Ritsuko Takada

The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway

Background: Ror2 is an orphan receptor, belonging to the Ror family of receptor tyrosine kinases. Although Ror2 has been shown to play crucial roles in developmental morphogenesis, the precise signalling events that Ror2 mediates remain elusive. Since Ror2 possesses an extracellular cysteine‐rich domain (CRD) that resembles the Wnt‐binding sites of the Frizzled (Fz) proteins, it is conceivable that Ror2 interacts with members of the Wnt family.

JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates

Recent genetic studies in Drosophila identified a novel non‐canonical Wnt pathway, the planar cell polarity (PCP) pathway, that signals via JNK to control epithelial cell polarity in Drosophila. Most recently, a pathway regulating convergent extension movements during gastrulation in vertebrate embryos has been shown to be a vertebrate equivalent of the PCP pathway. However, it is not known whether the JNK pathway functions in this non‐canonical Wnt pathway to regulate convergent extension movements in vertebrates. In addition, it is not known whether JNK is in fact activated by Wnt stimulation. Here we show that Wnt5a is capable of activating JNK in cultured cells, and present evidence that the JNK pathway mediates the action of Wnt5a to regulate convergent extension movements in Xenopus. Our results thus demonstrate that the non‐canonical Wnt/JNK pathway is conserved in both vertebrate and invertebrate and define that JNK has an activity to regulate morphogenetic cell movements.

Cytoskeletal reorganization by soluble Wnt-3a protein signalling

Wnt‐3a is an intercellular signalling molecule that is involved in a variety of morphogenetic events. However, the molecular mechanisms underlying Wnt‐3a signalling are poorly understood. We have sought to establish in vitro systems to assay the activity of this protein and investigate its biological roles.

Axin prevents Wnt-3a-induced accumulation of β-catenin

When Axin, a negative regulator of the Wnt signaling pathway, was expressed in COS cells, it coeluted with glycogen synthase kinase-3β (GSK-3β), β-catenin, and adenomatous polyposis coli protein (APC) in a high molecular weight fraction on gel filtration column chromatography. In this fraction, GSK-3β, β-catenin, and APC were co-precipitated with Axin. Although β-catenin was detected in the high molecular weight fraction in L cells on gel filtration column chromatography, addition of conditioned medium expressing Wnt-3a to the cells increased β-catenin in the low molecular weight fraction. However, Wnt-3a-dependent accumulation of β-catenin was greatly inhibited in L cells stably expressing Axin. Axin also suppressed Wnt-3a-dependent activation of Tcf-4 which binds to β-catenin and acts as a transcription factor. These results suggest that Axin forms a complex with GSK-3β, β-catenin, and APC, resulting in the stimulation of the degradation of β-catenin and that Wnt-3a induces the dissociation of β-catenin from the Axin complex and accumulates β-catenin.

Identification of the laminar-inducing factor: Wnt-signal from the anterior rim induces correct laminar formation of the neural retina in vitro.

To study the molecular mechanism that controls the laminar organization of the retina, we utilized reaggregation cultures of dissociated retinal cells prepared from chicken embryos. These cells cannot generate laminated structures by themselves and, instead, form rosettes within the reaggregates. However, the dissociated cells can organize into a correctly laminated structure when cultured in the presence of a putative laminar inducing factor coming from particular tissue or cells, but its molecular identity of this factor has long remained elusive. In this study, we found that the anterior rim of the retina sends a signal to rearrange the rosette-forming cells into a neuroepithelial structure characteristic of the undifferentiated retinal layer. This activity of the anterior rim was mimicked by Wnt-2b expressed in this tissue, and was neutralized by a soluble form of Frizzled, which works as a Wnt antagonist. Furthermore, the neuroepithelial structure induced by Wnt-2b subsequently developed into correctly laminated retinal layers. These observations suggest that the anterior rim functions as a layer-organizing center in the retina, by producing Wnt-2b.

Analysis of combinatorial effects of Wnts and Frizzleds on beta-catenin/armadillo stabilization and Dishevelled phosphorylation.

Both Wnt ligands and Frizzled (Fz) receptors each constitute a large family in vertebrates, but the receptor specificity of each Wnt has remained largely unknown. Here, we examined the receptor specificity of two typical Wnts, Wnt‐3a and Wnt‐5a, in signal transmission. To investigate systematically the combinatorial effects of these Wnts, various Fzs on canonical Wnt/β‐catenin signaling, we analyzed the ability of these Wnt proteins to increase stability of armadillo/β‐catenin proteins in Drosophila S2 cells expressing vertebrate Fzs. Wnt‐3a increases the amount of armadillo proteins in cells expressing Fzs 4, 5 and 8, but not Fzs 3 and 6; whereas Wnt‐5a does not increase it in any cell line. In contrast, both Wnt‐3a and Wnt‐5a increase the phosphorylation of Dsh in combination with most of the Fzs. This Dsh phosphorylation is abrogated by decreasing the levels of casein kinase I α by double‐stranded RNA‐mediated translational interference. These observations indicate that both Wnt proteins can interact with the majority of Fz receptors and elicit signaling reactions exemplified by Dsh phosphorylation but that the stabilization of β‐catenin/armadillo proteins in the Wnt/β‐catenin signaling occurs only when specific combinations of Wnt and Fz meet.

Cellular FLIP Inhibits β-Catenin Ubiquitylation and Enhances Wnt Signaling

ABSTRACT Cellular FLIP (cFLIP) is a close homologue of caspase 8 without caspase activity that inhibits Fas signaling. The cFLIP protein is often expressed in human tumors and is believed to suppress antitumor immune responses involving the Fas system. Here, we report that a long form of cFLIP (cFLIP-L) inhibits β-catenin ubiquitylation and increases endogenous cytosolic β-catenin, which results in translocation of β-catenin into nuclei and induction of β-catenin-dependent gene expression in cFLIP-L-expressing cells. When cells stably expressing cFLIP-L were stimulated with Wnt3a, enhanced Wnt signaling was observed compared with the control cells. Conversely, depletion of endogenous cFLIP results in reduced Wnt signaling. Furthermore, cFLIP-L increases secondary-body axis formation when coinjected with suboptimal doses of β-catenin into early Xenopus embryos. Down-regulation of FADD by RNA-mediated interference abolishes the β-catenin-dependent gene expression induced by cFLIP-L. These results indicate that cFLIP-L, in cooperation with FADD, enhances canonical Wnt signaling by inhibiting proteasomal degradation of β-catenin, thus suggesting an additional mechanism involved with tumorgenesis, in addition to inhibiting Fas signaling.

Determinative role of Wnt signals in dorsal iris-derived lens regeneration in newt eye

We have previously shown that lens regeneration from the pigmented epithelium of the dorsal iris in the adult newt eye proceeds in two steps after lens removal or intraocular FGF2 injection. The FGF2-dependent proliferation of iris pigmented epithelium and activation of early lens genes that occur over the entire circumference of the iris comprise the first step, while subsequent dorsally confined lens development marks the second step. Here, we investigated the expression of Wnt and Wnt receptor Frizzled genes in lens-regenerating iris tissues. Wnt2b and Frizzled4 were activated only in the dorsal half of the iris in synchrony with the occurrence of the second step, whereas Wnt5a and Frizzled2 were activated in both halves throughout the period of the first and second steps. Cultured explants of the iris-derived pigmented epithelium in the presence of FGF2 underwent dorsal-specific lens development fully recapitulating the in vivo lens regeneration process. Under these conditions, Wnt inhibitors Dkk1, which specifically inhibits the canonical signal pathway, and/or sFRP1 repressed the lens development, while exogenous Wnt3a, which generally activates the canonical pathway like Wnt2b, stimulated lens development from the dorsal iris epithelium and even caused lens development from the ventral iris epithelium, albeit at a reduced rate. Wnt5a did not elicit lens development from the ventral epithelium. These observations indicate that dorsal-specific activation of Wnt2b determines the dorsally limited development of lens from the iris pigmented epithelium.

Modulation of Wnt signaling by the nuclear localization of cellular FLIP-L.

Cellular FLIP (cFLIP) inhibits the apoptosis signaling initiated by death receptor ligation. We previously reported that a long form of cFLIP (cFLIP-L) enhances Wnt signaling via inhibition of β-catenin ubiquitylation. In this report, we present evidence that cFLIP-L translocates into the nucleus, which could have a role in modulation of Wnt signaling. cFLIP-L has a functional bipartite nuclear localization signal (NLS) at the C-terminus. Wild-type cFLIP-L (wt-FLIP-L) localizes in both the nucleus and cytoplasm, whereas NLS-mutated cFLIP-L localizes predominantly in the cytoplasm. cFLIP-L also has a nuclear export signal (NES) near the NLS, and leptomycin B, an inhibitor of CRM1-dependent nuclear export, increases the nuclear accumulation of cFLIP-L, suggesting that it shuttles between the nucleus and cytoplasm. Expression of mutant cFLIP-L proteins with a deletion or mutations in the NLS and NES confers resistance to Fas-mediated apoptosis, as does wt-FLIP-L, but they do not enhance Wnt signaling, which suggests an important role of the C-terminus of cFLIP-L in Wnt-signaling modulation. When wt-FLIP-L is expressed in the cytoplasm by conjugation with exogenous NES (NES-FLIP-L), Wnt signaling is not enhanced, whereas the NES-FLIP-L increases cytoplasmic β-catenin as efficiently as wt-FLIP-L. cFLIP-L physically interacts with the reporter plasmid for Wnt signaling, but not with the control plasmid. These results suggest a role for nuclear cFLIP-L in the modulation of Wnt signaling.

Impairment of the ubiquitin‐proteasome system by cellular FLIP

Cellular FLIP (cFLIP) is a homologue of caspase‐8 without protease activity that inhibits the apoptosis signaling initiated by death receptor ligation. We previously reported that a long form of cFLIP (cFLIP‐L) inhibits ubiquitylation of β‐catenin and enhances Wnt signaling. Here we show that cFLIP‐L impairs the function of the ubiquitin‐proteasome system (UPS), and increases the accumulation of various short‐lived proteins, such as GFP conjugated with destabilization sequence, β‐catenin and HIF1α, that are subjected to rapid ubiquitylation and degradation by proteasomes. Accordingly, β‐catenin‐ and HIF1α‐mediated gene expressions are induced in the cFLIP‐L‐expressing cells. Exogenously expressed cFLIP‐L accumulates in aggregates at the peri‐nuclear region in the cells, and the cFLIP‐L aggregates are refractory to solubilization. Like exogenously expressed cFLIP‐L, the endogenous cFLIP in A549 lung cancer cells displays particulate distribution in the cells and more than 60% of cFLIP‐L is refractory to solubilization. Down‐regulation of cFLIP in A549 cells by RNA‐mediated interference reduced β‐catenin‐ and HIF1α‐mediated gene expression. These results suggest that cFLIP‐L is prone to aggregate and impairs UPS function, which could be involved in the pathological function of cFLIP‐L expressed in certain cancer cells.