Wnt signaling: A complex issue

BART HENDRIKS AND ERNST REICHMANN*

Fetal and Pediatric Surgical Research Unit, Department of Surgery
University Children's Hospital Zürich. Steinwiesstr. 75, CH-8032 Zürich, Switzerland

ABSTRACT

The development of tissues and organs in multicellular organisms is controlled by the interplay of several signaling pathways that cross-talk to provide positional information and induce cell fate specification. Together with other families of secreted factors such as TGFßs, FGFs, Hedgehog and Notch proteins, Wnt growth factors are crucially implicated in these processes. Here, we will first discuss molecular mechanisms and then consider some biological consequences of Wnt signaling.

Key terms: Wnts, frizzled, LRP 5/6, ß-catenin, APC, TCF, stem cells, cancer

INTRODUCTION

The first Wnt gene, mouse Wnt-1 (Int-1), was discovered in 1982 as a proto oncogene in mammary tumors activated by integration of the mouse mammary tumor virus (Nusse and Varmus, 1982). As a consequence, the potential involvement of Wnt genes in cancer became a major area of research in the 1980s (Nusse and Varmus, 1992). With the molecular identification of the Drosophila segment polarity gene wingless (wg) as the orthologue of Wnt-1 (Cabrera et al., 1987; Rijsewijk et al., 1987) and the phenotypic analysis of Wnt-1 mutations in the mouse (McMahon and Bradley, 1990; Thomas and Capecchi, 1990), it became clear that Wnt genes are important regulators of many developmental decisions (Parr and McMahon, 1994; Cadigan and Nusse, 1997) At present, close to 100 Wnt genes have been isolated from species ranging from human to the nematode Caenorhabditis elegans and the simple metazoan Hydra. Presumably, all Wnt proteins are secreted from cells and act through cell surface receptors either on the producing cell or on adjacent cells to determine cell fate or other differentiation parameters.

Within target cells, Wnt signaling involves the control of several dynamically interacting protein complexes. In that context, the protein ß-catenin is of particular importance since this multitude of protein-protein interactions is employed to control ß-catenin levels via phosphorylation and degradation. Wnt signaling, in essence, liberates ß-catenin from these complexes thereby favoring translocation to the nucleus, where ß-catenin associates with the T cell factor (TCF) to activate transcription of target genes.

OLECULAR MECHANISMS THAT UNDERLIE WNT SIGNALING

Wnt-genes and growth factors

The Wnt genes encode a large family of secreted protein growth factors that have been identified in animals from hydra to humans. The name Wnt (pronounced `wint') denotes the relationship of this family to the Drosophila segment polarity gene `wingless' and to its vertebrate ortholog, int-1, a mouse protooncogene. Transcription of Wnt family genes appears to be developmentally regulated in a precise temporal and spatial manner (see also Aybar et al., 2002). All of the known vertebrate Wnt genes encode for 38- to 43-kDa cysteine-rich putative glycoproteins, which have features typical of secreted growth factors: a hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence, and 22 conserved cysteine residues whose relative spacing is maintained. In humans, 19 WNT proteins have been identified that share 27% to 83% amino-acid sequence identity and a conserved pattern of 23 or 24 cysteine residues. During development, Wnts have diverse roles in governing cell fate, proliferation, migration, polarity, and death. In adults, Wnts function in homeostasis, and inappropriate activation of the Wnt pathway is implicated in a variety of cancers.

Wnt-Receptors

Wnt ligands signal via seven transmembrane spanning receptors of the Frizzled family (ten members in the human genome) together with the recently identified LRP5 and LRP6 coreceptors, which are members of the low-density lipoprotein receptor-related protein family (LRP) (Wodarz and Nusse, 1998; Tamai et al., 2000). LRP6 associates with Frizzled in a Wnt-dependent manner (Fig. 1). A mutant of LRP6 lacking the intracellular domain blocks signaling by Wnt in Xenopus embryos. Deletion of the LRP6 gene in mice results in developmental defects that are strikingly similar to those caused by mutations of individual Wnt genes. Wnt stimulation of LRP5 has been shown to induce interaction with Axin and subsequent activation of Wnt target genes (Mao et al., 2001).

It has been shown that Wnt signaling is inhibited by the secreted protein Dickkopf 1 (Dkk1), a member of a multigene family, which induces head formation in amphibian embryos (Glinka et al., 1998) (Fig. 1). Dkk1 has been demonstrated to inhibit Wnt signaling by binding to and antagonizing LRP5/6 (Mao et al., 2001). There is recent evidence that the transmembrane proteins Kremen 1 and Kremen2 are high-affinity Dkk1 receptors that functionally cooperate with Dkk1 to block Wnt signaling (Fig. 1). Kremen 2 forms a ternary complex with Dkk1 and LRP6, and induces rapid endocytosis and removal of the LRP6 from the plasma membrane. From these data it is concluded that Kremen 1 and Kremen 2 are components of a membrane complex which modulates canonical Wnt signaling through LRP6 in vertebrates (Mao et al., 2002).

Fig. 1. Diversification of the Wnt signaling pathway and the dual role role of ß-catenin. See text for details. This scheme is adapted from Figure 1 in Current Opinion in Genetics & Development 2001, 11:547-553 by J. Huelsken and W. Birchmeier

The canonical Wnt pathway

The canonical Wnt pathway (ß-catenin pathway) leads to activation of target genes in the nucleus (Fig.1). In this pathway ß-Catenin/Armadillo is stabilized by preventing its degradation in proteasomes. Activated Frizzled receptors signal through a conserved motif (Umbhauer et al., 2000) to Dishevelled but the mechanism of Dishevelled activation is still ill-defined. Casein kinase Ie has recently been identified as an essential positive regulator that acts downstream of Dishevelled and regulates ß-catenin stability (Sakanaka et al., 1999; Peters et al., 1999). Axin/Conductin, in cooperation with the tumor suppressor gene product APC, promote ß-catenin degradation (Fig. 1). This involves serine-threonine phosphorylation of the amino-terminus of ß-catenin by GSK3ß (Fig. 1) and subsequent ubiquitination (Behrens et al., 1998; Polakis, 2001). Stabilized ß-catenin accumulates in the cytoplasm and is translocated to the nucleus, where it interacts with members of the LEF/TCF family of transcription factors and activates gene expression (Behrens et al., 1996; Molenaar et al., 1996) (Fig. 1).

It has recently been shown that the Drosophila segment polarity gene legless (lgs), a homolog of human BCL 9, functions as an adaptor protein to physically link Pygo to the ß-catenin-TCF complex (Fig. 1). This recruitment of Pygo is required for ß-catenin to function as a transcriptional coactivator and hence to enable the Wnt/Wg system to induce target genes (Kramps et al., 2002) (Fig. 1).

LEF/TCF can prevent transcriptional activation when bound to transcriptional co-repressors like CREB-binding protein (CBP), CtBP or members of the Groucho family (see Fig. 1 and Sharpe et al., 2001 for a review). Other results indicate that chromatin remodeling may be an important aspect of Wnt-induced gene regulation (Sharpe et al., 2001; Collins and Treisman, 2000; Sawa et al., 2000).

THE TCF/LEF FAMILY OF TRANSCRIPTION FACTORS

The founding members of the TCF/LEF family of transcription factors, TCF-1 and LEF-1, were identified in screens for T cell-specific transcription factors. TCF-1 was identified by its ability to bind to the CD3e enhancer, whereas LEF-1 was found in a screen for proteins binding to the TCRa enhancer and a site in the HIV LTR (Oosterwegel et al., 1991; van de Wetering et al., 1991; Travis et al., 1991). In more recent years, two additional family members were identified in mammals: TCF-3 and TCF-4 (Korinek et al., 1998). The Drosophila genome only contains one TCF gene, called dtcf or pangolin (Brunner et al., 1997; van de Wetering et al., 1997). Similarly, a single gene, Pop-1, resides in the genome of the nematode Caenorhabditis elegans (Lin et al., 1995). Proteins of the TCF/LEF family contain an 80-amino-acid high mobility group (HMG) box. HMG boxes bind DNA as monomers and can do so in a sequence-specific manner (Grosschedl et al., 1994; Laudet et al., 1993). However, the HMG box not only recognizes specific DNA sequences, but also induces a dramatic bend in the DNA structure (Giese et al., 1992; Dooijes et al., 1993). In doing so, HMG boxes may coordinate the binding of other transcription factors (Grosschedl et al., 1994; Giese et al., 1995; Bianchi and Beltrame, 1998).

This "DNA-bending" activity of TCF/LEFs, in conjunction with the observation that these factors cannot directly activate transcription in reporter assays, suggests that TCF/LEF family members primarily serve an architectural function. LEF-1 appears unique in that it contains a context-dependent activation domain (CAD) (Carlsson et al., 1993), which can activate transcription in the presence of the coactivator ALY (Bruhn et al., 1997). The other TCF family members do not appear to contain a CAD domain (Van de Wetering et al., 1996).

TCF/Lef mRNAs undergo extensive alternative splicing. In addition, the best studied gene of this family, TCF-1, also exhibits alternative promoter usage. Over 100 TCF-1 isoforms may theoretically be produced. However, Western blotting has revealed the predominant expression of only eight different TCF-1 isoforms (Van de Wetering et al., 1996). LEF-1 exhibits two splice variants (Waterman et al., 1991) and also possesses two promoters (Hovanes et al., 2001). Several C-terminal splice variants of TCF-4 exist that exhibit strong sequence homology to similar TCF-1 mRNAs (Korinek et al., 1997; Duval et al., 2000) (Fig. 1). While the functional relevance of alternative splice products from TCF/Lef genes is currently unclear, alternative promoter usage of both genes generates protein isoforms that either contain or lack the N-terminal ß-catenin interaction domain.

Target genes of Wnt signaling

In most models, Wnt signaling is predominantly implicated in control of cell fate by altering the transcriptional program of target cells in an instructive fashion. Consistent with these models, mutation of Wnt genes or inappropriate expression of Wnts usually lead to changes in cell fate as a consequence of altered gene expression. Not surprisingly, many genes regulated directly or indirectly by Wnts are transcription factors or secreted signaling molecules thought to represent key players in the plethora of genes controlling development. Among the best-studied examples are members of the homeobox gene family, including engrailed (en), ultrabithorax (ubx) and siamois. For two of these genes, ubx and siamois, there is evidence for direct transcriptional activation by binding of an Arm/ß-catenin complex together with HMG-box proteins to specific sites in the target gene promoter region (Brannon et al., 1997; Riese et al., 1997). Both these studies found that transcriptional activation by Wnt signaling alone is not sufficient to explain the normal expression pattern of target genes. Other sources of patterning information appear to be integrated at the level of the target gene promoter, giving rise to the final transcriptional pattern. Such combinatorial gene activation may explain how Wnt signaling can regulate a large number of different target genes and participate in apparently unrelated processes depending on the cellular context in which signaling takes place. One example for tissue-specific differences in target gene regulation is the effect of Wg on the expression of achaete (ac), a proneural gene. In the wing imaginal disc, Wg activates ac expression (Couso et al., 1994), whereas in the eye imaginal disc, Wg acts as a repressor of ac (Cadigan and Nusse, 1996). Cooperation with other signaling pathways at the level of target gene promoters may also explain why ubiquitous expression of Wg does not lead to ubiquitous expression of Wg target genes (Noordermeer et al., 1992; Sampedro et al., 1993; Baylies et al., 1995).

The generation of transcriptionally active TCF/ß-catenin complexes in tumor cells results in the inappropriate activation of TCF target genes, including c-myc and cyclin D1, (He et al., 1998; Tetsu and McCormick, 1999) which may ultimately lead to cancer. The expression of c-myc was found to be changed after induction of wild-type APC in an APC-deficient colon carcinoma cell line. Subsequent analysis showed that the c-myc promoter was activated by TCF-4/ß-catenin via specific binding sites, and that of endogenous c-myc could be blocked by a dominant-negative TCF-4 (He et al., 1998). Because c-myc overexpression previously had been linked to colorectal tumorigenesis, it is possible that TCF-4/ß-catenin induces neoplastic growth via activation of c-myc. Furthermore, this study indicates that several other genes associated with tumorigenesis are up- or down-regulated by APC. Components of the c-jun family and the metalloproteinase matrilysin were also shown to be targets of TCF/ß-catenin (Mann et al., 1999; Crawford et al., 1999).

Diversification of the Wnt signal

More recently, at least four different branches are being distinguished within the Wnt pathway. These include the canonical Wnt pathway, the planar cell polarity pathway, the spindle orientation and asymmetric cell division pathway and the Wnt/Ca²⁺ pathway. These branches will be discussed in the order indicated. The canonical Wnt pathway has been described above.

In the planar cell polarity pathway Wnt signaling activates Jun-N-terminal kinase (JNK) and directs asymmetric cytoskeletal organization and coordinated polarization of cell morphology within the plane of epithelial sheets. Examples of Wnt-mediated JNK activation are seen during eye and wing development of Drosophila and gastrulation movements in Zebrafish and Xenopus (Mlodzik, 2000; Heisenberg et al., 2000). This pathway branches at the level of Dishevelled (Fig. 1) and involves downstream components like the small guanosine triphosphatase Rho and a kinase cascade including Misshapen (a Ste20 homologue), JNK kinase and JNK (Mlodzik, 2000; Wallingford et al., 2000). Drosophila Rho-associated kinase (Drok) was identified recently as a target of the pathway that regulates the activity of myosin and hence provides a direct link to the cytoskeleton (Winter et al., 2001).

The mechanism underlying Wnt signaling divergence into either the canonical or the planar cell polarity pathway is presently the subject of intense investigation. Particular Wnts and Frizzled receptors, like Wnt11 or Frizzled 7, specifically activate the JNK pathway, as described for convergent extension movements of the mesoderm during Xenopus and zebrafish gastrulation (Heisenberg et al., 2000). The Wnt target gene naked/naked cuticle was identified recently as an antagonist for Wnt signaling. Naked protein binds to Dishevelled and blocks ß-catenin but stimulates the JNK pathway (Zeng et al., 2000; Yan et al., 2001; Rousset et al., 2001).

Several members of the Wnt-signaling pathway like GSK3ß or APC have been implicated in the pathway which regulates spindle orientation and asymmetric cell division in C. elegans and Drosophila. In the central nervous system of Drosophila, neuroepithelial cells divide symmetrically along the planar axis. Neuroblasts originate from this layer by asymmetric divisions and undergo further differentiation (Lu et al., 2001). The integrity of adherens junctions in the neuroepithelium was shown to provide a planar polarity cue required for symmetrical division, and APC associated with adherens junctions as well as microtubule-associated EB1 are necessary for this process (Lu et al., 2001).

The Wnt/Ca²⁺ pathway leads to release of intracellular calcium, possibly via a G-protein mediated process (Fig. 1). This pathway involves activation of phospholipase C, protein kinase C and calmodulin-dependent kinase II and is implicated in Xenopus ventralization and in the regulation of convergent extension movements (Kuhl et al., 2000; Wallingford et al., 2001).

BIOLOGICAL ASPECTS OF WNT SIGNALING

Wnt signaling in body axis formation and mesoderm patterning

ß-Catenin and other components of the canonical Wnt pathway have previously been found to be essential for body axis formation in Xenopus and Zebrafish (Moon and Kimelman, 1998; Solnica-Krezel, 1999). In Xenopus, accumulation of ß-catenin on the dorso-anterior side of the embryo is the earliest sign of axis formation and precedes gastrulation (Schneider et al., 1996; Larabell et al., 1997). Overexpression of ß-catenin induces formation of an additional embryonic axis (Heasman et al., 1994). Interestingly, members of the Wnt-signaling cascade have also been implicated recently in establishing the body axis in Hydra, a member of the evolutionary ancient metazoan phylum Cnidaria (Hobmayer et al., 2000). This suggests that Wnt signaling is an evolutionarily conserved process important for axial differentiation in all multicellular animals.

Wnt signaling in cell fate specification and stem cell control

Stem cells are pluripotent cells found in many tissues, that can adopt various fates. When exposed to particular growth factors and cytokines, they generate progenitors that proliferate transiently and then withdraw from the cell cycle and terminally differentiate (Fuchs and Segre, 2000). Currently, a great deal of research is directed towards identifying molecules that maintain stem cells and that control their commitment to particular lineages. Recently, the Wnt/ß-catenin signaling pathway was implicated in the control of stem cell specification in the skin (Huelsken et al., 2001). Skin stem cells reside in the bulges of hair follicles and are bipotent, as they give rise to both keratinocytes of the hair follicle and the interfollicular epidermis (Cotsarelis et al., 1999; Taylor et al., 2000; Jensen et al., 1999). These bulge stem cells represent the reservoir that allows both self-renewal of the epidermis and cyclic regeneration of hair follicles. Keratin 14-Cre mediated conditional ablation of the ß-catenin gene in the skin of mice blocked the differentiation of stem cells into the follicular lineages, since keratinocytes could only adopt the epidermal fate (Huelsken et al., 2001). In wild-type skin, ß-catenin might cooperate in the stem cells with LEF-1 and/or TCF-3 transcription factors to control these decisions over cell fate (van Genderen et al., 1994; DasGupta and Fuchs, 1999; Merrill et al., 2001). Detailed analysis revealed that, in the absence of ß-catenin, skin stem cells fail to initiate follicular morphogenesis but do give rise to dermal cysts that exhibit features of epidermal differentiation, while lacking markers of follicular keratinocytes. These cysts and the associated stem cells remain observable for many weeks in the skin of knock out mice. The data imply that ß-catenin is necessary for instructing stem cells to form follicular keratinocytes. ß-catenin alone, however, may be insufficient to induce follicular differentiation of keratinocytes, since additional mesenchymal signals are likely to be required.

Wnt signaling in cancer

Wnts do not appear to play a dominant role in human cancer. Rather, mutations in downstream components of the Wnt cascade are a major cause of several types of cancer. APC is mutated at both alleles in 80% of all colon carcinomas (Polakis et al., 1999). These mutations almost invariably lead to a truncation of APC, removing its interaction domains for ß-catenin and axin. As a consequence, the activity of the destruction complex is impaired and cytosolic ß-catenin is no longer phosphorylated and degraded. Thus accumulated ß-catenin in turn inappropriately activates one of the TCF/LEF family members, TCF4, that is specifically expressed in the intestinal epithelium (Korinek et al., 1997). In a significant fraction of sporadic colon tumors lacking APC mutations, mutations were found in the CTNNB1 (ß-catenin) gene. These mutations occur in or around the third exon, which encodes the putative phosphorylation sites for GSK-3ß and remove the target residues of this kinase (Morin et al., 1997; Polakis et al., 1999). Mutations in ß-catenin are not only found in intestinal tumors. The CTNNB1 gene has been shown to be extensively mutated in various other tumors. As the exception that perhaps confirms the rule, mutations in Axin/Conduction have rarely been found in colon tumors and other types of malignancies (Webster et al., 2000).

Taken together, mutations in APC, axin, and ß-catenin share a common denominator, in that they all promote formation of ß- catenin/TCF complexes. This implies that inappropriate activation of TCF target genes represents the primary event in cellular transformation of epithelial cells (reviewed in Polakis, 2000; Fodde and Smits, 2001).

PERSPECTIVES

Despite the impressive recent advances that have substantially improved our understanding of Wnt signaling, many gaps remain to be filled before we can hope to get a glimpse of the complete picture. Open questions include:

Given the importance of the signaling pathway and the multitude of experimental systems being used to study Wnt signaling, answers to remaining questions can be expected to come from many different directions at an accelerated pace.

ACKNOWLEDGEMENTS

Our research is supported by the Swiss National Science Foundation and the Swiss Cancer League.

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Corresponding author: Ernst Reichmann. tel.: +41 1 266 7493 (7111). fax: +41 1 266 7170. e-mail: Ernst.Reichmann@kispi.unizh.ch

Received: July 02, 2002. In revised form: July 09, 2002. Accepted: July 16, 2002