laevis activates the canonical Wnt pathway [79]. Perhaps these apparently contradictory effects of Wise reflect how the environment can shape the activation response of specific signaling pathways. Sclerostin is the product of the SOST locus, which encodes a secreted glycoprotein that has an inhibitory effect on Wnt signaling [74,81]. Mutation in the SOST locus causes abnormal bone formation

Newly found Wnt antagonists, such as Notum and Tiki, have drawn attention because of their distinct enzymatic mechanism of inhibition of the Wnt signaling pathway (Fig. 1C). Instead of just blocking Wnt signaling by binding to either Wnt receptor or ligand, they modify the Wnt protein in the extracellular stratum, minimizing their ability to bind receptors. Notum is an α/β hydrolase family member that includes peptidases, lipases, esterases and other hydrolytic enzymes. It was first described as a Wnt inhibitor in D. melanogaster. Mutations at the Notum locus produced wingless-like gain-of-function phenotypes [100,101]. Notum modifies the ability of glypicans (forms of heparin sulfate proteoglycans) to bind Wingless at the cell surface [101]. It also inhibits Wnt signaling in zebrafish development, which depends on its interactions with glypican-3. Notum is pointed to inhibit Wnt signaling thereby promoting planarian head regeneration [102,103].The mechanism by which Notum inhibits Wnt was recently elucidated

4. WNT antagonists Wnts have another level of regulation that ensures confinement of the signal to specific locations. Molecules that either bind to the Wnt ligand, preventing receptor binding and activation include Wnt inhibitory factor (WIF1), secreted Frizzled related protein (sFRP1–5), Cerberus, Tiki and Notum, or molecules that bind to the Wnt receptors preventing them from responding to Wnt ligands, such as Sclerostin, Dickkopf (DKK1–4) and Wise [34,35,73,74]. 4.1. Dickkopf (DKK)

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introduce some intracellular players of this pathway.

[104,105]. Notum acts as a Wnt deacylase that removes palmitoleic acid in the extracellular space through hydrolysis, inactivating the ability of Wnt to mediate in cell signaling. Wnt3a and Wnt5a depalmitoleoylated by Notum form large non-functional Wnt oligomers [104]. Notum is maternally expressed in Xenopus, and overexpression dorsally results in head enlargement, indicative of a role in axis specification [104,105]. Tiki was recently described as an extracellular modulator of Wnt activity, being a metalloprotease that cleaves the amino termini of mature Wnt proteins, thereby inactivating the Wnt ligand [106–109]. Blocking translation of Tiki in Xenopus embryo suppresses head formation genes and causes anterior defects, such as microcephaly and small eyes [106]. Tiki seems to be highly conserved among vertebrates [108]. Sequence homology indicates that it may have evolved from an ancient bacterial family of proteases [107].

6.1. Dishevelled (Dvl) Dishevelled (Dvl) is a cytosolic protein that carry out key roles in the non-canonical, β-catenin-independent as well as the canonical (βcatenin-dependent) Wnt signaling pathways [122,123]. Dishevelled allele was first identified as a recessive mutation in D. melanogaster that phenocopies Frizzled phenotype, indicating some sort of epistatic relationship between these 2 genes [123,124]. There are 3 homologues of the Dishevelled gene in vertebrates (Dvl1–3) [123]; Dishevelled comprises modular proteins raging in length from 500 to 600 amino acids. Near its N-terminus, it contains a DIX (Dishevelled Interaction with Axin) domain that mediates interaction with Axin [122,125,126]. In the central part of the protein lies a PDZ domain, required for interaction with Frizzled receptors and other PDZ-containing proteins [30,122]. The proximal part of the C-terminal domain contains a DEP domain, which also mediates protein-protein interactions, including Dishevelled itself and pleckstrin [127,128]. In mice, knockout experiments have questioned its primary requirement for activation of the canonical Wnt signaling. Because activation of this pathway takes place even in the absence of 2 out of the 3 Dishevelled isoforms [29]. A plausible explanation for this relies on some degree of redundancy among all 3 isoforms of Dishevelled in mice. A Dishevelled triple knockout mutant would shed light on this issue, but has yet to be generated.

5. β‑Catenin and the destruction complex β-Catenin is a protein of major importance in canonical Wnt signaling, being the key factor that transduces proximal events from the plasma membrane to the nuclear compartment [1,25,110]. Mouse βcatenin contains 781 amino acids with a molecular weight of 88 kDa. Its structure consists of an N-terminal region of 150 amino acids that contains phosphorylation sites for casein kinase 1α (CK1α) and glycogen synthase kinase 3 beta (GSK3-β), a central part of ~520 amino acids that contains 12 armadillo (arm) repeats, known to mediate protein-protein interactions, and a 100 residue C-terminus that contains some predicted phosphorylation sites that might also regulate proteinprotein interactions [111,112]. In the absence of Wnt ligands, β‑catenin exists mainly as part of the adherents junction complex, and its cytosolic concentrations are kept low by the β-catenin destruction complex [1,113,114]; this complex readily phosphorylates any β-catenin that detaches from the adherents junction, and this event tags β-catenin to the proteasome-mediated degradation pathway [110,115] (Fig. 2A–D). The basic scaffold of complex comprises 4 proteins, although it may have others (discussed below). Axin, a scaffolding protein, serves as an anchor site for β-catenin and the other proteins of the complex; it is encoded by a fused locus in mice [116,117]. Adenomatous polyposis coli (APC), a binding protein that brings together β-catenin and Axin, CK1α and GSK3-β, are 2 kinases that sequentially phosphorylate β-catenin, tagging it for ubiquitination and subsequent degradation [114,115,118]. Each component will be discussed below. Although the exact order of β-catenin phosphorylation remains unclear, it seems that phosphorylation of S45 by CK1α permits subsequent phosphorylation of S33, S37 and T41 by GSK3-β [112,115,119].

6.2. Axin A classical view of the activation of the canonical Wnt pathway dictates that binding of a Wnt ligand to its cognate receptors (Frizzled and LRP5/6) would lead to a phosphorylation-mediated activation of cytosolic pools of Dishevelled protein [27,129]. Phosphorylated Dishevelled binds Axin and recruits it to the plasma membrane, resulting in dissolution of the β-catenin destruction complex, Axin being at its core [27,28]. Thus, Axin appears as a pivotal scaffold protein for the assembly of the destruction complex (Fig. 2A–D). Its expression is considerably low compared to other components of the destruction complex [130,131]. Axin, at limiting concentrations may, in part, regulate the amount of β-catenin targeted to the proteasome-mediated degradation pathway. In support of this Axin dose-dependent contention, in vivo inactivation of Axin promotes the constitutive activation of the canonical Wnt pathway [131,132]. Conversely, Axin overexpression inhibits canonical Wnt signaling [116,131,132]. There may be a threshold of Axin expression that ensures robust activation of the canonical Wnt pathway upon Wnt stimulation but not in its absence [130]. In fact, not only the amount, but also conformation changes in Axin, seem to be important to control the functional capability of the destruction complex [133] and that will be further discussed below.

6. Mechanics of activation of the canonical (β-catenin-dependent) WNT pathway The canonical or β-catenin-dependent Wnt pathway is the most extensively studied branch of the Wnt signaling pathway [17,73], briefly defined by the following: 1) binding of a Wnt ligand to frizzled and LRP5/6 receptors at the plasma membrane; 2) activation of intracellular machinery that blocks β-catenin degradation, leading to its cytosolic accumulation and nuclear translocation; and 3) nuclear accumulation of β-catenin, which, in turn, leads to changes in the transcription rate of target genes in a T-cell factor/lymphoid enhancer factor (TCF/LEF) dependent manner [1,73,114,118,120,121]. The dynamics of the system implies that any β-catenin that breaks loose from cellular junctions is readily captured by the destruction complex and degraded in a proteasome-mediated manner. Thus, how does the binding of the Wnt ligand to its cognate receptors, at the plasma membrane inhibit the β-catenin destruction complex in the cytosol? The answer is not simple and the underlying mechanisms remain partially unclear. To better comprehend it, we will need to

6.3. Adenomatous Polyposis Coli (APC) Adenomatous Polyposis Coli (APC) is another large protein component of this multiprotein destruction complex that keeps cytosolic levels of β-catenin low [117,134]. It appears to be a multifunctional protein because of its roles in interacting with E-cadherins, which are involved in cellular adhesions and polarizing activity seen in cell migration [135,136]. Two different genes, APC1 and APC2, encode the APC protein. There is a high degree of APC conservation in most organisms regarding gene numbers and protein structures [134,137–139]. Human APC has 2843 amino acids, with an estimated mass of ~312 kDa. It contains 3 Axin-binding motifs interspersed with stretches of 15–20 amino acid repeats that bind β-catenin [134,139]. Although its functional importance in the destruction complex is unquestionable, details of its mechanism of action remain unsolved. 34

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Fig. 2. Evolution of activation of canonical Wnt signaling. Four models for the activation of the canonical Wnt pathway are chronologically presented. The first model depicts the β-catenin captured by the destruction complex and degraded in a proteasome-mediated manner, which in turn prevents β-catenin from entering the nucleus (A, Wnt OFF). When the Wnt ligand is present, the destruction complex somehow falls apart or is inactivated, and β‑catenin accumulates in the cytosol (A, Wnt ON). Note that there were not many scientific insights into how this process indeed occurs. The second model, proposed in 2010, suggests that, upon Wnt ligand binding to its receptors, β-catenin is captured by the destruction complex and both β-catenin and the whole destruction complex undergo endocytosis, forming an early endosome vesicle that eventually fuses into a multivesicular body. This step results in the destruction complex being trapped inside 2 lipid bilayers separated from newly synthetized cytosolic β-catenin, leading to its accumulation and translocation to the nucleus (B). The third model suggests that, upon Wnt ligand binding, the destruction complex is immobilized in an inactive form at the plasma membrane, clogged with phosphorylated β-catenin and trapping β-TrCP, thereby allowing β-catenin to freely accumulate in the cytosol (C). The last model was proposed in 2013, and is based on conformational changes of the scaffold protein, Axin, which dictates the activity of the destruction complex. In the absence of Wnt, the destruction complex stays in the cytosol where it captures and marks β-catenin for destruction. In the presence of Wnt ligand, Axin changes its conformation, abolishing the activity of the destruction complex, thus leading to accumulation of β-catenin in the cytosol, where it can further translocate to the nucleus and act as a cotranscription activator (D).

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enough to activate the canonical Wnt signaling pathway remains unclear.

6.4. Wilms' tumor protein on the X chromosome (WTX) Another protein that may have a structural role in the destruction complex is the Wilms' tumor protein on the X chromosome (WTX) also referred as AMER1 (APC membrane recruitment protein 1). WTX is mutated in a substantial amount of Wilms' tumor cases, a rare infantile kidney cancer [140]. It interacts with proteins in the destruction complex, as determined by high-affinity protein purification and mass spectrometry [141]. Interestingly, experiments on its activity indicate that WTX favors β-catenin degradation, therefore suggesting a mechanism by which WTX antagonizes the canonical Wnt pathway [141,142]. However, WTX's specific role in the destruction complex, or even whether it is present in the destruction complex in all cell types, remains debatable. Recently, it has been reported that production of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at the plasma membrane seems to be required for LRP6 phosphorylation by the dual kinase system (GSK3β and CK1α) and for signalosome formation [143]. Dishevelled protein appears to be a key regulator of this process, because it binds and activates phosphatidylinositol 4-kinase type IIα (PI4KIIα) and phosphatidylinositol-4-phosphate 5-kinase type Iβ (PIP5KIβ) enzymes that produce PtdIns [4,5] P2 [143,144]. Perhaps part of the missing link relies on the ability of WTX protein to recruit the destruction complex to the plasma membrane in a PtdIns [4,5] P2-dependent fashion [145]. This recruitment leads to inactivation of the destruction complex and thus, accumulation of β-catenin in the cytosol [142–145]. It is important not to mistake WTX/AMER1 by WT1. Wilm's tumor protein 1 (WT1) is a transcription factor that plays important roles for development, cell survival and urogenital tract formation [146,147]. It binds to specific G-rich DNA motifs increasing or decreasing the rate of transcription of target genes in a cell type dependent manner [146,148]. A growing pool of evidence has linked WT1 with Wnt signaling. First, Wnt signaling is required for kidney development and, loss-of-function mutations in this pathway lead to kidney agenesis or malformation [149,150]. Second, WT1 appears to modulate the transcription of Wnt target genes [147,148]. Third, WTX modulate WT1 function by directing binding resulting in modulation of WT1 transcriptional ability [151]. Thus, this body of evidence favors a conclusion that Wnt signaling and Wilms tumor protein 1 act together to govern several functions, among them normal kidney development and perhaps its regeneration.

7. Destruction complex inhibition mechanism: old vs new 7.1. Classical biochemical model A “classical” model for activation of the canonical Wnt signaling, namely Biochemical Model (Fig. 2A) predicts that, upon Wnt ligand binding, an intracellular event brings the destruction complex close to the plasma membrane in a Dishevelled-dependent manner. This proximity allows GSK3-β to phosphorylate LRP5/6 at the PPPSP repeats in its cytosolic tail. These phosphorylations create docking sites for GSK3-β catalytic pocket. The phosphorylated PPPSP repeats behave as pseudosubstrate of GSK3-β, describing a classical competitive inhibition kinetics [41,43]. Phosphorylated LRP co-receptors physical interaction with GSK3-β, not only inhibits the kinase but also traps the inactivated destruction complex at the plasma membrane, preventing it from further phosphorylate β-catenin. This event leads to cytosolic stabilization of β-catenin and its translocation to the nucleus, where it acts as a co-transcriptional factor [17,76,157–159]. This model is largely based in biochemical analysis (in vitro), utilizing fused proteins and overexpression of genes otherwise kept at low transcription rates inside the cell [28]. Thus, it raises a question of how much it recapitulates the cytosolic environment of a regular cell. Furthermore, the biochemical model predicts that the β-catenin destruction complex would either stay at the plasma membrane in an inactivated state or fall apart, so that it can no longer phosphorylate βcatenin. However, i) the complex does not fall apart, and endocytosis of the complex (that is not predicted to occurs) has been observed during activation of the Wnt signaling pathway [160];_ii) The proposed destabilization mechanism for inhibition of GSK3-β seems inefficient and it does not appear to be a reliable mechanism for GSK3-β inhibition [159,161]. The direct inhibition of GSK3-β by phosphorylated PPPSP LRP5/6 repeats also cannot be reliable since it has been measured as a low affinity event (Ki 1.3 × 10− 5 M) [159]; iii) The activity of GSK3-β is unaffected in detergent permeabilized cells, even though this model would predict that this the inhibition should occur [160,162]; iv) Wnt/ LRP signalosomes (not expected to form in the biochemical model) have been observed in the cytosol of cells previously treated with Wnt ligand [163] and v) not only LRP-mediated GSK3-β inhibition but the internalization of the destruction complex itself appears to be needed for signaling [160].

6.5. Glycogen synthase kinase 3 beta (GSK3-β) and casein kinase 1 alpha (CK 1α)

7.2. The cell biological model Two kinases are central to the role of the destruction complex, GSK3-β [152–154] and CK 1α [129,155]. Both are serine/threonine kinases that phosphorylate the N-terminal portion of cytosolic β-catenin, driving it to proteasome-mediated degradation [27,115,156]. Interestingly, CK phosphorylation at serine 45 appears permissive for GSK phosphorylation at residues T41, S37 and S33 [115,129]. Although most studies refer to GSK3-β and CK1-α, it is now accepted that other isoforms of these 2 kinases show some degree of redundancy [115,129].

Analyzing these findings and reflecting about the resemblance with activation of signaling pathways initiated with growth factors such as EGF (which internalization and formation of a multivesicular body seems to be a requirement) [164,165], in 2010 a new model for activation of the canonical Wnt signaling was proposed [162] (Fig. 2B). This model is sometimes referred as the cell biological model [166]. In this model, the first steps remain the same, i.e. in the presence of Wnt ligand, the β-catenin destruction complex is recruited to the plasma membrane in a Dishevelled-dependent manner. Once at the plasma membrane, the β-catenin destruction complex and the WntFrizzled-LRP complex undergo endocytosis, forming an early endosome vesicle that eventually fuses with a multivesicular body [162]. This results in separation of the β-catenin destruction complex from the cytosolic environment by a double membrane layer that prevents GSK3β to phosphorylates β-catenin, preventing its degradation from occurring, leading to β-catenin accumulation and translocation to the nucleus, where it functions as a co-transcription activator [162]. It has been argued that this model could explain a long-term rather than a short-term inhibition mechanism mediated by the classical model [166]. Thus, one could imagine two distinctive mechanisms that can

6.6. β-Transducin repeat-containing protein (β-TrCP) The sequential phosphorylation of β-catenin creates recognition sites for the β-transducin repeat-containing protein (β-TrCP), a dedicated E3 ubiquitin ligase. β-TrCP carries out poly-ubiquitination of βcatenin, driving it to the proteasome-mediated degradation pathway [110,129,153]. At the plasma membrane level, GSK3-β also has other important phosphorylation targets, the LRP5/6 co-receptors. GSK3-β-mediated phosphorylation of LRP5/6 in PPPSP repeats create docking sites for physical interactions between GSK3-β and LRP5/6 [41,43]. This interaction may partially inhibit GSK3-β, but whether this inhibition is 36

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CK1α, APC and Dvl) [28,178]. Axin-1 is the least abundant protein of the destruction complex, as mentioned, appears to be pivotal to assembly and disassembly of the destruction complex and it acts as a negative regulator of Wnt signaling [116,117,132]. One of the first targets of the activation of the Wnt signaling is the locus of Axin-2/ conductin that also negatively regulates Wnt signaling, also acting as a scaffolding protein for the formation of another destruction complex [179]. Degradation of Axin-1 in Wnt-probed cells have been proposed to be at the core of the activation of the canonical Wnt signaling [157,167]. Although Axin-1 is particularly important for destruction complex function in all three previous models there was not an analysis of the relevance of its post-translational modifications for its function. Phosphorylation/dephosphorylation of target proteins is among the most common post-translational modification in Eukaryotes. So, does the phosphorylation state of Axin play a role for Wnt signaling? Canonical Wnt pathway activation leads to phosphorylation of Lrp5/6, the scaffolding protein Axin-1 and β-catenin in a GSK3- β and CK1α dependent manner [174,180,181]. On the other hand, dephosphorylation carried out by protein phosphatases, such as protein phosphatase 1 countered protein kinases activity, i.e.: they remove the phosphate group added by protein kinases. Protein Phosphatase I (PP1) dephosphorylates Axin-1, leading to a conformational change in its structure, resulting in an inactive form that is not capable of trapping βcatenin in the destruction complex, leading to β-catenin cytosolic accumulation [182]. Thus, PP1 stimulation correlates with activation of the canonical Wnt signaling. Corroborating to this idea, pharmacological treatment of Wnt competent cells with phosphatase the inhibitor tautomycin prevented Wnt-induced Axin dephosphorylation and β-catenin stabilization [133]. So, how is the activity of PP1 regulated in a cellular context? And how PP1 regulation interferes with Wnt signaling? In the cell, protein phosphatases are majorly regulated by their binding partners, the inhibitors of protein phosphatase, such as I1 and I2 [183]. Inhibitor of protein phosphatase 2 (I2), is a cellular partner of PP1 that negatively modulates PP1 activity. Interestingly, overexpression of I2 leads to β-catenin degradation and inactivation of the canonical Wnt pathway [133]. Conversely, depletion of endogenous pool of I2 leads to β-catenin stabilization and activation of Wnt pathway [184]. These observations link Wnt-induced stabilization of βcatenin to Axin phosphorylation/dephosphorylation states. In the model proposed in 2012 by Li et al. [177], the authors did not focus their analysis in this crucial piece of information. Therefore, they overlooked possible Axin phosphorylation-mediated conformational changes importance for activation of the canonical Wnt pathway. Axin transition between phosphorylation and dephosphorylation states appears to be part of the processes of Wnt stimulation that leads to βcatenin cytosolic accumulation. β-catenin phosphorylation is the first step to drive it to ubiquitination and further proteasome-mediated destruction. There is a positive correlation between Axin phosphorylation in the absence of Wnt ligand as well as Axin dephosphorylation and βcatenin stabilization in the presence of Wnt ligand [129,133,185]. Supporting this view, Wnt stimulation leads to a weakening of the interaction between Axin-β-catenin, measured by an increase in the dissociation constant (kd) [133,186]. So, how does β-catenin phosphorylation state timely correlated with the activity of the destruction complex? β-catenin phosphorylation state is highly dependent of the activity of the destruction complex. The total amount of cellular β-catenin increases upon Wnt stimulation from 15 to 30 min, and it is kept above control for several hours. By contrast, GSK3-β mediated phosphorylation of β-catenin is decreased by 80% in 15–30 min and then returned to its initial concentration after 2 h [133,185]. The use of agents that promoted either inhibition or knock down of specific components of the destruction complex mimic Wnt stimulation [185] thus, again giving support the idea that a balance in the phosphorylation states of β-catenin and perhaps players of the destruction complex affect the ability of getting β-catenin stabilized in the

explain short-term versus long-term inhibition, altering PPPSP LRP repeat-mediated GSK3-β inhibition versus GSK3-β sequestration events to explain long term inhibition (Fig. 2B). The main idea of this review is to contextualize the evolution of thinking regarding the activation of the canonical Wnt pathway. The major differences between these two models (Biochemical model versus Cell Biology model) have already been analyzed in deep elsewhere [166]. Over the last two decades, a poll of evidence favoring the biochemical model to explain β-catenin stabilization upon Wnt stimulation appeared in the literature. Recent data have demonstrated that Axin degradation promotes β-catenin stabilization and Wnt signaling [157,167,168]. Dissociation of the destruction complex leading to stabilization of β-catenin [17,169–171].Inhibition of GSK3-β towards βcatenin phosphorylation and degradation [159,172]. Dynamics of phosphorylation and dephosphorylation of β-catenin upon Wnt stimulation [173]. Also, in the last few years, evidences favoring the cell biology model emerged in the literature, such as: membrane sequestration and trapping of the destruction complex [76,174]. Formation of multivesicular body containing GSK3-β [162]. Formation of signalosomes containing LRP co-receptors and the destruction complex [163]. Requirement of endocytosis for Wnt signaling to occurs [160,175,176]. Thus, the activation of the canonical Wnt signaling seems to be a rather intriguing event regulated by biochemical and cellular events. 7.3. The destruction complex clogged model It is worth noting that Axin-1, a pivotal protein for destruction complex formation, exists inside the cell in very low levels of expression and studies aided by overexpression of this protein (or others) may not recapitulated normal intracellular conditions of Wnt signaling. With that thinking in mind, in 2012, experiments in which the endogenous proteins levels were maintained and their post-translational modifications analyzed in the presence of Wnt ligand, another model for activation of the canonical Wnt signaling was proposed [177]. It differs from the previous view in the mechanism of destruction complex inhibition. In this model, in the absence of Wnt ligand, the destruction complex is active and stays in the cytosol, where it binds, phosphorylates and polyubiquitinates β-catenin, thus driving β-catenin to the proteasome-mediated degradation pathway. The proteasome activity recycles the complex by degrading β-catenin and releasing the binding site for another β-catenin to bind (Fig. 2C). However, in the presence of Wnt ligand, the whole destruction complex migrates to the plasma membrane, and interacts and phosphorylated PPPSP repeats in the cytosolic tail of LRP co-receptors (this step is dependent on Dvl). At the plasma membrane, the destruction complex, loaded with β-catenin, gets trapped and β-catenin cannot longer be polyubiquitinated by the E3 ubiquitin ligase β-TrCP neither can it be degraded by the proteasome. Thereby, the destruction complex stay clogged at the plasma membrane, incapable of promoting further degradation of β-catenin. This event results in stabilization of newly synthetizing β-catenin in the cytosol [177]. This model would predict that the destruction complex can maintain its integrity and ability to phosphorylate β-catenin even in the presence of Wnt ligand. The biding of the Wnt ligand to its cognate receptors would interfere with the ability of the complex to drive β-catenin polyubiquitination and proteasome-mediated degradation, and not inhibition of GSK3-β phosphorylation. Note that, endocytosis could still be occurring after inhibition, but in this scenario (Fig. 2C), endocytosis would not be the cause of inhibition and β-catenin stabilization but rather a physical impairment by the unreleased β-catenin from the destruction complex. 7.4. The Axin auto-inhibition model Axin seems to be rate-limiting protein for the destruction complex assembly and activity, because it is at the core of the destruction complex and keep together all other major players (β-catenin, GSK3-β, 37

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[28,114,190,199].The β-catenin nuclear complex involves a number of co-factors binding to its C-terminus with the ability to modify the structure of the surrounding chromatin, including CREB-binding protein (CBP), Brg1, SET Domain Containing 1 (SET1), coactivator-associated arginine methyltransferase (CARM), TATA-box binding protein and associated factors [201–207], transcription elongation factor and RNA polymerase 2 [208]. B-cell lymphoma 9 (BCL-9/B9L/legless) is yet another protein recruited to the β-catenin-mediated transcriptional complex. It was identified in B-cell cancers as a gene commonly upregulated due to chromosome translocations [209,210]. Legless, the D. melanogaster orthologue of BCL-9, could be a binding partner of Pygopus [211,212]. Pygopus is another binding partner of β-catenin; although required for wingless signaling in flies, its requirement for Wnt signaling in vertebrates remains uncertain [212–214]. Through its C-terminal, β-catenin binds to the histone acyltransferases, CB/p300, which promote chromatin unpacking and recruit RNA pol II to the site of transcription, allowing transcription of genes to start [202,215]. One of the most important phenomena controlled by β-catenin could be its regulation of the cell cycle and mitosis. Cell cycle genes, such as Cyclin D1 and c-Myc, are direct targets of β-catenin [216–218]. C-Myc encodes a transcription factor that upregulates cyclin D1 and represses p21 and p27 during G1 progression [219–221]. Cyclin D1 and c–Myc also are upregulated when β-catenin is abnormally active [218,222]. This occurs in many colorectal cancers because > 90% of them have mutations in one or more members of the Wnt signaling pathway, mainly APC (Cancer Genome Atlas Network, 2012). Wnt signaling controls cell cycle progression, but there is a reciprocal positive effect on Wnt signaling as cycling progresses [223,224]. This reciprocal regulation is better exemplified by the Wnt co-receptor, LRP6. LRP6 phosphorylation is under cell cycle control, peaking in the G2/M phase. This happens because Cyclin D1 phosphorylates the intracellular domain of LRP6, priming it to Wnt-dependent phosphorylation [225], GSK3-β presumably. These findings are interesting, since Wnt signaling peaks during mitosis, when transcription is thought to be silenced; thus this phosphorylation event may either have a post-translational role, such as preventing protein degradation, or prime cells to start transcription at only a few important loci after cell division has been complete, which may be needed in differentiation (but has yet to be investigated).

cytosol in a timely manner. Facing these observations Kim et al. [133] re-accessed the Wnt pathway and proposed a different mechanism to explain how the destruction complex is stopped from phosphorylating cytosolic β–catenin, thereby activating the Wnt signaling pathway (Fig. 2D). This new model is based on conformational changes of the scaffold protein, Axin, that dictates an elegant auto-inhibition mechanism. Events of phosphorylation and dephosphorylation govern whether Axin flips from an “open” conformation that is permissive for β-catenin and LRP5/6 binding and phosphorylation, to a “closed” state not permissive for binding, therefore, resulting in disassembly of the destruction complex and stabilization of β-catenin in the cytosol. This altering conformation change of Axin was shown by elegant FRET experiments [133] and with the use of small molecules targeting this pathway [187]. This model explains how Axin, and thus the destruction complex, oscillate between at least 2 conformational states in the presence vs absence of Wnt ligand, and how the intracellular levels of β–catenin correlates with the phosphorylation state of Axin-1. It is important to notice that the balance between a kinase and a phosphatase activity, namely GSK3-β and PP1 respectively, is at the core of this model. Only time and the rigor of science will set what model better explain the intracellular events that mediate activation of the canonical Wnt signaling. 8. β-Catenin and activation of the transcriptional machinery β-Catenin inside the nuclear compartment is important in assembling the nuclear protein complex required for transcriptional activation of Wnt target genes [121,188]. The primary DNA-binding proteins in this complex are TCF/LEF family members [28,189]. In vertebrates, the TCF/LEF family comprises 4 genes; however, due to the existence of several splice variants, there are numerous slightly different protein isoforms [121,189]. TCFs are members of the high mobility group (HMG) box proteins that, upon binding, cause DNA bending thought to facilitate recruitment of other factors by making DNA more accessible to the transcriptional machinery [121,189,190]. All TCF/LEF family members bind to a conserved DNA sequence CCTTTGWW (“W” means weak “A” or “T” bases at any given position) at the core of a 16 base-pair motif called the TCF-binding site [189–191]. Loss-of-function experiments have been used to sort out specific requirements of each TCF/LEF family member [29]. TCF1 and LEF1 Double knockout in mice yields a phenotype similar to deletion of Wnt3a (i.e. defects in paraxial mesoderm, limb-bud development and other problems with neural tube development) [192]. TCF3 knockout embryos have defects in formation of the A-P axis, such as axial duplication and duplication of structures (node and notochord) [193]. In vivo CHIP assay has shown occupancy of the Wnt3 promoter by β–catenin at a TCF-binding site rich region [194], showing the importance of this β-catenin shuttling between the cytosol and the nucleus in vivo. Recent studies reported that TCF4 is involved in neural apoptosis and proliferation of microglia cells after traumatic brain injury [195]. Also signaling mediated by TCF4 appears to be present in cancer proliferation in a specific type of brain cancer, gliomas [196]. These findings suggest participation of TCF4 in several processes of cell biology and development, including normal neurobiology and cancer. β-Catenin has major importance for TCF-mediated signaling, in the absence of β-catenin, TCF proteins form complexes with transducinglike enhancer of split (TLE/groucho) repressor [189,190,197–199]. The TLE/TCF complex recruits agents, such as histone deacetylases (HDAC), leading to chromatin condensation and gene silencing [199,200]. Conversely, when the canonical Wnt pathway is activated, β-catenin is translocated into the nucleus where it displaces TLE and forms a complex with TCF proteins bound to DNA; this complex recruits transcription activators to that particular DNA site [28,114,199]. It is noteworthy that binding of β‑catenin to TCFs is insufficient to start transcription, but rather a first step that leads to recruitment of factors that come together to create a transcription “hot-spot”

9. Concluding remarks Activation of the Wnt signaling pathway is a process that depends on a specific cellular context. It includes presence of receptors, Wnt ligands, and intracellular effectors that drive changes in the transcriptional rate of the cell. In unorganized cellular states like cancer the set of Wnt genes actively transcribed is altered and it affects normal cellular behavior. The discovered of pharmaceutical compounds that specifically target Wnt signaling in cancer is slowed down due to the lack of a in depth comprehension of the intracellular events that mediate Wnt signaling. Several groups are performing drug or genetic screenings to search for new modulators of the Wnt signaling pathway [89,148,187,226,227]. Wnt signaling has been a major subject of research to treat cancers, such as colorectal cancer. Most of the different types of colorectal cancer have mutations on the Wnt machinery complex, leading to an over activation of this pathway [134,139,228,229]. On the other hand, controlled re-activation of Wnt signaling on brains affected by neurodegenerative diseases may prevent the progression of the neural disease [230,231]. When in balance, spatial and temporal Wnt signals help to guide the responses towards development and regeneration of a complete organism. During development, a plethora of Wnt and Wnt-associated genes govern normal cell responses, such as cell division, differentiation, maturation and regeneration [118,120]. 38

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[8] F. Rijsewijk, M. Schuermann, E. Wagenaar, P. Parren, D. Weigel, R. Nusse, The Drosophila homology of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless, Cell 50 (1987) 649–657. [9] R. Nusse, A. Brown, J. Papkoff, P. Scambler, G. Shackleford, A. McMahon, et al., A new nomenclature for int-1 and related genes: the Wnt gene family, Cell 64 (1991) 231. [10] Y. Nakamura, C.D. Tsiairis, S. Özbek, T.W. Holstein, Autoregulatory and repressive inputs localize Hydra Wnt3 to the head organizer, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 9137–9142. [11] A. Sidow, Diversification of the Wnt gene family on the ancestral lineage of vertebrates, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 5098–5102. [12] S.-J. Cho, Y. Vallès, V.C. Giani, E.C. Seaver, D.A. Weisblat, Evolutionary dynamics of the wnt gene family: a lophotrochozoan perspective, Mol. Biol. Evol. 27 (2010) 1645–1658. [13] S.A. Nichols, W. Dirks, J.S. Pearse, N. King, Early evolution of animal cell signaling and adhesion genes, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 12451–12456. [14] M. Almuedo-Castillo, M. Sureda-Gómez, T. Adell, Wnt signaling in planarians: new answers to old questions, Int. J. Dev. Biol. 56 (2012) 53–65. [15] J. Heasman, Patterning the early Xenopus embryo, Development 133 (2006) 1205–1217. [16] P. Herr, G. Hausmann, K. Basler, WNT secretion and signalling in human disease, Trends Mol. Med. 18 (2012) 483–493. [17] C.Y. Logan, R. Nusse, The Wnt signaling pathway in development and disease, Annu. Rev. Cell Dev. Biol. 20 (2004) 781–810. [18] C. Kemp, E. Willems, S. Abdo, L. Lambiv, L. Leyns, Expression of all Wnt genes and their secreted antagonists during mouse blastocyst and postimplantation development, Dev. Dyn. 233 (2005) 1064–1075. [19] M. Kühl, L.C. Sheldahl, M. Park, J.R. Miller, R.T. Moon, The Wnt/Ca2 + pathway, Trends Genet. 16 (2000) 279–283. [20] M. Koyanagi, J. Haendeler, C. Badorff, R.P. Brandes, J. Hoffmann, P. Pandur, et al., Non-canonical Wnt signaling enhances differentiation of human circulating progenitor cells to cardiomyogenic cells, J. Biol. Chem. 280 (2005) 16838–16842. [21] Q. Tao, C. Yokota, H. Puck, M. Kofron, B. Birsoy, D. Yan, et al., Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos, Cell 120 (2005) 857–871. [22] M. Kofron, B. Birsoy, D. Houston, Q. Tao, C. Wylie, J. Heasman, Wnt11/betacatenin signaling in both oocytes and early embryos acts through LRP6-mediated regulation of axin, Development 134 (2007) 503–513. [23] S.P. Acebron, C. Niehrs, β-Catenin-independent roles of Wnt/LRP6 signaling, Trends Cell Biol. 26 (2016) 956–967. [24] J.R. Jessen, L. Solnica-Krezel, Axis formation–beta-catenin catches a Wnt, Cell 120 (2005) 736–737. [25] T.P. Rao, M. Kühl, An updated overview on wnt signaling pathways: a prelude for more, Circ. Res. 106 (2010) 1798–1806. [26] H.A. Kestler, M. Kuhl, From individual Wnt pathways towards a Wnt signalling network, Philos. Trans. R. Soc. B 363 (2008) 1333–1347. [27] H. Clevers, R. Nusse, Wnt/β-catenin signaling and disease, Cell 149 (2012) 1192–1205. [28] B.T. MacDonald, K. Tamai, X. He, Wnt/β-catenin signaling: components, mechanisms, and diseases, Dev. Cell 17 (2009) 9–26. [29] R. van Amerongen, A. Berns, C.Y. Logan, R. Nusse, R. Nusse, R.T. Moon, et al., Knockout mouse models to study Wnt signal transduction, Trends Genet. 22 (2006) 678–689. [30] G. Schulte, V. Bryja, The Frizzled family of unconventional G-protein-coupled receptors, Trends Pharmacol. Sci. 28 (2007) 518–525. [31] S.M. Foord, T.I. Bonner, R.R. Neubig, E.M. Rosser, J.-P. Pin, A.P. Davenport, et al., International Union of Pharmacology. XLVI. G protein-coupled receptor list, Pharmacol. Rev. 57 (2005) 279–288. [32] K.M. Cadigan, Y.I. Liu, Wnt signaling: complexity at the surface, J. Cell Sci. 119 (2006) 395–402. [33] C.Y. Janda, D. Waghray, A.M. Levin, C. Thomas, K.C. Garcia, Structural basis of Wnt recognition by Frizzled, Science 337 (2012) 59–64. [34] G. Schulte, International Union of Basic and Clinical Pharmacology. LXXX. The class Frizzled receptors, Pharmacol. Rev. 62 (2010) 632–667. [35] S. Angers, R.T. Moon, Proximal events in Wnt signal transduction, Nat. Rev. Mol. Cell Biol. 10 (2009) 468–477. [36] H.-C. Huang, P.S. Klein, The Frizzled family: receptors for multiple signal transduction pathways, Genome Biol. 5 (2004) 234. [37] H. Wang, T. Liu, C.C. Malbon, Structure-function analysis of Frizzleds, Cell. Signal. 18 (2006) 934–941. [38] J.-P. Rey, D.L. Ellies, Wnt modulators in the biotech pipeline, Dev. Dyn. 239 (2010) 102–114. [39] X. He, K. Tamai, M. Semenov, Y. Kato, R. Spokony, C. Liu, et al., LDL-receptorrelated proteins in Wnt signal transduction, Nature 407 (2000) 530–535. [40] S. DiNardo, M. Wehrli, S.T. Dougan, K. Caldwell, L. O'Keefe, Schwartz S et al. arrow encodes an LDL-receptor-related protein essential for Winglesssignalling, Nature 407 (2000) 527–530. [41] M. Wehrli, S.T. Dougan, K. Caldwell, L. O'Keefe, S. Schwartz, D. Vaizel-Ohayon, et al., Arrow encodes an LDL-receptor-related protein essential for Wingless signalling, Nature 407 (2000) 527–530. [42] O.G. Kelly, K.I. Pinson, W.C. Skarnes, The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice, Development 131 (2004) 2803–2815. [43] X. He, M. Semenov, K. Tamai, X. Zeng, LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way, Development 131 (2004) 1663–1677. [44] J.-C. Hsieh, L. Lee, L. Zhang, S. Wefer, K. Brown, C. DeRossi, et al., Mesd encodes

Wnt signaling has evolved as a pivotal signaling pathway to the development of an organism. We think that a clear view of how Wnt signaling works will emerge with time, but it will require cooperative efforts from researchers with different scientific backgrounds. This review only tackles a small part of this multifaceted machinery that cells use to communicate with one another and behave in distinctive ways. Imbalances in Wnt signaling leads to morphological catastrophes during embryonic development and cancers in adult organisms [1,27–29,73,118,120,232]. Therefore, to understand this pathway would be advantageous in order to find new drugs for therapeutic purposes as in cancer. Furthermore, it should not scape one's knowledge that, given the roles of Wnts through development; the understanding of such pathway will improve our knowledge of stem cell biology and tissue regeneration as well, allowing the manipulation of the pathway for medicine regenerative goals. Currently, several efforts have been employed on the development of organoids (i.e.: in vitro mini organs that resembles the architecture of the normal adult tissue). Wnt signaling has been shown to be essential for maintenance of such structures [233]. Some of the organoids-generation protocols, have as an initial signal, activation of the Wnt pathway, as in the case of Kidney organoids [234,235], due to the Wnt role for mesoderm induction [194,236,237]. Brain organoids, however, the initial signal appears to be the blockage of Wnt signaling, inhibiting the formation of mesoderm, favoring ectoderm [238—240]. Finally, the Wnt signaling pathway is a key regulator of animal development. In the near future, new therapies to treat cancer targeting the Wnt signaling pathway will be bring hope to patients who are now running out of options. Screenings to find such compound are already been carried out [204,226,241—244]. However, as we discussed above parts of this intriguing pathway are still not clear. Therefore, considering the importance of this pathway, new research is needed to shed light on old questions and perhaps propel the development of new and more efficient cancer therapies. Some questions remain wide open to research, such as: are there other cellular regulators of Wnt signaling to be found? How does the cell control re-activation of Wnt signaling in regenerative processes? Can we safely target control pharmacological re-activation of Wnt signaling for regenerative proposes? Will the Axin auto-inhibition model be definitive? Answers for these questions might guide researchers to better understanding of Wnt signaling and its functions. Conflict of interest The authors declare no conflict of interest. Acknowledgments GGT is a National postdoctoral fellow supported by CAPES. RRR is supported by a PhD fellowship from CAPES. This work was by CNPq (462073/2014-9) and FAPERJ (E-26/202.964/2015). References [1] K.M. Cadigan, R. Nusse, Wnt signaling: a common theme in animal development, Genes Dev. 11 (1997) 3286–3305. [2] D. Kimelman, Mesoderm induction: from caps to chips, Nat. Rev. Genet. 7 (2006) 360–372. [3] A. Klaus, W. Birchmeier, Wnt signalling and its impact on development and cancer, Nat. Rev. Cancer 8 (2008) 387–398. [4] C.P. Petersen, P.W. Reddien, A wound-induced Wnt expression program controls planarian regeneration polarity, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 17061–17066. [5] E.M. Tanaka, P.W. Reddien, The cellular basis for animal regeneration, Dev. Cell 21 (2011) 172–185. [6] R.P. Sharma, Wingless a new mutant in Drosophila melanogaster, Drosoph. Inf. Serv. 50 (1973) 134. [7] R. Nusse, A. van Ooyen, D. Cox, Y.K. Fung, H. Varmus, Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15, Nature 307 (1984) 131–136.

39

Cellular Signalling 40 (2017) 30–43

G.G. Tortelote et al.

[45]

[46]

[47]

[48] [49] [50] [51] [52]

[53]

[54]

[55] [56]

[57]

[58]

[59] [60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

[71]

[72] [73] [74] [75] [76]

protein 6 is a receptor for Dickkopf proteins, Nature 411 (2001) 321–325. [77] A. Bafico, G. Liu, A. Yaniv, A. Gazit, S.A. Aaronson, Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/arrow, Nat. Cell Biol. 3 (2001) 683–686. [78] B. Mao, W. Wu, G. Davidson, J. Marhold, M. Li, B.M. Mechler, et al., Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling, Nature 417 (2002) 664–667. [79] N. Itasaki, C.M. Jones, S. Mercurio, A. Rowe, P.M. Domingos, J.C. Smith, et al., Wise, a context-dependent activator and inhibitor of Wnt signalling, Development 130 (2003) 4295–4305. [80] X. Li, Y. Zhang, H. Kang, W. Liu, P. Liu, J. Zhang, et al., Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling, J. Biol. Chem. 280 (2005) 19883–19887. [81] M.E. Brunkow, J.C. Gardner, J. Van Ness, B.W. Paeper, B.R. Kovacevich, S. Proll, et al., Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein, Am. J. Hum. Genet. 68 (2001) 577–589. [82] E. Piters, E. Boudin, W. Van Hul, Wnt signaling: a win for bone, Arch. Biochem. Biophys. 473 (2008) 112–116. [83] X. Li, M.S. Ominsky, Q.-T. Niu, N. Sun, B. Daugherty, D. D'Agostin, et al., Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength, J. Bone Miner. Res. 23 (2008) 860–869. [84] S.E. Weidauer, P. Schmieder, M. Beerbaum, W. Schmitz, H. Oschkinat, T.D. Mueller, NMR Structure of the Wnt Modulator Protein Sclerostin, (2009), http://dx.doi.org/10.1016/j.bbrc.2009.01.062. [85] C. MacNabb, D. Patton, J.S. Hayes, Sclerostin antibody therapy for the treatment of osteoporosis: clinical prospects and challenges, J. Osteoporos. 2016 (2016) 6217286. [86] E.M. Lewiecki, Role of sclerostin in bone and cartilage and its potential as a therapeutic target in bone diseases, Ther. Adv. Musculoskelet. Dis. 6 (2014) 48–57. [87] C.-M. Cruciat, C. Niehrs, Secreted and transmembrane wnt inhibitors and activators, Cold Spring Harb. Perspect. Biol. 5 (2013) a015081. [88] T. Malinauskas, A.R. Aricescu, W. Lu, C. Siebold, E.Y. Jones, Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1, Nat. Struct. Mol. Biol. 18 (2011) 886–893. [89] M. Kansara, M. Tsang, L. Kodjabachian, N.A. Sims, M.K. Trivett, M. Ehrich, et al., Wnt inhibitory factor 1 is epigenetically silenced in human osteosarcoma, and targeted disruption accelerates osteosarcomagenesis in mice, J. Clin. Invest. 119 (2009) 837–851. [90] J.C. Hsieh, L. Kodjabachian, M.L. Rebbert, A. Rattner, P.M. Smallwood, C.H. Samos, et al., A new secreted protein that binds to Wnt proteins and inhibits their activities, Nature 398 (1999) 431–436. [91] E. Liepinsh, L. Bányai, L. Patthy, G. Otting, NMR structure of the WIF domain of the human Wnt-inhibitory factor-1, J. Mol. Biol. 357 (2006) 942–950. [92] Y. Mii, M. Taira, Secreted Wnt & quot;inhibitors & quot; are not just inhibitors: regulation of extracellular Wnt by secreted Frizzled-related proteins, Develop. Growth Differ. 53 (2011) 911–923. [93] R. Surana, S. Sikka, W. Cai, E.M. Shin, S.R. Warrier, H.J.G. Tan, et al., Secreted frizzled related proteins: implications in cancers, Biochim. Biophys. Acta, Rev. Cancer 2014 (1845) 53–65. [94] J. Yan, H. Jia, Z. Ma, H. Ye, M. Zhou, L. Su, et al., The evolutionary analysis reveals domain fusion of proteins with frizzled-like CRD domain, Gene 533 (2014) 229–239. [95] T. Bouwmeester, S. Kim, Y. Sasai, B. Lu, E.M. De Robertis, Cerberus is a headinducing secreted factor expressed in the anterior endoderm of Spemann's organizer, Nature 382 (1996) 595–601. [96] Y. Kawano, Secreted antagonists of the Wnt signalling pathway, J. Cell Sci. 116 (2003) 2627–2634. [97] Y. Kawano, R. Kypta, Secreted antagonists of the Wnt signalling pathway, J. Cell Sci. 116 (2003) 2627–2634. [98] J.J.H. Pearce, G. Penny, J. Rossant, A mouse Cerberus/Dan-related gene family, Dev. Biol. 209 (1999) 98–110. [99] J.A. Belo, D. Bachiller, E. Agius, C. Kemp, A.C. Borges, S. Marques, et al., Cerberuslike is a secreted BMP and nodal antagonist not essential for mouse development, Genesis 26 (2000) 265–270. [100] O. Gerlitz, K. Basler, Wingful, an extracellular feedback inhibitor of wingless, Genes Dev. 16 (2002) 1055–1059. [101] A.J. Giráldez, R.R. Copley, S.M. Cohen, HSPG modification by the secreted enzyme notum shapes the wingless morphogen gradient, Dev. Cell 2 (2002) 667–676. [102] G.P. Flowers, J.M. Topczewska, J. Topczewski, A zebrafish notum homolog specifically blocks the Wnt/β-catenin signaling pathway, Development 139 (2012) 2416–2425. [103] C.P. Petersen, P.W. Reddien, Polarized notum activation at wounds inhibits Wnt function to promote planarian head regeneration, Science 332 (2011) 852–855. [104] S. Kakugawa, P.F. Langton, M. Zebisch, S.A. Howell, T.-H. Chang, Y. Liu, et al., Notum deacylates Wnt proteins to suppress signalling activity, Nature 519 (2015) 187–192. [105] X. Zhang, S.-M. Cheong, N.G. Amado, A.H. Reis, B.T. MacDonald, M. Zebisch, et al., Notum is required for neural and head induction via Wnt deacylation, oxidation, and inactivation, Dev. Cell 32 (2015) 719–730. [106] X. Zhang, J.G. Abreu, C. Yokota, B.T. MacDonald, S. Singh, K.L.A. Coburn, et al., Tiki1 is required for head formation via Wnt cleavage-oxidation and inactivation, Cell 149 (2012) 1565–1577. [107] J.F. Bazan, B. MacDonald, X. He, The TIKI/TraB/PrgY family: a common protease fold for cell signaling from bacteria to metazoa? Dev. Cell 25 (2013) 225–227. [108] A.H. Reis, B.T. Macdonald, K. Feistel, J.M. Brito, N.G. Amado, C. Xu, et al.,

an LRP5/6 chaperone essential for specification of mouse embryonic polarity, Cell 112 (2003) 355–367. A. Meindl, W. Berger, T. Meitinger, D. van de Pol, H. Achatz, C. Dörner, et al., Norrie disease is caused by mutations in an extracellular protein resembling Cterminal globular domain of mucins, Nat. Genet. 2 (1992) 139–143. W. Berger, D. van de Pol, M. Warburg, A. Gal, L. Bleeker-Wagemakers, H. de Silva, et al., Mutations in the candidate gene for Norrie disease, Hum. Mol. Genet. 1 (1992) 461–465. Z.Y. Chen, R.W. Hendriks, M.A. Jobling, J.F. Powell, X.O. Breakefield, K.B. Sims, et al., Isolation and characterization of a candidate gene for Norrie disease, Nat. Genet. 1 (1992) 204–208. W. Berger, Molecular dissection of Norrie disease, Cells Tissues Organs 162 (1998) 95–100. B.M. Braunger, E.R. Tamm, The Different Functions of Norrin, Springer, US, 2012, pp. 679–683. A. Ohlmann, R. Merkl, E.R. Tamm, Focus on molecules: Norrin, Exp. Eye Res. 102 (2012) 109–110. C. Niehrs, Norrin and Frizzled: a new vein for the eye, Dev. Cell 6 (2004) 453–454. J. Ke, K.G. Harikumar, C. Erice, C. Chen, X. Gu, L. Wang, et al., Structure and function of Norrin in assembly and activation of a Frizzled 4-Lrp5/6 complex, Genes Dev. 27 (2013) 2305–2319. Q. Xu, Y. Wang, A. Dabdoub, P.M. Smallwood, J. Williams, C. Woods, et al., Vascular development in the retina and inner ear: control by Norrin and Frizzled4, a high-affinity ligand-receptor pair, Cell 116 (2004) 883–895. S. Xu, F. Cheng, J. Liang, W. Wu, J. Zhang, Maternal xNorrin, a canonical Wnt signaling agonist and TGF-β antagonist, controls early neuroectoderm specification in Xenopus, PLoS Biol. 10 (2012) e1001286. C. Deng, P. Reddy, Y. Cheng, C.-W. Luo, C.-L. Hsiao, A.J.W. Hsueh, Multi-functional norrin is a ligand for the LGR4 receptor, J. Cell Sci. 126 (2013) 2060–2068. K.-A. Kim, M. Wagle, K. Tran, X. Zhan, M.A. Dixon, S. Liu, et al., R-Spondin family members regulate the Wnt pathway by a common mechanism, Mol. Biol. Cell 19 (2008) 2588–2596. M.E. Binnerts, K.-A. Kim, J.M. Bright, S.M. Patel, K. Tran, M. Zhou, et al., RSpondin1 regulates Wnt signaling by inhibiting internalization of LRP6, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 14700–14705. T. Kamata, K. Katsube, M. Michikawa, M. Yamada, S. Takada, H. Mizusawa, Rspondin, a novel gene with thrombospondin type 1 domain, was expressed in the dorsal neural tube and affected in Wnts mutants, Biochim. Biophys. Acta Gene Struct. Expr. 2004 (1676) 51–62. W.B.M. de Lau, B. Snel, H.C. Clevers, The R-spondin protein family, Genome Biol. 13 (2012) 242. J.-S. Nam, T.J. Turcotte, J.K. Yoon, Dynamic Expression of R-Spondin Family Genes in Mouse Development, (2007), http://dx.doi.org/10.1016/j.modgep.2006. 08.006. X. Rong, C. Chen, P. Zhou, Y. Zhou, Y. Li, L. Lu, et al., R-Spondin 3 regulates dorsoventral and anteroposterior patterning by antagonizing Wnt/β-catenin signaling in zebrafish embryos, PLoS One 9 (2014) e99514. A. Glinka, C. Dolde, N. Kirsch, Y.-L. Huang, O. Kazanskaya, D. Ingelfinger, et al., LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling, EMBO Rep. 12 (2011) 1055–1061. K.S. Carmon, X. Gong, Q. Lin, A. Thomas, Q. Liu, R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 11452–11457. N. Barker, J.H. van Es, J. Kuipers, P. Kujala, M. van den Born, M. Cozijnsen, et al., Identification of stem cells in small intestine and colon by marker gene Lgr5, Nature 449 (2007) 1003–1007. M. Shimokawa, Y. Ohta, S. Nishikori, M. Matano, A. Takano, M. Fujii, et al., Visualization and targeting of LGR5(+) human colon cancer stem cells, Nature 545 (2017) 187–192. W. de Lau, N. Barker, T.Y. Low, B.-K. Koo, V.S.W. Li, H. Teunissen, et al., Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling, Nature 476 (2011) 293–297. K.-A. Kim, M. Kakitani, J. Zhao, T. Oshima, T. Tang, M. Binnerts, et al., Mitogenic influence of human R-spondin1 on the intestinal epithelium, Science 309 (2005) 1256–1259. T. Sato, J.H. van Es, H.J. Snippert, D.E. Stange, R.G. Vries, M. van den Born, et al., Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts, Nature 469 (2011) 415–418. H.F. Farin, J.H. Van Es, H. Clevers, Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth cells, Gastroenterology 143 (2012) 1518–1529.e7. R. Aoki, M. Shoshkes-Carmel, N. Gao, S. Shin, C.L. May, M.L. Golson, et al., Foxl1expressing mesenchymal cells constitute the intestinal stem cell niche, Cell. Mol. Gastroenterol. Hepatol. 2 (2016) 175–188. A.-A. Chassot, S.T. Bradford, A. Auguste, E.P. Gregoire, E. Pailhoux, D.G. de Rooij, et al., WNT4 and RSPO1 together are required for cell proliferation in the early mouse gonad, Development 139 (2012) 4461–4472. W. de Lau, W.C. Peng, P. Gros, H. Clevers, The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength, Genes Dev. 28 (2014) 305–316. A.J. Chien, W.H. Conrad, R.T. Moon, A Wnt survival guide: from flies to human disease, J. Invest. Dermatol. 129 (2009) 1614–1627. T. Malinauskas, E.Y. Jones, Extracellular modulators of Wnt signalling, Curr. Opin. Struct. Biol. 29 (2014) 77–84. C. Niehrs, Function and biological roles of the Dickkopf family of Wnt modulators, Oncogene 25 (2006) 7469–7481. B. Mao, W. Wu, Y. Li, D. Hoppe, P. Stannek, A. Glinka, et al., LDL-receptor-related

40

Cellular Signalling 40 (2017) 30–43

G.G. Tortelote et al.

[109]

[110]

[111] [112] [113] [114]

[115] [116]

[117]

[118] [119]

[120] [121]

[122] [123]

[124]

[125]

[126] [127]

[128]

[129]

[130]

[131]

[132]

[133]

[134] [135]

[136] [137] [138] [139]

[140]

[141] M.B. Major, N.D. Camp, J.D. Berndt, X. Yi, S.J. Goldenberg, C. Hubbert, et al., Wilms tumor suppressor WTX negatively regulates WNT/beta-catenin signaling, Science 316 (2007) 1043–1046. [142] X.H. Yannik Regimbald-Dumas, Wnt signalling: what the X@# is WTX? EMBO J. 30 (2011) 1415. [143] W. Pan, S.-C. Choi, H. Wang, Y. Qin, L. Volpicelli-Daley, L. Swan, et al., Wnt3amediated formation of phosphatidylinositol 4,5-bisphosphate regulates LRP6 phosphorylation, Science 321 (2008) 1350–1353. [144] Y. Qin, L. Li, W. Pan, D. Wu, Regulation of phosphatidylinositol kinases and metabolism by Wnt3a and Dvl, J. Biol. Chem. 284 (2009) 22544–22548. [145] K. Tanneberger, A.S. Pfister, K. Brauburger, J. Schneikert, M.V. Hadjihannas, V. Kriz, et al., Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation, EMBO J. 30 (2011) 1433–1443. [146] T.B. Hamilton, K.C. Barilla, P.J. Romaniuk, High affinity binding sites for the Wilms' tumour suppressor protein WT1, Nucleic Acids Res. 23 (1995) 277–284. [147] M.S. Kim, S.K. Yoon, F. Bollig, J. Kitagaki, W. Hur, N.J. Whye, et al., A novel Wilms tumor 1 (WT1) target gene negatively regulates the WNT signaling pathway, J. Biol. Chem. 285 (2010) 14585–14593. [148] M.K.-H. Kim, T.J. McGarry, O Broin P, Flatow JM, Golden AA-J, Licht JD., An integrated genome screen identifies the Wnt signaling pathway as a major target of WT1, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 11154–11159. [149] K. Halt, S. Vainio, Coordination of kidney organogenesis by Wnt signaling, Pediatr. Nephrol. 29 (2014) 737–744. [150] K. Pulkkinen, S. Murugan, S. Vainio, Wnt signaling in kidney development and disease, Organ 4 (2008) 55–59. [151] M.N. Rivera, W.J. Kim, J. Wells, A. Stone, A. Burger, E.J. Coffman, et al., The tumor suppressor WTX shuttles to the nucleus and modulates WT1 activity, Proc. Natl. Acad. Sci. 106 (2009) 8338–8343. [152] B.W. Doble, S. Patel, G.A. Wood, L.K. Kockeritz, J.R. Woodgett, Functional redundancy of GSK-3alpha and GSK-3beta in Wnt/beta-catenin signaling shown by using an allelic series of embryonic stem cell lines, Dev. Cell 12 (2007) 957–971. [153] H. Aberle, A. Bauer, J. Stappert, A. Kispert, R. Kemler, β-Catenin is a target for the ubiquitin-proteasome pathway, EMBO J. 16 (1997) 3797–3804. [154] C. Yost, G.H. Farr, S.B. Pierce, D.M. Ferkey, M.M. Chen, D. Kimelman, GBP, an inhibitor of GSK-3, is implicated in Xenopus development and oncogenesis, Cell 93 (1998) 1031–1041. [155] S. Amit, A. Hatzubai, Y. Birman, J.S. Andersen, E. Ben-Shushan, M. Mann, et al., Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway, Genes Dev. 16 (2002) 1066–1076. [156] E.M. Verheyen, C.J. Gottardi, Regulation of Wnt/beta-catenin signaling by protein kinases, Dev. Dyn. 239 (2010) 34–44. [157] J. Mao, J. Wang, B. Liu, W. Pan, G.H. Farr, C. Flynn, et al., Low-density lipoprotein receptor-related protein-5 binds to axin and regulates the canonical Wnt signaling pathway, Mol. Cell 7 (2001) 801–809. [158] K. Mi, P.J. Dolan, G.V.W. Johnson, The low density lipoprotein receptor-related protein 6 interacts with glycogen synthase kinase 3 and attenuates activity, J. Biol. Chem. 281 (2006) 4787–4794. [159] C.S. Cselenyi, K.K. Jernigan, E. Tahinci, C.A. Thorne, L.A. Lee, E. Lee, LRP6 transduces a canonical Wnt signal independently of axin degradation by inhibiting GSK3's phosphorylation of -catenin, Proc. Natl. Acad. Sci. 105 (2008) 8032–8037. [160] J.T. Blitzer, R. Nusse, C. Logan, R. Nusse, R. Moon, B. Bowerman, et al., A critical role for endocytosis in Wnt signaling, BMC Cell Biol. 7 (2006) 28. [161] G. Wu, H. Huang, J. Garcia Abreu, X. He, Inhibition of GSK3 phosphorylation of beta-catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6, PLoS One 4 (2009) e4926. [162] V.F. Taelman, R. Dobrowolski, J.-L. Plouhinec, L.C. Fuentealba, P.P. Vorwald, I. Gumper, et al., Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes, Cell 143 (2010) 1136–1148. [163] J. Bilic, Y.-L. Huang, G. Davidson, T. Zimmermann, C.-M. Cruciat, M. Bienz, et al., Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation, Science 316 (2007) 1619–1622. [164] J. Gruenberg, H. Stenmark, Opinion: the biogenesis of multivesicular endosomes, Nat. Rev. Mol. Cell Biol. 5 (2004) 317–323. [165] J.A. McKanna, H.T. Haigler, S. Cohen, Hormone receptor topology and dynamics: morphological analysis using ferritin-labeled epidermal growth factor, Proc. Natl. Acad. Sci. U. S. A. 76 (1979) 5689–5693. [166] C. Metcalfe, M. Bienz, S. Angers, R.T. Moon, M. Bienz, H. Clevers, et al., Inhibition of GSK3 by Wnt signalling—two contrasting models, J. Cell Sci. 124 (2011) 3537–3544. [167] N.S. Tolwinski, M. Wehrli, A. Rives, N. Erdeniz, S. DiNardo, E. Wieschaus, Wg/ Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity, Dev. Cell 4 (2003) 407–418. [168] E. Lee, A. Salic, R. Krüger, R. Heinrich, M.W. Kirschner, The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway, PLoS Biol. 1 (2003) e10. [169] X. Liu, J.S. Rubin, A.R. Kimmel, Rapid, Wnt-induced changes in GSK3beta associations that regulate beta-catenin stabilization are mediated by Galpha proteins, Curr. Biol. 15 (2005) 1989–1997. [170] C.C. Malbon, H.-Y. Wang, Dishevelled: a mobile scaffold catalyzing development, Curr. Top. Dev. Biol. 72 (2006) 153–166. [171] D.M. Roberts, M.I. Pronobis, J.S. Poulton, J.D. Waldmann, E.M. Stephenson, S. Hanna, et al., Deconstructing the βcatenin destruction complex: mechanistic roles for the tumor suppressor APC in regulating Wnt signaling, Mol. Biol. Cell 22 (2011) 1845–1863. [172] S. Piao, S.-H. Lee, H. Kim, S. Yum, J.L. Stamos, Y. Xu, et al., Direct inhibition of GSK3beta by the phosphorylated cytoplasmic domain of LRP6 in Wnt/beta-

Expression and evolution of the Tiki1 and Tiki2 genes in vertebrates, Int. J. Dev. Biol. 58 (2014) 355–362. X. Zhang, B.T. MacDonald, H. Gao, M. Shamashkin, A.J. Coyle, R.V. Martinez, et al., Characterization of Tiki, a new family of Wnt-specific metalloproteases, J. Biol. Chem. 291 (2016) 2435–2443. Y. Marikawa, R.P. Elinson, β-TrCP is a negative regulator of the Wnt/β-catenin signaling pathway and dorsal axis formation in Xenopus embryos, Mech. Dev. 77 (1998) 75–80. L. Shapiro, W.I. Weis, Structure and biochemistry of cadherins and catenins, Cold Spring Harb. Perspect. Biol. 1 (2009) a003053. F.J.T. Staal, M. van Noort, G.J. Strous, H.C. Clevers, Wnt signals are transmitted through N-terminally dephosphorylated beta-catenin, EMBO Rep. 3 (2002) 63–68. K.M. Cadigan, TCFs and Wnt/β-catenin signaling: more than one way to throw the switch, Curr. Top. Dev. Biol. 98 (2012) 1–34. H.C. Archbold, Y.X. Yang, L. Chen, K.M. Cadigan, How do they do Wnt they do?: regulation of transcription by the Wnt/β-catenin pathway, Acta Physiol (Oxford) 204 (2012) 74–109. J.L. Stamos, W.I. Weis, The β-catenin destruction complex, Cold Spring Harb. Perspect. Biol. 5 (2013) a007898. L. Zeng, F. Fagotto, T. Zhang, W. Hsu, T.J. Vasicek, W.L. Perry, et al., The mouse fused locus encodes axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation, Cell 90 (1997) 181–192. M.J. Hart, R. de los Santos, I.N. Albert, B. Rubinfeld, P. Polakis, Downregulation of β-catenin by human axin and its association with the APC tumor suppressor, βcatenin and GSK3β, Curr. Biol. 8 (1998) 573–581. F. Verkaar, K.M. Cadigan, R. van Amerongen, et al., Sci. Signal. 5 (2012) mr2. N.-C. Ha, T. Tonozuka, J.L. Stamos, H.-J. Choi, W.I. Weis, S. Amit, et al., Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation, Mol. Cell 15 (2004) 511–521. A. Klaus, W. Birchmeier, Wnt signalling and its impact on development and cancer, Nat. Rev. Cancer 8 (2008) 387–398. K.M. Cadigan, M.L. Waterman, TCF/LEFs and Wnt signaling in the nucleus, Cold Spring Harb. Perspect. Biol. 4 (2012), http://dx.doi.org/10.1101/cshperspect. a007906. C. Gao, Y.-G. Chen, Dishevelled: the hub of Wnt signaling, Cell. Signal. 22 (2010) 717–727. J.B. Wallingford, R. Habas, The developmental biology of Dishevelled: an enigmatic protein governing cell fate and cell polarity, Development 132 (2005) 4421–4436. A. Penton, A. Wodarz, R. Nusse, A mutational analysis of Dishevelled in Drosophila defines novel domains in the dishevelled protein as well as novel suppressing alleles of axin, Genetics 161 (2002) 747–762. T. Schwarz-Romond, M. Fiedler, N. Shibata, P.J.G. Butler, A. Kikuchi, Y. Higuchi, et al., The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization, Nat. Struct. Mol. Biol. 14 (2007) 484–492. T. Schwarz-Romond, C. Metcalfe, M. Bienz, Dynamic recruitment of axin by Dishevelled protein assemblies, J. Cell Sci. 120 (2007) 2402–2412. J. Zheng, H.C. Wong, J. Mao, J.T. Nguyen, S. Srinivas, W. Zhang, et al., Structural basis of the recognition of the Dishevelled DEP domain in the Wnt signaling pathway, Nat. Struct. Biol. 7 (2000) 1178–1184. H.-C. Wong, A. Bourdelas, A. Krauss, H.-J. Lee, Y. Shao, D. Wu, et al., Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of Frizzled, Mol. Cell 12 (2003) 1251–1260. C. Liu, Y. Li, M. Semenov, C. Han, G.H. Baeg, Y. Tan, et al., Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism, Cell 108 (2002) 837–847. Z. Wang, O. Tacchelly-Benites, E. Yang, C.A. Thorne, H. Nojima, E. Lee, et al., Wnt/wingless pathway activation is promoted by a critical threshold of axin maintained by the tumor suppressor APC and the ADP-ribose polymerase tankyrase, Genetics 203 (2016) 269–281. K. Willert, C.Y. Logan, A. Arora, M. Fish, R. Nusse, H. Aberle, et al., A Drosophila axin homolog, Daxin, inhibits Wnt signaling, Development 126 (1999) 4165–4173. F. Hamada, Y. Tomoyasu, Y. Takatsu, M. Nakamura, S. Nagai, A. Suzuki, et al., Negative regulation of wingless signaling by D-axin, a Drosophila homolog of axin, Science 283 (1999) 1739–1742. S.-E. Kim, H. Huang, M. Zhao, X. Zhang, A. Zhang, M.V. Semonov, et al., Wnt stabilization of β-catenin reveals principles for morphogen receptor-scaffold assemblies, Science 340 (2013) 867–870. C.L. Day, T. Alber, Crystal structure of the amino-terminal coiled-coil domain of the APC tumor suppressor, J. Mol. Biol. 301 (2000) 147–156. I. Nishisho, Y. Nakamura, Y. Miyoshi, Y. Miki, H. Ando, A. Horii, et al., Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients, Science 253 (1991) 665–669. S. Etienne-Manneville, APC in cell migration, Adv. Exp. Med. Biol. 656 (2009) 30–40. A.I.M. Barth, W.J. Nelson, What can humans learn from flies about adenomatous polyposis coli? BioEssays 24 (2002) 771–774. Y. Ahmed, A. Nouri, E. Wieschaus, Drosophila Apc1 and Apc2 regulate wingless transduction throughout development, Development 129 (2002) 1751–1762. D.P. Minde, M. Radli, F. Forneris, M.M. Maurice, S.G.D. Rüdiger, Large extent of disorder in adenomatous polyposis coli offers a strategy to guard Wnt signalling against point mutations, PLoS One 8 (2013) e77257. M.N. Rivera, W.J. Kim, J. Wells, D.R. Driscoll, B.W. Brannigan, M. Han, et al., An X chromosome gene, WTX, is commonly inactivated in Wilms tumor, Science 315 (2007) 642–645.

41

Cellular Signalling 40 (2017) 30–43

G.G. Tortelote et al.

modules, Mol. Cell 10 (2002) 573–584. [205] N. Barker, The chromatin remodelling factor Brg-1 interacts with beta-catenin to promote target gene activation, EMBO J. 20 (2001) 4935–4943. [206] S.S. Koh, H. Li, Y.-H. Lee, R.B. Widelitz, C.-M. Chuong, M.R. Stallcup, Synergistic coactivator function by coactivator-associated arginine methyltransferase (CARM) 1 and -catenin with two different classes of DNA-binding transcriptional activators, J. Biol. Chem. 277 (2002) 26031–26035. [207] J. Sierra, The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes, Genes Dev. 20 (2006) 586–600. [208] C. Mosimann, G. Hausmann, K. Basler, Parafibromin/hyrax activates Wnt/Wg target gene transcription by direct association with beta-catenin/armadillo, Cell 125 (2006) 327–341. [209] T. Itoyama, G. Nanjungud, W. Chen, V.G. Dyomin, J. Teruya-Feldstein, S.C. Jhanwar, et al., Molecular cytogenetic analysis of genomic instability at the 1q12-22 chromosomal site in B-cell non-Hodgkin lymphoma, Genes Chromosom. Cancer 35 (2002) 318–328. [210] T.G. Willis, I.R. Zalcberg, L.J. Coignet, I. Wlodarska, M. Stul, D.M. Jadayel, et al., Molecular cloning of translocation t(1;14)(q21;q32) defines a novel gene (BCL9) at chromosome 1q21, Blood 91 (1998) 1873–1881. [211] T. Kramps, O. Peter, E. Brunner, D. Nellen, B. Froesch, S. Chatterjee, et al., Wnt/ wingless signaling requires BCL9/legless-mediated recruitment of Pygopus to the nuclear β-catenin-TCF complex, Cell 109 (2002) 47–60. [212] F.M. Townsley, B. Thompson, M. Bienz, Pygopus residues required for its binding to legless are critical for transcription and development, J. Biol. Chem. 279 (2004) 5177–5183. [213] R. Hoffmans, R. Städeli, K. Basler, Pygopus and legless provide essential transcriptional coactivator functions to armadillo/β-catenin, Curr. Biol. 15 (2005) 1207–1211. [214] M. de la Roche, M. Bienz, Wingless-independent association of Pygopus with dTCF target genes, Curr. Biol. 17 (2007) 556–561. [215] J. Li, C. Sutter, D.S. Parker, T. Blauwkamp, M. Fang, K.M. Cadigan, CBP/p300 are bimodal regulators of Wnt signaling, EMBO J. 26 (2007) 2284–2294. [216] M. Shtutman, J. Zhurinsky, I. Simcha, C. Albanese, M. D'Amico, R. Pestell, et al., The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 5522–5527. [217] T.C. He, A.B. Sparks, C. Rago, H. Hermeking, L. Zawel, L.T. da Costa, et al., Identification of c-MYC as a target of the APC pathway, Science 281 (1998) 1509–1512. [218] F. McCormick, O. Tetsu, Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells, Nature 398 (1999) 422–426. [219] K.B. Marcu, S.A. Bossone, A.J. Patel, Myc function and regulation, Annu. Rev. Biochem. 61 (1992) 809–860. [220] W.P. Tansey, Mammalian MYC proteins and cancer, New J. Sci. 2014 (2014), http://dx.doi.org/10.1155/2014/757534. [221] N. Meyer, L.Z. Penn, Reflecting on 25 years with MYC, Nat. Rev. Cancer 8 (2008) 976–990. [222] E.A. Klein, R.K. Assoian, Transcriptional regulation of the cyclin D1 gene at a glance, J. Cell Sci. 121 (2008) 3853–3857. [223] D. Olmeda, S. Castel, S. Vilaró, A. Cano, Beta-catenin regulation during the cell cycle: implications in G2/M and apoptosis, Mol. Biol. Cell 14 (2003) 2844–2860. [224] C. Niehrs, S.P. Acebron, Mitotic and mitogenic Wnt signalling, EMBO J. 31 (2012) 2705–2713. [225] G. Davidson, J. Shen, Y.-L. Huang, Y. Su, E. Karaulanov, K. Bartscherer, et al., Cell cycle control of wnt receptor activation, Dev. Cell 17 (2009) 788–799. [226] B.T. Dickinson, J. Kisiel, D.A. Ahlquist, W.M. Grady, Molecular markers for colorectal cancer screening, Gut 64 (2015) 1485–1494. [227] C. Jamieson, M. Sharma, B.R. Henderson, Targeting the β-catenin nuclear transport pathway in cancer, Semin. Cancer Biol. 27 (2014) 20–29. [228] L. Vermeulen, E. De Sousa, F. Melo, M. van der Heijden, K. Cameron, J.H. de Jong, T. Borovski, et al., Wnt activity defines colon cancer stem cells and is regulated by the microenvironment, Nat. Cell Biol. 12 (2010) 468–476. [229] N.G. Amado, D. Predes, B.F. Fonseca, D.M. Cerqueira, A.H. Reis, A.C. Dudenhoeffer, et al., Isoquercitrin suppresses colon cancer cell growth in vitro by targeting the Wnt/β-catenin signaling pathway, J. Biol. Chem. 289 (2014) 35456–35467. [230] C.A. Oliva, J.Y. Vargas, N.C. Inestrosa, Wnts in adult brain: from synaptic plasticity to cognitive deficiencies, Front. Cell. Neurosci. 7 (2013) 224. [231] G.V. De Ferrari, M.A. Chacón, M.I. Barría, J.L. Garrido, J.A. Godoy, G. Olivares, et al., Activation of Wnt signaling rescues neurodegeneration and behavioral impairments induced by β-amyloid fibrils, Mol. Psychiatry 8 (2003) 195–208. [232] J.D. Holland, A. Klaus, A.N. Garratt, W. Birchmeier, Wnt signaling in stem and cancer stem cells, Curr. Opin. Cell Biol. 25 (2013) 254–264. [233] N. Tüysüz, L. van Bloois, S. van den Brink, H. Begthel, M.M.A. Verstegen, L.J. Cruz, et al., Lipid-mediated Wnt protein stabilization enables serum-free culture of human organ stem cells, Nat. Commun. 8 (2017) 14578. [234] R. Morizane, A.Q. Lam, B.S. Freedman, S. Kishi, M.T. Valerius, J.V. Bonventre, Nephron organoids derived from human pluripotent stem cells model kidney development and injury, Nat. Biotechnol. 33 (2015) 1193–1200. [235] A.Q. Lam, B.S. Freedman, R. Morizane, P.H. Lerou, M.T. Valerius, J.V. Bonventre, Rapid and efficient differentiation of human pluripotent stem cells into intermediate mesoderm that forms tubules expressing kidney proximal tubular markers, J. Am. Soc. Nephrol. 25 (2014) 1211–1225. [236] Y. Yoon, T. Huang, G.G. Tortelote, M. Wakamiya, A.-K. Hadjantonakis, R.R. Behringer, et al., Extra-embryonic Wnt3 regulates the establishment of the primitive streak in mice, Dev. Biol. 403 (2015) 80–88. [237] P. Liu, M. Wakamiya, M.J. Shea, U. Albrecht, R.R. Behringer, A. Bradley,

catenin signaling, PLoS One 3 (2008) e4046. [173] Y. Su, C. Fu, S. Ishikawa, A. Stella, M. Kojima, K. Shitoh, et al., APC is essential for targeting phosphorylated beta-catenin to the SCFbeta-TrCP ubiquitin ligase, Mol. Cell 32 (2008) 652–661. [174] X. Zeng, K. Tamai, B. Doble, S. Li, H. Huang, R. Habas, et al., A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation, Nature 438 (2005) 873–877. [175] C. Niehrs, S.P. Acebron, Wnt signaling: multivesicular bodies hold GSK3 captive, Cell 143 (2010) 1044–1046. [176] F. Port, M. Kuster, P. Herr, E. Furger, C. Bänziger, G. Hausmann, et al., Wingless secretion promotes and requires retromer-dependent cycling of Wntless, Nat. Cell Biol. 10 (2008) 178–185. [177] V.S.W. Li, S.S. Ng, P.J. Boersema, T.Y. Low, W.R. Karthaus, J.P. Gerlach, et al., Wnt signaling through inhibition of β-catenin degradation in an Intact Axin1 complex, Cell 149 (2012) 1245–1256. [178] H. Clevers, R. Nusse, H. Aberle, A. Bauer, J. Stappert, A. Kispert, et al., Wnt/βcatenin signaling and disease, Cell 149 (2012) 1192–1205. [179] E. Jho, T. Zhang, C. Domon, C.-K. Joo, J.-N. Freund, F. Costantini, Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway, Mol. Cell. Biol. 22 (2002) 1172–1183. [180] C. Liu, Y. Li, M. Semenov, C. Han, G.-H. Baeg, Y. Tan, et al., Control of β-catenin phosphorylation/degradation by a dual-kinase mechanism, Cell 108 (2002) 837–847. [181] H. Yamamoto, S. Kishida, M. Kishida, S. Ikeda, S. Takada, A. Kikuchi, Phosphorylation of axin, a Wnt signal negative regulator, by glycogen synthase kinase-3beta regulates its stability, J. Biol. Chem. 274 (1999) 10681–10684. [182] W. Luo, A. Peterson, B.A. Garcia, G. Coombs, B. Kofahl, R. Heinrich, et al., Protein phosphatase 1 regulates assembly and function of the beta-catenin degradation complex, EMBO J. 26 (2007) 1511–1521. [183] D.M. Virshup, S. Shenolikar, From promiscuity to precision: protein phosphatases get a makeover, Mol. Cell 33 (2009) 537–545. [184] C. Niehrs, Regionally specific induction by the Spemann-Mangold organizer, Nat. Rev. Genet. 5 (2004) 425–434. [185] A.R. Hernández, A.M. Klein, M.W. Kirschner, Kinetic responses of β-catenin specify the sites of Wnt control, Science 338 (2012) 1337–1340. [186] K. Willert, S. Shibamoto, R. Nusse, Wnt-induced dephosphorylation of axin releases beta-catenin from the axin complex, Genes Dev. 13 (1999) 1768–1773. [187] S. Wang, J. Yin, D. Chen, F. Nie, X. Song, C. Fei, et al., Small-molecule modulation of Wnt signaling via modulating the axin-LRP5/6 interaction, Nat. Chem. Biol. 9 (2013) 579–585. [188] B. Thompson, F. Townsley, R. Rosin-Arbesfeld, H. Musisi, M. Bienz, A new nuclear component of the Wnt signalling pathway, Nat. Cell Biol. 4 (2002) 367–373. [189] L. Arce, N.N. Yokoyama, M.L. Waterman, Diversity of LEF/TCF action in development and disease, Oncogene 25 (2006) 7492–7504. [190] D. Hrckulak, M. Kolar, H. Strnad, V. Korinek, TCF/LEF transcription factors: an update from the internet resources, Cancers (Basel) 8 (2016), http://dx.doi.org/ 10.3390/cancers8070070. [191] F.A. Atcha, A. Syed, B. Wu, N.P. Hoverter, N.N. Yokoyama, J.-H.T. Ting, et al., A unique DNA binding domain converts T-cell factors into strong Wnt effectors, Mol. Cell. Biol. 27 (2007) 8352–8363. [192] J. Galceran, I. Fariñas, M.J. Depew, H. Clevers, R. Grosschedl, Wnt3a−/−−like phenotype and limb deficiency in Lef1(−/−)Tcf1(−/−) mice, Genes Dev. 13 (1999) 709–717. [193] B.J. Merrill, H.A. Pasolli, L. Polak, M. Rendl, M.J. García-García, K.V. Anderson, et al., Tcf3: a transcriptional regulator of axis induction in the early embryo, Development 131 (2004) 263–274. [194] G.G. Tortelote, J.M. Hernández-Hernández, A.J.C. Quaresma, J.A. Nickerson, A.N. Imbalzano, J.A. Rivera-Pérez, Wnt3 function in the epiblast is required for the maintenance but not the initiation of gastrulation in mice, Dev. Biol. 374 (2013) 164–173. [195] X. Liu, Y. Huang, Y. Zhang, X. Li, C. Liu, S. Huang, et al., T-cell factor (TCF/LEF1) binding elements (TBEs) of FasL (Fas ligand or CD95 ligand) bind and cluster Fas (CD95) and form complexes with the TCF-4 and b-catenin transcription factors in vitro and in vivo which result in triggering cell death and/or cell a, Cell. Mol. Neurobiol. 36 (2016) 1001–1013. [196] X. Gu, L. Yao, G. Ma, L. Cui, Y. Li, W. Liang, et al., TCTP promotes glioma cell proliferation in vitro and in vivo via enhanced?-catenin/TCF-4 transcription, Neuro-Oncology 16 (2014) 217–227. [197] H. Brantjes, All Tcf HMG box transcription factors interact with Groucho-related co-repressors, Nucleic Acids Res. 29 (2001) 1410–1419. [198] H. Song, P. Hasson, Z. Paroush, A.J. Courey, Groucho oligomerization is required for repression in vivo, Mol. Cell. Biol. 24 (2004) 4341–4350. [199] G. Chen, A.J. Courey, Groucho/TLE family proteins and transcriptional repression, Gene 249 (2000) 1–16. [200] B.H. Jennings, D. Ish-Horowicz, S. Stifani, C. Blaumueller, N. Redhead, R. Hill, et al., The Groucho/TLE/Grg family of transcriptional co-repressors, Genome Biol. 9 (2008) 205. [201] A. Hecht, C.M. Litterst, O. Huber, R. Kemler, Functional characterization of multiple Transactivating elements in -catenin, some of which interact with the TATAbinding protein in vitro, J. Biol. Chem. 274 (1999) 18017–18025. [202] A. Hecht, The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates, EMBO J. 19 (2000) 1839–1850. [203] K.-I. Takemaru, R.T. Moon, The transcriptional coactivator Cbp interacts with βcatenin to activate gene expression, J. Cell Biol. 149 (2000) 249–254. [204] D.L. Daniels, W.I. Weis, ICAT inhibits β-catenin binding to Tcf/Lef-family transcription factors and the general coactivator p300 using independent structural

42

Cellular Signalling 40 (2017) 30–43

G.G. Tortelote et al.

[238]

[239] [240]

[241]

notch, hedgehog, and Wnt pathways in cancer stem cells: clinical update, Nat. Rev. Clin. Oncol. 12 (2015) 445–464. [242] P.N. Le, J.D. McDermott, A. Jimeno, Targeting the Wnt pathway in human cancers: therapeutic targeting with a focus on OMP-54F28, Pharmacol. Ther. 146 (2015) 1–11. [243] A.M. Lebensohn, R. Dubey, L.R. Neitzel, O. Tacchelly-Benites, E. Yang, C.D. Marceau, et al., Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling, elife 5 (2016), http://dx.doi.org/10. 7554/eLife.21459. [244] M. Dahlmann, D. Kobelt, W. Walther, G. Mudduluru, U. Stein, S100A4 in cancer metastasis: Wnt signaling-driven interventions for metastasis restriction, Cancers (Basel) 8 (2016), http://dx.doi.org/10.3390/cancers8060059.

Requirement for Wnt3 in vertebrate axis formation, Nat. Genet. 22 (1999) 361–365. M.A. Lancaster, M. Renner, C.-A. Martin, D. Wenzel, L.S. Bicknell, M.E. Hurles, et al., Cerebral organoids model human brain development and microcephaly, Nature 501 (2013) 373–379. M.A. Lancaster, J.A. Knoblich, Generation of cerebral organoids from human pluripotent stem cells, Nat. Protoc. 9 (2014) 2329–2340. M. Eiraku, K. Watanabe, M. Matsuo-Takasaki, M. Kawada, S. Yonemura, M. Matsumura, et al., Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals, Cell Stem Cell 3 (2008) 519–532. N. Takebe, L. Miele, P.J. Harris, W. Jeong, H. Bando, M. Kahn, et al., Targeting

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