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The Wnt Signaling Network: From Biology to Medicine
Summary: Randall Moon studies the highly conserved Wnt signal transduction pathways. Wnts are secreted ligands that activate receptor-mediated pathways that regulate cell proliferation, cell fate, and cell behavior in development, and stem and progenitor cells in adults. His first goal is to identify the roles of Wnt signaling at the level of the organism, with a focus on regenerative processes in adults. His second goal is to elucidate the mechanisms by which Wnts signal. His third goal is to understand how Wnt signaling is linked to diseases. This has led to a better understanding of Wnt signaling in a retinal disease and in Alzheimer's disease and to identification of a promising new approach to treatment of metastatic melanoma.
Wnts are a family of secreted proteins that activate receptor-mediated signal transduction pathways. During embryonic development, Wnt signaling is involved in specifying cell proliferation, cell fate, and cell behavior. In the adult, Wnt signaling regulates progenitor pools of cells required for homeostasis and for responses to injury, including regeneration. In the Wnt/β-catenin pathway, the secreted Wnt signal acts on Frizzled receptors of responding cells, resulting in the post-translational stabilization and nuclear accumulation of β-catenin, and to transcriptional regulation. Our lab is focused on three goals: identification of the normal roles of Wnt signaling in embryos and adults, dissection of the molecular and cellular mechanisms by which this signaling occurs, and application of this knowledge to understand Wnt signaling in disease.
Goal 1: The Roles of Wnt/β-Catenin Signaling in Regenerative Processes
Wnt/β-catenin signaling participates in a wide range of processes in both embryos and adults. In the past we showed, for example, that Wnt/β-catenin signaling specifies the dorsoventral axis of vertebrate embryos, patterns the mesoderm and the developing nervous system during and after gastrulation, and functions in organogenesis. A current challenge is to identify the roles of Wnt signaling in adults, which would shed light on whether the control of cellular processes in embryos differs from that in adults.
To consider the potential roles for Wnt signaling in adults, we have focused on regeneration, which surprisingly had not been addressed. We initially examined the zebrafish tail fin, as this structure is highly regenerative (Figure 1). Previously we had found that Wnt signaling is required for normal tail formation during development. More recently, we found that there is a striking requirement for Wnt/β-catenin signaling for the adult zebrafish tail fin to regenerate after injury and that activating signaling accelerates tail fin regeneration. We also found that regeneration of the adult zebrafish heart and the Xenopus tadpole hindlimb depends on Wnt/β-catenin signaling. Moreover, in collaboration with Leonard Zon (HHMI, Children's Hospital Boston), we showed that liver regeneration requires β-catenin signaling. We have also found that a β-catenin–independent "noncanonical" Wnt pathway antagonizes the regeneration of the tail fin, consistent with prior reports that this Wnt pathway antagonizes β-catenin in other contexts.
Goal 2: The Mechanisms of Wnt Signaling
In the broadest sense, it is clear that signal transduction pathways operate via large nonlinear protein networks, which are often only partially elucidated by genetics. We have taken full advantage of advanced screening technologies to identify complex networks of proteins that regulate β-catenin signaling, with the expectation that this broad approach will capture components of the "noncanonical" Wnt pathway that functionally antagonize β-catenin signaling.
In collaboration with Rosetta/Merck, we first used pools of small interfering RNAs (siRNAs) directed against more than 30,000 target RNAs to ask whether reduction of any of the target RNAs would modulate our optimized β-catenin–responsive luciferase reporter in human cells. We next validated and characterized each candidate gene that emerged from this primary screen. This highly effective screen identified numerous new components of the Wnt/β-catenin signaling pathway.
In a second screen, we purified protein complexes and followed that with mass spectrometry. We started with known components of Wnt pathways and worked progressively through new interacting proteins to generate a global protein-protein interaction network for the Wnt/β-catenin signal transduction pathway. This approach identified novel proteins, as well as the expected known proteins in the pathway. Overlaying the proteomic data (which define complexes of proteins but not protein function) with the siRNA data (which identify functional genes but not how the encoded proteins work) allows us to greatly accelerate the process of defining the Wnt signaling network (Figure 2).
The third screen (conducted in collaboration with the Broad Institute at MIT/Harvard University) tests diverse small molecules for their ability to activate or inhibit our optimized β-catenin–responsive reporter in cultured cells. We expect that validated small-molecule hits will provide us with new tools for reversibly activating or inhibiting Wnt signaling in short time frames.
Goal 3: Wnt Signaling in Disease
In the short term, linking mutations in humans to specific diseases enables us to study the roles of specific Wnt pathway genes in humans. For example, we have found that an allele of LRP6, a Wnt coreceptor, is linked to Alzheimer's disease and displays attenuated β-catenin signaling in cultured cells, which suggests that Wnt signaling is important in the adult brain.
As a second example, in collaboration with Michael MacCoss (University of Washington), we used mass spectrometry of β-catenin complexes to identify a novel protein that is required for promoting the degradation of β-catenin. After the publication by another group of the cloning of the WTX gene, a tumor suppressor that is mutated in 30 percent of Wilm's tumors, we realized that our novel protein is actually WTX. This connection provides insights into the underlying causes of this disease and points toward candidate therapies. Third, in a prior collaboration we had found that a Wnt receptor, Frizzled 4, is mutated in the retinal disease familial exudative vitreoretinopathy (FEVR). Independently, we had found that Frizzleds are constitutively oligomeric. When we tested a mutant allele of Frizzled 4 found in FEVR, we discovered that it binds wild-type Frizzleds, blocks their trafficking to the membrane, and thereby reduces Wnt signaling. Thus, analysis of this disease has helped validate the oligomeric nature of Frizzled proteins and revealed a plausible explanation for why this FEVR allele is genetically dominant.
In summary, our laboratory is focused on identifying the functions and mechanisms of action of Wnt signaling networks in vertebrates. We use these insights to simultaneously explore the normal roles of Wnts in development and adults, as well as potential roles of Wnt signaling in human diseases. Work in progress is now revealing a potential new therapy for one form of cancer, which is an advance made possible by first focusing on the normal biology of Wnts in vertebrates.
Grants from the National Institutes of Health and awards from the Alzheimer's Association and the Department of Defense provided partial support for some of these projects.
Last updated June 25, 2008
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