Biochimica et Biophysica Acta (BBA) - General Subjects
ReviewOn the role of Wnt/β-catenin signaling in stem cells☆
Highlights
► Wnt proteins are secreted glycoproteins that can activate different intracellular signaling pathways. ► Wnt signaling supports self-renewal and pluripotency of embryonic stem cells. ► Wnt signaling can improve reprogramming of somatic cells towards iPS. ► Wnt signaling is important for maintenance of intestinal stem cells.
Introduction
Wnt proteins were discovered about 30 years ago [1]. They are secreted glycoproteins acting as growth factors that regulate diverse processes such as cell proliferation, differentiation, migration, polarity and asymmetric cell division. The family comprises 19 members that are characterized by a conserved pattern of cysteine residues. Wnt proteins can also be lipid modified [2], [3], [4]. To initiate intracellular signal transduction, they interact with seven transmembrane receptors of the Frizzled (Fzd) family [5] and with single pass transmembrane co-receptors such as Lrp5/6 [6], [7], Ror2 [8], and Ryk [9]. The Frizzled family of Wnt receptors contains 10 members [10] which are named according to the International Union of Basic and Clinical Pharmacology (IUPHAR) as FZD1–10 and according to the human genome organization (HUGO) as FZD1–10. In non-mammalian organisms such as Caenorhabditis elegans, Drosophila melanogaster, zebrafish or Xenopus laevis divergent nomenclatures are in use for Frizzled receptors. Interaction between Wnt ligands and Wnt receptors results in an activation of different intracellular signaling pathways that are highly interconnected including the Wnt/β-catenin, the Wnt/JNK and the Wnt/calcium pathway [11].
In this review, we mainly focus on the best characterized Wnt signaling branch, the Wnt/β-catenin pathway also often referred to as canonical Wnt pathway (Fig. 1). Main feature of this pathway is the regulation of cytoplasmic β-catenin protein stability. In the absence of a Wnt ligand, cytoplasmic β-catenin is degraded upon interaction with the destruction complex formed by the three proteins APC, Axin and GSK3β. Axin is the limiting component in this complex [12] and regulation of Axin stability thus has a direct impact on the destruction complex. Axin can be ADP ribosylated by tankyrase which results in its degradation [13].
A key event in β-catenin degradation in this process is the phosphorylation of β-catenin at multiple serine and threonine residues in a dual kinase mechanism by CKIε and GSK3β [14]. Initial CKI phosphorylation occurs at Ser45 which primes the molecule for subsequent phosphorylation by GSK3β on Thr41, Ser37 and Ser33. In particular the final phosphorylation at Ser33 by GSK3β is of particular importance as Ser33 phosphorylated β-catenin is polyubiquitinated and degraded by the proteasome. In the presence of a Wnt ligand, GSK3β is inhibited by a not yet fully understood mechanism resulting in unphosphorylated β-catenin at Ser33. Earlier data suggested that the destruction complex is disassembled upon an incoming Wnt signaling whereas more recent data suggested that the complex is translocated to the membrane with simultaneous inhibition of β-catenin ubiquitination. In this model the destruction complex is saturated by phosphorylated β-catenin [15]. In both models, β-catenin is therefore not degraded anymore and accumulates in the cytoplasm.
Activation of the Wnt/β-catenin pathway involves binding of a Wnt ligand to Frizzled and Lrp5/6 forming a heterotrimeric complex. Subsequent molecular events that result in the inactivation of the destruction complex include the phosphorylation and translocation of Disheveled to Frizzled receptors and the phosphorylation of Lrp5/6 by CKIγ and GSK3β. This process requires Wnt triggered formation of phosphatidylinositol-4,5-bisphosphate (PIP2), a process mediated by Disheveled activated phosphatidylinositol 4-kinase type IIα (PI4KIIα) and phosphatidylinositol-4-phosphate 5-kinase type Iβ (PIP5KIβ) [16], [17]. PIP2 is recognized by Amer which recruits CKIγ and GSK3β for Lrp phosphorylation [18].
Although Frizzleds are seven transmembrane receptors the involvement of heterotrimeric G-proteins was debated for a long time, in particular with respect to Wnt/β-catenin signaling. Increasing biochemical and genetic evidence however suggests that all Wnt signaling pathways involve G-proteins [10], [19], [20], [21] at least in some conditions. With respect to Wnt/β-catenin signaling it is notable that Disheveled interacts with Gβγ [22] and that Gβγ promotes Lrp phosphorylation by activating and recruiting GSK3β to the membrane [23].
β-catenin is composed of an N-terminus, a central region with multiple armadillo repeats, and a C-terminal region that harbors a transcriptional activator domain. Upon stabilization, β-catenin enters the nucleus where it interacts with transcription factors of the TCF/LEF family. Through this interaction, these transcription factors turn into transcriptional activators (Fig. 1). Four members of the TCF/LEF family have been described so far, LEF1, TCF1, TCF3, and TCF4. Multiple splice variants of these transcription factors have also been identified [24]. β-catenin has an additional function in cell adhesion. It can interact with the intracellular domain of cell adhesion molecules of the cadherin family. These are calcium dependent single pass transmembrane proteins forming a family of cell adhesion molecules. They prefer homophilic interactions to establish firm cell contacts. Through interaction with α-catenin, β-catenin bridges the cytoplasmic tail of cadherins to the actin cytoskeleton (Fig. 1). An analysis of β-catenin function in any cell therefore has to consider two different aspects, Wnt signal transduction and cadherin mediated cell adhesion.
Other Wnt signaling pathways are meant to be independent of β-catenin signaling and include, among others, the Wnt/JNK and the Wnt/calcium pathway, respectively [11]. The Wnt/JNK pathway is characterized by the activation of jun-N-terminal kinase (JNK) which involves small GTPases of the rho family such as RhoA, Rac or Cdc42. Despite Frizzled receptors, Ror2 has been shown to be involved in this pathway [25], [26]. Finally, Wnt binding to Frizzled receptors can trigger intracellular calcium release and the activation of calcium dependent kinases such as protein kinase C (PKC), calcium–calmodulin dependent kinase II (CamKII) or the calcium dependent phosphatase calcineurin (CaCN) [27]. This Wnt pathway apparently does not involve any co-receptor. Of note, these pathways are highly interconnected by sharing individual molecules or cross-regulating each other. Rac1 and JNK2 for example have been shown to be required for β-catenin signaling in some cells [28] and the Wnt/calcium pathway has been shown to antagonize Wnt/β-catenin signaling [29], [30].
The rather high number of Wnt ligands, Frizzled receptors and co-receptors raises the question of signaling specificity and which ligand/receptor combinations are preferentially formed. No systematic and complete analysis has been performed to this end [31]. It is widely accepted that some Wnt proteins such as Wnt1 or Wnt3a preferentially signal through the Wnt/β-catenin pathway. In contrast, other Wnts such as Wnt5a or Wnt11 are considered to activate β-catenin independent pathways in many situations. However, regulation of Wnt signaling specificity is complex and not yet understood. Wnt3a has also been shown to activate β-catenin independent signaling [32] and Wnt5a can activate β-catenin signaling [33], [34]. This indicates that the combination of Wnt ligands, Frizzled receptors and co-receptors determines the intracellular signaling output. This might also include Frizzled homo- and heterodimers [31], [35]. Of high interest is also the fact that different Wnt signaling branches can be activated in the same cell at the same time. A first example was the discovery that Wnt3a simultaneously activates β-catenin signaling and PKCδ during bone formation [36]. More recently it was suggested that the type of pathway activated by the same ligand is concentration dependent [11], [37]. Whereas low concentrations of Wnt3a activated Wnt/calcium signaling, higher concentrations were found to trigger Wnt/β-catenin signaling.
These findings should be kept in mind when analyzing the function of Wnt proteins in stem cells as discussed below.
Chemicals and drugs that inhibit the catalytic activity of GSK3β are widely used as activators of the Wnt/β-catenin pathway. Whereas in earlier work often LiCl has been used, today more specific inhibitors such as BIO or CHIR99021 are in use. It should be kept in mind, however, that GSK3β is by far not specific for Wnt/β-catenin signaling. An inhibition of GSK3β thus might elicit phenotypes that are not necessarily linked to Wnt/β-catenin signaling. Other substances interfere with TCF/LEF/β-catenin complex formation [38]. IWR and XAV939 [13], [39] are tankyrase inhibitors resulting in an increase in Axin amount and thus enhanced β-catenin degradation. These substances therefore are inhibitors of Wnt/β-catenin signaling. The extracellular Wnt inhibitor Dkk1 [40] is often used to block Wnt/β-catenin signaling by adding Dkk1 to the culture media. Other extracellular inhibitors of Wnt signaling such as secreted Frizzled related proteins (Sfrp) or Wnt inhibiting factor WIF are not in routine use. For functional analyses of the Wnt/β-catenin pathway, a S33A mutation of β-catenin is widely used that represents a stabilized version of β-catenin.
An important matter which has not yet received full attention is the fact that activation of Wnt/β-catenin signaling by inhibiting GSK3β certainly is not equivalent to a Wnt stimulation of the same cell. As discussed above, Wnt signaling rather reflects a signaling network than a linear pathway and inhibiting at the level of GSK3β does not stimulate other signaling branches and further upstream that might be activated in parallel. Analyzing the effect of Wnt signaling in any context thus always should include an analysis of which of the signaling mediators are activated.
Stem cells are defined by two properties: i) the ability to stay in an undifferentiated state, a feature called self-renewal, and ii) the ability to differentiate into different cell types, a feature named potency. Basically, two stem cell types can be distinguished, embryonic stem (ES) cells and adult stem cells. ES cells are pluripotent stem cells that are of embryonic character and are characterized by their ability to differentiate into derivatives of all three germ layers. In contrast, adult stem cells possess a lower potential to differentiate and are only multi-, oligo- or unipotent and are located in the adult organism. ES cells represent a transitory state during embryonic development and are able to recapitulate development in vitro whereas adult stem cells are particularly involved in tissue homeostasis in the adult organism.
In the first part of this review, we summarize our knowledge on Wnt signaling in embryonic stem cells and discuss contradicting results. With respect to embryonic stem cells, we also concentrate on the impact of Wnt signaling during reprogramming of somatic cells and generation of induced pluripotent (iPS) cells. We do not, however, summarize the effect of Wnt proteins during differentiation of ES cells due to space restriction. In the second part, we will focus on the role of Wnt signaling in adult stem cell behavior.
Stem cells with an embryonic character can occur in two different states called naive or primed, respectively [41]. Embryonic stem cells represent the naive state which is characterized by a particular open chromatin structure with relatively few epigenetic marks. Murine embryonic stem cells are derived from the inner cell mass of an E3.5 embryo. In contrast, primed pluripotent stem cells are represented by epiblast stem cells (EpiSC). They have already activated the epigenetic machinery that later on allows differentiation into the different embryonic cell types and are derived from the epiblast of E5.5–E6.5 embryos (Fig. 2).
The pluripotent state is characterized and maintained by the expression of some key transcription factors, namely Oct4, Nanog and Sox2 [42]. These factors on the one hand regulate the expression of each other in a cooperative manner. On the other hand they repress the expression of genes required for differentiation into the derivatives of the three germ layers. During early embryonic development, these factors are, however, also involved in specific differentiation processes [43].
Pluripotent stem cells require particular culture conditions that prevent differentiation. Embryonic stem cells have been cultured on murine embryonic fibroblast as feeder cells. Those can be replaced by medium containing serum and the leukemia inhibiting factor LIF. As serum contains unknown factors and varies from batch to batch, uniform culture conditions are required with serum free medium and defined supplements to study in particular the effect of individual factors. Of note, human ES cells do not require LIF but depend on activin A, a feature which has also been attributed to EpiS cells. It has been debated thus whether human ES cells might represent a later developmental stage than murine ES cells [41]. Other signaling molecules such as Wnts (see Section 2 of this review), BMPs and FGFs were also reported to be of relevance in murine or human ES cells, respectively. Of note, these corresponding signaling pathways are interconnected and function in concert although the precise cross-talk has not been fully deciphered [44], [45], [46], [47].
In ES cell cultures, often both cell types, ES and EpiS cells, are present in parallel [48]. This heterogeneity in ES cell cultures makes it difficult to unequivocally determine the role of Wnt signaling in naive versus primed pluripotent cells. Furthermore, it has to be kept in mind that the different genetic background of ES cell lines in use as well as different culture conditions might have an impact on experimental results. Due to these facts, no uniform model has yet been established that takes into account all aspects of Wnt signaling on self-renewal, pluripotency and differentiation of ES cells.
Section snippets
Wnt/β-catenin signaling in embryonic stem cells
Multiple studies suggested a role of Wnt/β-catenin signaling in murine ES cells. The potential role of β-catenin independent Wnt signaling in ES cells has not been investigated so far which has to be kept in mind when summarizing the influence of Wnt signaling on ES cells. The impact of Wnt/β-catenin signaling on stem cells of embryonic character can best be described by raising several questions:
- -
Is ectopic Wnt signaling able to maintain pluripotency?
- -
Is there a role of Wnt signaling during
Adult stem cells and the concept of the stem cell niche
Adult stem cells are found in most regenerative tissues of adult organisms such as the bone marrow, the skin and the intestine just to name few. They are located in the so called stem cell niches which are required to maintain stem cell properties of adult stem cells by extrinsic and intrinsic signals. Beside stem cells, components of the stem cell niche are stroma cells, endothelial cells, neurons as well as the extracellular matrix and the extracellular milieu within the niche. Upon exit out
Outlook
We have summarized here our current knowledge of Wnt/β-catenin signaling in two different populations of stem cells, embryonic stem cells and intestinal stem cells. In both populations of cells, Wnt/β-catenin signaling is required to maintain the major properties of stem cells, self-renewal and pluripotency or multipotency, respectively. Many important questions still need to be addressed and solved. We here highlight some of them. (1) Additional studies will indicate which of the effects
References (123)
- et al.
Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion
Dev. Cell
(2006) - et al.
Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism
Cell
(2002) - et al.
Wnt Signaling through Inhibition of beta-catenin degradation in an intact axin1 complex
Cell
(2012) - et al.
Regulation of phosphatidylinositol kinases and metabolism by Wnt3a and Dvl
J. Biol. Chem.
(2009) - et al.
Wnt-5A/Ror2 regulate expression of XPAPC through an alternative noncanonical signaling pathway
Dev. Cell
(2007) - et al.
The Wnt/Ca2 + pathway: a new vertebrate Wnt signaling pathway takes shape
Trends Genet.
(2000) - et al.
Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling
Cell
(2008) - et al.
Antagonistic regulation of convergent extension movements in Xenopus by Wnt/beta-catenin and Wnt/Ca2 + signaling
Mech. Dev.
(2001) - et al.
Selective activation mechanisms of Wnt signaling pathways
Trends Cell Biol.
(2009) - et al.
Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation
Dev. Cell
(2007)
Small-molecule antagonists of the oncogenic Tcf/beta-catenin protein complex
Cancer Cell
Structure-activity relationship studies of small-molecule inhibitors of Wnt response
Bioorg. Med. Chem. Lett.
Naive and primed pluripotent states
Cell Stem Cell
Pluripotency factors in embryonic stem cells regulate differentiation into germ layers
Cell
Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states
Cell Stem Cell
Wnt3a but not Wnt11 supports self-renewal of embryonic stem cells
Biochem. Biophys. Res. Commun.
Generation of multipotential mesendodermal progenitors from mouse embryonic stem cells via sustained Wnt pathway activation
J. Biol. Chem.
Capture of authentic embryonic stem cells from rat blastocysts
Cell
Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors
Cell Stem Cell
Beta-catenin signaling contributes to stemness and regulates early differentiation in murine embryonic stem cells
FEBS Lett.
A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo
Dev. Cell
Beta-catenin enhances Oct-4 activity and reinforces pluripotency through a TCF-independent mechanism
Cell Stem Cell
Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells
Biochem. Biophys. Res. Commun.
Regulation of TCF3 by Wnt-dependent phosphorylation during vertebrate axis specification
Dev. Cell
Phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2
J. Biol. Chem.
An Oct4-centered protein interaction network in embryonic stem cells
Cell Stem Cell
An expanded Oct4 interaction network: implications for stem cell biology, development, and disease
Cell Stem Cell
Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors
Cell
Wnt signaling promotes reprogramming of somatic cells to pluripotency
Cell Stem Cell
Periodic activation of Wnt/beta-catenin signaling enhances somatic cell reprogramming mediated by cell fusion
Cell Stem Cell
Wnt7a activates the planar cell polarity pathway to drive the symmetric expansion of satellite stem cells
Cell Stem Cell
Wnt proteins are self-renewal factors for mammary stem cells and promote their long-term expansion in culture
Cell Stem Cell
De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated beta-catenin in skin
Cell
Three decades of Wnts: a personal perspective on how a scientific field developed
EMBO J.
Wnt proteins are lipid-modified and can act as stem cell growth factors
Nature
Post-translational palmitoylation and glycosylation of Wnt-5a are necessary for its signalling
Biochem. J.
A Drosophila tissue polarity locus encodes a protein containing seven potential transmembrane domains
Nature
Arrow encodes an LDL-receptor-related protein essential for wingless signalling
Nature
LDL-receptor-related proteins in Wnt signal transduction
Nature
Wnt5a induces homodimerization and activation of Ror2 receptor tyrosine kinase
J. Cell. Biochem.
Wnt-mediated axon guidance via the Drosophila derailed receptor
Nature
International Union of Basic and Clinical Pharmacology. LXXX. The class Frizzled receptors
Pharmacol. Rev.
From individual Wnt pathways towards a Wnt signalling network
Philos. Trans. R. Soc. Lond. B Biol. Sci.
The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway
PLoS Biol.
Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling
Nature
Wnt3a-mediated formation of phosphatidylinositol 4,5-bisphosphate regulates LRP6 phosphorylation
Science
Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation
EMBO J.
Wnt signalling: the case of the ‘missing’ G-protein
Biochem. J.
Wnt3a stimulation elicits G-protein-coupled receptor properties of mammalian Frizzled proteins
Biochem. J.
Kinases and G proteins join the Wnt receptor complex
Bioessays
Cited by (0)
- ☆
This article is part of a Special Issue entitled Biochemistry of Stem Cells.