Tiki1 is required for head formation via Wnt cleavage-oxidation and inactivation.

Secreted Wnt morphogens are signaling molecules essential for embryogenesis, pathogenesis, and regeneration and require distinct modifications for secretion, gradient formation, and activity. Whether Wnt proteins can be posttranslationally inactivated during development and homeostasis is unknown. Here we identify, through functional cDNA screening, a transmembrane protein Tiki1 that is expressed specifically in the dorsal Spemann-Mangold Organizer and is required for anterior development during Xenopus embryogenesis. Tiki1 antagonizes Wnt function in embryos and human cells via a TIKI homology domain that is conserved from bacteria to mammals and acts likely as a protease to cleave eight amino-terminal residues of a Wnt protein, resulting in oxidized Wnt oligomers that exhibit normal secretion but minimized receptor-binding capability. Our findings identify a Wnt-specific protease that controls head formation, reveal a mechanism for morphogen inactivation through proteolysis-induced oxidation-oligomerization, and suggest a role of the Wnt amino terminus in evasion of oxidizing inactivation. TIKI proteins may represent potential therapeutic targets.

Head formation requires Dishevelled degradation that is mediated by March2 in concert with Dapper1.

Dishevelled (Dvl/Dsh) is a key scaffold protein that propagates Wnt signaling essential for embryogenesis and homeostasis. However, whether the antagonism of Wnt signaling that is necessary for vertebrate head formation can be achieved through regulation of Dsh protein stability is unclear. Here, we show that membrane-associated RING-CH2 (March2), a RING-type E3 ubiquitin ligase, antagonizes Wnt signaling by regulating the turnover of Dsh protein via ubiquitin-mediated lysosomal degradation in the prospective head region of Xenopus We further found that March2 acquires regional and functional specificities for head formation from the Dsh-interacting protein Dapper1 (Dpr1). Dpr1 stabilizes the interaction between March2 and Dsh in order to mediate ubiquitylation and the subsequent degradation of Dsh protein only in the dorso-animal region of Xenopus embryo. These results suggest that March2 restricts cytosolic pools of Dsh protein and reduces the need for Wnt signaling in precise vertebrate head development.

Dab2 is pivotal for endothelial cell migration by mediating VEGF expression in cancer cells.

Although angiogenesis is crucial for tumor growth and metastasis, the molecular mechanisms controlling this process are not clearly understood. Here, we explore the role of Dab2 in tumor angiogenesis. We found that Dab2 is expressed in several cancer cells, including A549 lung cancer cells, but it is hardly detectable in SW480 colon cancer cells. Migration and Erk phosphorylation were enhanced in human umbilical vein endothelial cells (HUVECs) treated with the conditioned medium obtained from Dab2-overexpressing SW480 stable cells. In addition, vascular endothelial growth factor (VEGF) protein was strongly detected in conditioned medium derived from Dab2-overexpressing SW480 cells, and Erk phosphorylation enhanced by Dab2(+) CM was restored by VEGF inhibition. Moreover, Dab2 depletion in A549 cells led to a decrease in HUVEC migration and Erk phosphorylation. Furthermore, we show that Dab2 is required for the TGFß-induced gene expression of angiogenic factors such as VEGF and FGF2. Taken together, these results suggest that Dab2, which is expressed in cancer cells, is pivotal for endothelial cell migration by affecting VEGF expression.

Identification of a novel negative regulator of activin/nodal signaling in mesendodermal formation of Xenopus embryos.

Phosphotyrosine binding (PTB) domains, which are found in a large number of proteins, have been implicated in signal transduction mediated by growth factor receptors. However, the in vivo roles of these PTB-containing proteins remain to be investigated. Here, we show that Xdpcp (Xenopus dok-PTB containing protein) has a pivotal role in regulating mesendoderm formation in Xenopus, and negatively regulates the activin/nodal signaling pathway. We isolated cDNA for xdpcp and examined its potential role in Xenopus embryogenesis. We found that Xdpcp is strongly expressed in the animal hemisphere at the cleavage and blastula stages. The overexpression of xdpcp RNA affects activin/nodal signaling, which causes defects in mesendoderm formation. In addition, loss of Xdpcp function by injection of morpholino oligonucleotides leads to the expansion of the mesodermal territory. Moreover, we found that axis duplication by ventrally forced expression of activin is recovered by coexpression with Xdpcp. In addition, Xdpcp inhibits the phosphorylation and nuclear translocation of Smad2. Furthermore, we also found that Xdpcp interacts with Alk4, a type I activin receptor, and inhibits activin/nodal signaling by disturbing the interaction between Smad2 and Alk4. Taken together, these results indicate that Xdpcp regulates activin/nodal signaling that is essential for mesendoderm specification.

Xenopus Dab2 is required for embryonic angiogenesis.

BACKGROUND: The molecular mechanisms governing the formation of the embryonic vascular system remain poorly understood. Here, we show that Disabled-2 (Dab2), a cytosolic adaptor protein, has a pivotal role in the blood vessel formation in Xenopus early embryogenesis. RESULTS: Xenopus Disabled-2 (XDab2) is spatially localized to the blood vessels including the intersomitic veins (ISV) in early embryos. Both antisense morpholino oligonucleotide (MO)-mediated knockdown and overexpression of XDab2 inhibit the formation of ISV, which arise from angiogenesis. In addition, we found that activin-like signaling is essential for this angiogenic event. Functional assays in Xenopus animal caps reveal that activin-like signals induce VEGF expression and this induction can be inhibited by XDab2 depletion. However, XDab2 MO has no effects on the induction of other target genes by activin-like signals. Furthermore, we show that the disruption of the sprouting ISV in XDab2-depleted embryos can be rescued by coexpression of VEGF. CONCLUSION: Taking together, we suggest that XDab2 regulates the embryonic angiogenesis by mediating the VEGF induction by activin-like signaling in Xenopus early development.

Notum is required for neural and head induction via Wnt deacylation, oxidation, and inactivation.

Secreted Wnt morphogens are essential for embryogenesis and homeostasis and require a lipid/palmitoleoylate modification for receptor binding and activity. Notum is a secreted Wnt antagonist that belongs to the α/ß hydrolase superfamily, but its mechanism of action and roles in vertebrate embryogenesis are not fully understood. Here, we report that Notum hydrolyzes the Wnt palmitoleoylate adduct extracellularly, resulting in inactivated Wnt proteins that form oxidized oligomers incapable of receptor binding. Thus, Notum is a Wnt deacylase, and palmitoleoylation is obligatory for the Wnt structure that maintains its active monomeric conformation. Notum is expressed in naive ectoderm and neural plate in Xenopus and is required for neural and head induction. These findings suggest that Notum is a prerequisite for the "default" neural fate and that distinct mechanisms of Wnt inactivation by the Tiki protease in the Organizer and the Notum deacylase in presumptive neuroectoderm orchestrate vertebrate brain development.

Identification and characterization of a small-molecule inhibitor of Wnt signaling in glioblastoma cells.

Glioblastoma multiforme (GBM) is the most common and prognostically unfavorable form of brain tumor. The aggressive and highly invasive phenotype of these tumors makes them among the most anatomically damaging human cancers with a median survival of less than 1 year. Although canonical Wnt pathway activation in cancers has been historically linked to the presence of mutations involving key components of the pathway (APC, ß-catenin, or Axin proteins), an increasing number of studies suggest that elevated Wnt signaling in GBM is initiated by several alternative mechanisms that are involved in different steps of the disease. Therefore, inhibition of Wnt signaling may represent a therapeutically relevant approach for GBM treatment. After the selection of a GBM cell model responsive to Wnt inhibition, we set out to develop a screening approach for the identification of compounds capable of modulating canonical Wnt signaling and associated proliferative responses in GBM cells. Here, we show that the small molecule SEN461 inhibits the canonical Wnt signaling pathway in GBM cells, with relevant effects at both molecular and phenotypic levels in vitro and in vivo. These include SEN461-induced Axin stabilization, increased ß-catenin phosphorylation/degradation, and inhibition of anchorage-independent growth of human GBM cell lines and patient-derived primary tumor cells in vitro. Moreover, in vivo administration of SEN461 antagonized Wnt signaling in Xenopus embryos and reduced tumor growth in a GBM xenograft model. These data represent the first demonstration that small-molecule-mediated inhibition of Wnt signaling may be a potential approach for GBM therapeutics.

Xenopus Wntless and the retromer complex cooperate to regulate XWnt4 secretion.

Wnt signaling is implicated in a variety of developmental and pathological processes. The molecular mechanisms governing the secretion of Wnt ligands remain to be elucidated. Wntless, an evolutionarily conserved multipass transmembrane protein, is a dedicated secretion factor of Wnt proteins that participates in Drosophila melanogaster embryogenesis. In this study, we show that Xenopus laevis Wntless (XWntless) regulates the secretion of a specific Wnt ligand, XWnt4, and that this regulation is specifically required for eye development in Xenopus. Moreover, the Retromer complex is required for XWntless recycling to regulate the XWnt4-mediated eye development. Inhibition of Retromer function by Vps35 morpholino (MO) resulted in various Wnt deficiency phenotypes, affecting mesoderm induction, gastrulation cell movements, neural induction, neural tube closure, and eye development. Overexpression of XWntless led to the rescue of Vps35 MO-mediated eye defects but not other deficiencies. These results collectively suggest that XWntless and the Retromer complex are required for the efficient secretion of XWnt4, facilitating its role in Xenopus eye development.