Ken M. Cadigan

Wnt signaling: complexity at the surface

Wnts are secreted proteins that are essential for a wide array of developmental and physiological processes. They signal across the plasma membrane by interacting with serpentine receptors of the Frizzled (Fz) family and members of the low-density-lipoprotein-related protein (LRP) family. Activation of Fz-LRP promotes the stability and nuclear localization of β-catenin by compromising the ability of a multiprotein complex containing axin, adenomatosis polyposis coli (APC) and glycogen synthase kinase 3 (GSK3) to target it for degradation and block its nuclear import. The Fz-LRP receptor complex probably accomplishes this by generating multiple signals in the cytoplasm. These involve activation of Dishevelled (Dsh), possibly through trimeric G proteins and LRP-mediated axin binding and/or degradation. However, individual Wnts and Fzs can activate both β-catenin-dependent and -independent pathways, and Fz co-receptors such as LRP probably provide some of this specificity. Additional, conflicting data concern the role of the atypical receptor tyrosine kinase Ryk, which might mediate Wnt signaling independently of Fz and/or function as a Fz co-receptor in some cells.

Wnt Signaling from Development to Disease: Insights from Model Systems

One of the early surprises in the study of cell adhesion was the discovery that beta-catenin plays dual roles, serving as an essential component of cadherin-based cell-cell adherens junctions and also serving as the key regulated effector of the Wnt signaling pathway. Here, we review our current model of Wnt signaling and discuss how recent work using model organisms has advanced our understanding of the roles Wnt signaling plays in both normal development and in disease. These data help flesh out the mechanisms of signaling from the membrane to the nucleus, revealing new protein players and providing novel information about known components of the pathway.

TCF/LEFs and Wnt Signaling in the Nucleus

T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors are the major end point mediators of Wnt/Wingless signaling throughout metazoans. TCF/LEFs are multifunctional proteins that use their sequence-specific DNA-binding and context-dependent interactions to specify which genes will be regulated by Wnts. Much of the work to define their actions has focused on their ability to repress target gene expression when Wnt signals are absent and to recruit β-catenin to target genes for activation when Wnts are present. Recent advances have highlighted how these on/off actions are regulated by Wnt signals and stabilized β-catenin. In contrast to invertebrates, which typically contain one TCF/LEF protein that can both activate and repress Wnt targets, gene duplication and isoform complexity of the family in vertebrates have led to specialization, in which individual TCF/LEF isoforms have distinct activities.

The microRNA miR-8 is a conserved negative regulator of Wnt signaling

Wnt signaling plays many important roles in animal development. This evolutionarily conserved signaling pathway is highly regulated at all levels. To identify regulators of the Wnt/Wingless (Wg) pathway, we performed a genetic screen in Drosophila. We identified the microRNA miR-8 as an inhibitor of Wg signaling. Expression of miR-8 potently antagonizes Wg signaling in vivo, in part by directly targeting wntless, a gene required for Wg secretion. In addition, miR-8 inhibits the pathway downstream of the Wg signal by repressing TCF protein levels. Another positive regulator of the pathway, CG32767, is also targeted by miR-8. Our data suggest that miR-8 potently antagonizes the Wg pathway at multiple levels, from secretion of the ligand to transcription of target genes. In addition, mammalian homologues of miR-8 promote adipogenesis of marrow stromal cells by inhibiting Wnt signaling. These findings indicate that miR-8 family members play an evolutionarily conserved role in regulating the Wnt signaling pathway.

C-terminal-binding protein directly activates and represses Wnt transcriptional targets in Drosophila.

Regulation of Wnt transcriptional targets is thought to occur by a transcriptional switch. In the absence of Wnt signaling, sequence‐specific DNA‐binding proteins of the TCF family repress Wnt target genes. Upon Wnt stimulation, stabilized β‐catenin binds to TCFs, converting them into transcriptional activators. C‐terminal‐binding protein (CtBP) is a transcriptional corepressor that has been reported to inhibit Wnt signaling by binding to TCFs or by preventing β‐catenin from binding to TCF. Here, we show that CtBP is also required for the activation of some Wnt targets in Drosophila. CtBP is recruited to Wnt‐regulated enhancers in a Wnt‐dependent manner, where it augments Armadillo (the fly β‐catenin) transcriptional activation. We also found that CtBP is required for repression of a subset of Wnt targets in the absence of Wnt stimulation, but in a manner distinct from previously reported mechanisms. CtBP binds to Wnt‐regulated enhancers in a TCF‐independent manner and represses target genes in parallel with TCF. Our data indicate dual roles for CtBP as a gene‐specific activator and repressor of Wnt target gene transcription.

CBP/p300 are bimodal regulators of Wnt signaling.

Many Wnts influence cell behavior by a conserved signaling cascade that promotes the stabilization and nuclear accumulation of β‐catenin (β‐cat), which then associates with TCF family members to activate target genes. The histone acetyltransferase CREB binding protein (CBP) can bind to TCF and inhibit Wnt signaling in Drosophila. In contrast, studies in vertebrates indicate a positive role for CBP and the closely related protein p300 as β‐cat binding transcriptional co‐activators. We address this discrepancy by demonstrating that in addition to its negative role, CBP has an essential positive role in Wnt signaling in flies. CBP binds directly to the C‐terminus of Armadillo (Arm, the fly β‐cat) and is recruited to a Wnt‐regulated enhancer (WRE) in a Wnt‐ and Arm‐dependent manner. In a human colorectal cancer cell line, we show that CBP and p300 can inhibit Wnt signaling and demonstrate that human p300 can bind directly to TCF4 in vitro. Our results argue that CBP/p300 has an evolutionarily conserved role as a buffer regulating TCF‐β‐cat/Arm binding. Subsequent to this interaction, it also has an essential role in mediating the transactivation activity of β‐cat/Arm.

How do they do Wnt they do?: regulation of transcription by the Wnt/β‐catenin pathway

Wnt/β‐catenin signalling is known to play many roles in metazoan development and tissue homeostasis. Misregulation of the pathway has also been linked to many human diseases. In this review, specific aspects of the pathway’s involvement in these processes are discussed, with an emphasis on how Wnt/β‐catenin signalling regulates gene expression in a cell and temporally specific manner. The T‐cell factor (TCF) family of transcription factors, which mediate a large portion of Wnt/β‐catenin signalling, will be discussed in detail. Invertebrates contain a single TCF gene that contains two DNA‐binding domains, the high mobility group (HMG) domain and the C‐clamp, which increases the specificity of DNA binding. In vertebrates, the situation is more complex, with four TCF genes producing many isoforms that contain the HMG domain, but only some of which possess a C‐clamp. Vertebrate TCFs have been reported to act in concert with many other transcription factors, which may explain how they obtain sufficient specificity for specific DNA sequences, as well as how they achieve a wide diversity of transcriptional outputs in different cells.

A copper-regulated transporter required for copper acquisition, pigmentation, and specific stages of development in Drosophila melanogaster

The trace element copper is required for normal growth and development, serving as an essential catalytic co-factor for enzymes involved in energy generation, oxidative stress protection, neuropeptide maturation, and other fundamental processes. In yeast and mammals copper acquisition occurs through the action of the Ctr1 family of high affinity copper transporters. Here we describe studies using Drosophila melanogaster to investigate the role of copper acquisition through Ctr1 in normal growth and development. Three distinct Drosophila Ctr1 genes (Ctr1A, Ctr1B, and Ctr1C) have been identified, which have unique expression patterns over the course of development. Interestingly, Ctr1B, which is expressed exclusively during the late embryonic and larval stages of development, is transcriptionally activated in response to nutritionally induced copper deprivation and down-regulated in response to copper adequacy. The generation of Ctr1B mutant flies results in decreased larval copper accumulation, marked body pigmentation defects that parallel defects in tyrosinase activity, and specific developmental arrest under conditions of both nutritional copper limitation and excess. These studies establish that copper acquisition through the Drosophila Ctr1B transporter is crucial for normal growth and in early and specific stages of metazoan development.

Regulating morphogen gradients in the Drosophila wing

During development, diffusible ligands, known as morphogens, are thought to move across fields of cells, regulating gene expression in a concentration dependent manner. The case for morphogens has been convincingly made for the Decapentapleigic (Dpp), Wingless (Wg) and Hedgehog (Hh) proteins in the Drosophila wing. In each case, the concentration of the morphogens receptor plays an important role in shaping the morphogen gradient, through influencing ligand transport and/or stability. However, the relationships between each ligand/receptor pair are different. The role of heparan sulfated proteoglycans, endocytosis and novel exovesicles called argosomes in regulating morphogen distribution will also be discussed.

Wnt–β-catenin signaling

After stabilization, β-catenin translocates across the nuclear pore complex to the nucleoplasm. It is thought that this is an intrinsic property of β-catenin, although there are several factors that can influence its nuclear import or export. Once in the nucleus, β-catenin can act as a transcriptional co-regulator, binding to specific DNA-binding transcription factors. Although the list of proteins known to recruit β-catenin to target genes is growing — Foxo, PitX2, SOX9 and SOX17, for example — most cases of Wnt-mediated transcriptional regulation involve members of the TCF protein family.While the interaction of β-catenin and TCF is critical for activation of Wnt targets, it is also critical that these targets are not activated inappropriately by small amounts of nuclear β-catenin. Several different factors have been identified that act as nuclear β-catenin buffers — they compete with the β-catenin–TCF interaction by binding to either factor. ICAT, Chibby, Sox9 and CtBP-APC are among the factors that bind to β-catenin and inhibit it binding to TCF. Members of the TLE/Gro family do the same for TCFs (Figure 3Figure 3A). These nuclear factors raise the threshold of nuclear β-catenin required for its recruitment to Wnt targets via binding to TCF.Figure 3The TCF transcriptional switch mediating activation of Wnt targets.In the absence of signaling (A), TCF represses Wnt targets by recruiting co-repressors such as TLE/Gro. Other repressive complexes also contribute to this silencing. In addition, there are several factors that act as ‘nuclear β-catenin buffers’ which prevent β-catenin–TCF interaction when β-catenin is present at low concentrations. On Wnt signaling (B), the high level of nuclear β-catenin overcomes these buffers, and β-catenin displaces the repressors from the target gene chromatin. β-catenin dependent recruitment of a variety of co-activators allows transcription to proceed.View Large Image | View Hi-Res Image | Download PowerPoint SlideIn the absence of signaling, many Wnt targets are silenced by TCF-mediated repression. This occurs in part through TCF binding to TLE/Gro, which in turn recruits histone deacetylases (Figure 3Figure 3A). Other factors have also been implicated in silencing Wnt targets — for example, Kaiso and the chromatin remodeling complex ACF — though these act in parallel to TCF–TLE/Gro and do not function on all targets.When β-catenin reaches levels sufficient to bind to TCF, this interaction displaces TLE/Gro from Wnt target genes, relieving repression. In addition, β-catenin serves as a landing platform for a variety of transcriptional co-activators. These include Legless/Bcl9 and Pygopus (Pygo) which bind to the amino-terminal half of β-catenin, while the histone acetyl transferase CBP and Parafibromin/Hyrax bind to the carboxy-terminal portion. There are many other co-activators that bind to β-catenin and promote its ability to activate Wnt target genes; however, many of these factors are likely to be gene, cell-type or species-specific. Indeed, while pygo is essential for Wnt regulation of targets in flies, mice lacking both pygo genes have a much more modest reduction in Wnt target gene expression. At the general level, however, the idea that β-catenin switches a TCF from a transcriptional repressor to an activator is a useful way to think of Wnt-mediated regulation of many target genes. While invertebrate TCFs clearly contain both the repressive and activating activities — essential in flies and worms which only have one TCF each — it appears that some vertebrate TCFs have become more specialized, with TCF3 possessing mainly silencing activity and LEF1 functioning in the activation portion of the transcriptional switch.It should be noted that there are many genes that are downregulated in response to Wnt signaling, and in some cases it has been confirmed that a TCF–β-catenin complex directly mediates this repression. How many of the genes downregulated by Wnt–β-catenin signaling are directly repressed remains an important unanswered question. The mechanism of TCF–β-catenin repression has not been worked out in detail, and it appears to be different among the few genes studied in detail. The diversity of mechanisms by which β-catenin can regulate gene expression likely explains how this pathway can perform so many essential functions throughout the animal kingdom.