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Wnt5a regulates distinct signalling pathways by binding to Frizzled2

2009; Springer Nature; Volume: 29; Issue: 1 Linguagem: Inglês

10.1038/emboj.2009.322

1460-2075

Akira Sato, Hideki Yamamoto, Hiroshi Sakane, Hirofumi Koyama, Akira Kikuchi,

Plant Molecular Biology Research

Article12 November 2009free access Wnt5a regulates distinct signalling pathways by binding to Frizzled2 Akira Sato Akira Sato Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Search for more papers by this author Hideki Yamamoto Hideki Yamamoto Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Search for more papers by this author Hiroshi Sakane Hiroshi Sakane Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Search for more papers by this author Hirofumi Koyama Hirofumi Koyama Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Search for more papers by this author Akira Kikuchi Corresponding Author Akira Kikuchi Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Faculty of Medicine, Osaka University, Suita, Japan Search for more papers by this author Akira Sato Akira Sato Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Search for more papers by this author Hideki Yamamoto Hideki Yamamoto Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Search for more papers by this author Hiroshi Sakane Hiroshi Sakane Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Search for more papers by this author Hirofumi Koyama Hirofumi Koyama Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Search for more papers by this author Akira Kikuchi Corresponding Author Akira Kikuchi Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Faculty of Medicine, Osaka University, Suita, Japan Search for more papers by this author Author Information Akira Sato1, Hideki Yamamoto1, Hiroshi Sakane1, Hirofumi Koyama1 and Akira Kikuchi 1,2 1Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan 2Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Faculty of Medicine, Osaka University, Suita, Japan *Corresponding author. Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. Tel.: +81 82 257 5130; Fax: +81 82 257 5134; E-mail: [email protected] The EMBO Journal (2010)29:41-54https://doi.org/10.1038/emboj.2009.322 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Wnt5a regulates multiple intracellular signalling cascades, but how Wnt5a determines the specificity of these pathways is not well understood. This study examined whether the internalization of Wnt receptors affects the ability of Wnt5a to regulate its signalling pathways. Wnt5a activated Rac in the β-catenin-independent pathway, and Frizzled2 (Fz2) and Ror1 or Ror2 were required for this action. Fz2 was internalized through a clathrin-mediated route in response to Wnt5a, and inhibition of clathrin-dependent internalization suppressed the ability of Wnt5a to activate Rac. As another action of Wnt5a, it inhibited Wnt3a-dependent lipoprotein receptor-related protein 6 (LRP6) phosphorylation and β-catenin accumulation. Wnt3a-dependent phosphorylation of LRP6 was enhanced in Wnt5a knockout embryonic fibroblasts. Fz2 was also required for the Wnt3a-dependent accumulation of β-catenin, and Wnt5a competed with Wnt3a for binding to Fz2 in vitro and in intact cells, thereby inhibiting the β-catenin pathway. This inhibitory action of Wnt5a was not affected by the impairment of clathrin-dependent internalization. These results suggest that Wnt5a regulates distinct pathways through receptor internalization-dependent and -independent mechanisms. Introduction Wnt proteins constitute a large family of secreted ligands that control developmental processes in animals (Logan and Nusse, 2004). In mice and humans, there are 19 Wnt members, which exhibit unique expression patterns and distinct functions in development. Wnts control various cellular functions including proliferation, differentiation, apoptosis, survival, migration, and polarity, by regulating multiple intracellular signalling cascades. In humans and mice, the 10 members of the Frizzled (Fz) seven transmembrane receptor family have been identified as Wnt receptors (Wang et al, 2006). In addition to Fz proteins, single-pass transmembrane proteins, such as low-density lipoprotein receptor-related protein 5 (LRP5), LRP6, receptor tyrosine kinase-like orphan receptor 1 (Ror1), Ror2, and atypical tyrosine kinase receptor (Ryk), have been shown to act as Wnt receptors (Gordon and Nusse, 2006; Fukuda et al, 2008; Green et al, 2008; Kikuchi et al, 2009). The intracellular signalling pathway activated by Wnts was originally identified as a β-catenin-dependent signalling pathway that is highly conserved among various species (He et al, 2004; Kikuchi et al, 2009). In the β-catenin pathway, the binding of Wnts, such as Wnt1 and Wnt3a, to a cell surface receptor complex consisting of Fz and LRP5 or LRP6 induces the stabilization of cytoplasmic β-catenin and its entry into the nucleus, where it activates the transcription factor T-cell factor (Tcf) or lymphoid enhancer factor (Lef) and stimulates the transcription of target genes. Some Wnts, such as Wnt5a and Wnt11, activate β-catenin-independent pathways that modulate cell movement and polarity (Veeman et al, 2003; Kikuchi and Yamamoto, 2008). The β-catenin-independent pathways include multiple signalling cascades such as Rac/Jun N-terminal kinase (JNK), Rho/Rho-associated kinase (Rho-kinase), Ca2+/protein kinase C, and Ca2+/calmodulin-dependent protein kinase II. Wnt5a is representative of the Wnt proteins that activate the β-catenin-independent pathway, although the activation mechanism has not yet been clarified (Veeman et al, 2003; Kikuchi and Yamamoto, 2008). Many studies on signal transduction and membrane trafficking have suggested that the sorting of signalling molecules and their receptors to different membrane-bound compartments has a critical function in regulating signalling (Conner and Schmid, 2003). β-Arrestin mediates the internalization of the seven transmembrane receptor family in clathrin-coated pits by binding to clathrin, μ2-adaptin of adaptor protein-2 (AP-2), and other elements of the endocytic machinery (Lefkowitz and Shenoy, 2005). Wnt5a triggered the endocytosis of Fz4, which was mediated by the recruitment of Dvl2 and β-arrestin2 to the plasma membrane in HEK293 cells (Chen et al, 2003). Wnt5a also induced the internalization of Fz5 (Kurayoshi et al, 2007). Dvl2 interacted with AP-2, which also bound to clathrin, and this interaction was required for the internalization of Fz4 (Yu et al, 2007). In addition, reduction of β-arrestin2 levels in Xenopus embryos led to defects in convergent extension, which is believed to be mediated by the β-catenin-independent pathway (Kim and Han, 2007; Bryja et al, 2008). Therefore, it is possible to speculate that Wnt5a might induce the internalization of its receptors through a clathrin-mediated route, thereby activating the β-catenin-independent pathway. However, there is no direct evidence for this possibility at present. Another Wnt5a action is to inhibit the β-catenin pathway. An early experiment in Xenopus embryos showed that coexpression of XWnt5a with XWnt8 reduces the ability of XWnt8 to induce a secondary axis (Torres et al, 1996). Several possible mechanisms have been proposed to explain this inhibition. Wnt5a was reported to inhibit the transcriptional activity of Tcf downstream of β-catenin by the phosphorylation of Tcf-4 by Nemo-like kinase (NLK) (Ishitani et al, 2003). However, it was also reported that Wnt5a inhibits Tcf independently of the NLK pathway (Smit et al, 2004). Another possible mechanism is that Wnt5a induced the downregulation of β-catenin through the expression of Siah2, which acts as an E3 ubiquitin ligase for β-catenin (Topol et al, 2003). Thus, the inhibitory mechanism of the β-catenin pathway by Wnt5a remains to be clarified. In addition, it is unclear whether the internalization of receptors is involved in the Wnt5a-dependent inhibition. In this study, it was found that Wnt5a-dependent activation of Rac requires clathrin-mediated internalization of Fz2. It was also shown that Wnt5a inhibits the β-catenin pathway by competing with Wnt3a for binding to Fz2, and that the impairment of clathrin-mediated internalization does not affect this Wnt5a inhibitory action. These results suggest that Wnt5a regulates distinct pathways through the receptor internalization-dependent and -independent mechanisms. Results Fz2 is involved in the activation of both β-catenin-dependent and -independent pathways To identify the actions of Wnts, purified Wnt3a and Wnt5a proteins were used in all of the following experiments. First, it was determined whether which receptors mediate distinct Wnt signalling pathways. As Wnt receptors, HeLaS3 cells expressed mRNAs of Fz2, Fz5, and Fz6 and also mRNAs of LRP6, Ror1, and Ror2 (Supplementary Figure S1A and data not shown). These receptors, except for Fz5, were knocked down using small interference RNAs (siRNAs) (Supplementary Figure S1B), because Fz5 mRNA was reduced to only 50% even though 10 different siRNAs or their combinations were used to target Fz5. Individual siRNA did not show off-target effects on the expression of other receptors (Supplementary Figure S1B). Therefore, Fz2, Fz6, LRP6, Ror1, and Ror2 were analysed in this study. Among the β-catenin-independent pathways, the activation of Rac was observed clearly in HeLaS3, CHO, and L cells when they were stimulated with purified Wnt5a (Figure 1A; Supplementary Figure S2A). However, it was hard to detect the activation of JNK and Rho by Wnt5a and that of Rac by Wnt3a in these cells (Supplementary Figure S2B–D). Knockdown of Fz2, Ror1, and Ror2 but not that of Fz6 or LRP6 suppressed the Wnt5a-dependent Rac activation in HeLaS3 cells (Figure 1A). Wnt3a, but not Wnt5a, induced the phosphorylation of LRP6 and the accumulation of β-catenin in HeLaS3, CHO, and L cells (Yamamoto et al, 2006) (Figures 1B and 4B). Knockdown of Fz2 and LRP6 in HeLaS3 cells suppressed the Wnt3a-dependent phosphorylation of LRP6 and accumulation of β-catenin, but knockdown of Fz6, Ror1, or Ror2 did not (Figure 1B). Figure 1.Fz2 mediates both β-catenin-dependent and -independent pathways. (A) After HeLaS3 cells transfected with the indicated siRNAs were treated with 50 ng/ml Wnt5a for 1 h, the cells were incubated with GST-CRIB immobilized on glutathione-Sepharose. The total lysates (total Rac) and precipitates (active Rac) were probed with anti-Rac1 antibody. The signals of active Rac were quantified using NIH Image and expressed as arbitrary units as compared with the signal intensity in control cells without Wnt5a stimulation. The results shown are means±s.e. from four independent experiments. (B) HeLaS3 cells transfected with the indicated siRNAs were treated with 100 ng/ml Wnt3a for 1 h. The lysates were probed with the indicated antibodies. S1490P, anti-phospho-S1490 LRP6 antibody. β-Actin was used as a loading control. The signals of β-catenin and S1490P were quantified using NIH Image and expressed as arbitrary units as compared with the signal intensities in control cells with Wnt3a stimulation. The results shown are means±s.e. from four independent experiments. (C) Left panel, the indicated concentrations of Wnt3a or Wnt5a proteins were incubated with 0.5 nM (25 ng/ml) of FzCRD-IgG proteins or 0.5 nM (17.5 ng/ml) control IgG protein for 2 h. The precipitates were probed with anti-Wnt3a and anti-Wnt5a antibodies. Right panel, the signals of Wnt3a or Wnt5a bound to Fz2CRD-IgG were quantified using NIH Image and expressed as arbitrary units. Wnt3a or Wnt5a (10 ng) was loaded into the right-hand lane as a control. (D) The indicated concentrations of Wnt3a or Wnt5a were incubated with 2 nM (400 ng/ml) LRP6N-IgG protein or 2 nM (70 ng/ml) control IgG protein for 2 h. The precipitates were probed with anti-Wnt3a and anti-Wnt5a antibodies. Download figure Download PowerPoint To perform the ligand-receptor binding assay, the cysteine-rich domain (CRD) of Fz or the extracellular domain of LRP6 (LRP6N) fused to IgG (Fz2CRD-IgG, Fz6CRD-IgG, and LRP6N-IgG) were expressed in HEK293 cultured medium, and proteins were purified using protein A-Sepharose. Consistent with the results in Figure 1A and B, Wnt5a and Wnt3a bound to the CRD of Fz2 with Kd values of 2.3 nM (90 ng/ml)±0.7 nM and 2.8 nM (112 ng/ml)±0.9 nM, respectively, and neither of them bound to the CRD of Fz6 (Figure 1C). Wnt5a showed a weak-binding activity with LRP6N as compared with Wnt3a (Figure 1D). In contrast, Wnt5a, but not Wnt3a, has been reported to bind to Ror2 (Oishi et al, 2003). Thus, it is likely that Fz2 is involved in the activation of both β-catenin-dependent and -independent pathways. Clathrin-dependent internalization of receptors is required for Wnt5a-dependent Rac activation To examine whether the internalization of Fz2 is necessary for the activation of Wnt signalling pathways, the endocytic route of Fz2 was analysed. It has been reported that Wnt5a induces the internalization of Fz4 and Fz5 (Chen et al, 2003; Kurayoshi et al, 2007). Wnt5a also induced the internalization of FLAG-tagged Fz2 (FLAG-Fz2) in HeLaS3 cells (Figure 2A). Approximately 45% of the internalized Fz2 was colocalized with clathrin but not with caveolin in HeLaS3 cells (Figure 2B). Knockdown of either Ror1 or Ror2 suppressed Wnt5a-dependent internalization of Fz2, and knockdown of both Ror1 and Ror2 further inhibited it (Figure 2C; Supplementary Figure S3A). Knockdown of clathrin (Supplementary Figure S4) or treatments of the cells with monodansylcadaverine (MDC) or chlorpromazine, which suppressed the clathrin-dependent internalization of receptors, also inhibited the Wnt5a-induced internalization of Fz2 (Figure 2D; Supplementary Figure S3B). Although it has been reported that the protein levels of Dvl are reduced in the cells treated with hyperosmotic sucrose and potassium depletion that suppress the clathrin-dependent internalization (Bryja et al, 2007a), clathrin siRNA, MDC, or chlorpromazine did not affect Dvl levels (Supplementary Figure S5). In addition, knockdown of Dvl2 and β-arrestin2 also suppressed the Wnt5a-dependent internalization of Fz2 (Figure 2E; Supplementary Figure S4). These results suggest that Wnt5a induces the internalization of Fz2 through a clathrin-mediated endocytic route and that Ror1, Ror2, Dvl2, and β-arrestin have a function in this step. Figure 2.Wnt5a induces the internalization of Fz2 through a clathrin-mediated route. (A) HeLaS3 cells expressing FLAG-Fz2 were treated with 100 ng/ml Wnt5a for the indicated periods of time. Left panel, confocal images; right panel, quantification of internalized FLAG-Fz2. The results shown are means of four independent experiments. (B) Left panel, HeLaS3 cells expressing FLAG-Fz2 were treated with Wnt5a for 30 min, and then the cells were stained with anti-FLAG and anti-clathrin or anti-caveolin antibodies. In the merged images, FLAG-Fz2 is shown in red and clathrin or caveolin is in green. Colocalization of FLAG-Fz2 and clathrin appears as yellow. Right panel, the percentages of Fz2 colocalized with clathrin- or caveolin-positive vesicles were quantified. The results shown are means±s.e. from three independent experiments. Approximately 43% of 1365 Fz2 puncta were colocalized with clathrin-positive vesicles. (C) HeLaS3 cells expressing FLAG-Fz2 were transfected with siRNA for Ror1 or/and Ror2, and then the cells were treated with Wnt5a for 30 min. (D) HeLaS3 cells expressing FLAG-Fz2 were transfected with siRNA for clathrin, and then the cells were treated with Wnt5a for 30 min. (E) HeLaS3 cells expressing FLAG-Fz2 were transfected with siRNA for Dvl2 or β-arrestin2, and then the cells were treated with Wnt5a for 30 min. Scale bars, 5 μm. Download figure Download PowerPoint The knockdown of clathrin in HeLaS3 cells, but not that of caveolin, inhibited the Wnt5a-dependent Rac activation (Figure 3A; Supplementary Figures S4 and S6A). Treatment of the cells with MDC or chlorpromazine also inhibited Wnt5a-dependent Rac activation (Figure 3B), whereas that with Nystatin, which disrupts the lipid raft containing caveolin, did not (Supplementary Figure S6B). In addition, Wnt5a failed to activate Rac in Dvl2 or β-arrestin2 knockdown cells (Figure 3C). It has been shown that Epsin is involved in the clathrin-dependent internalization of receptors for trasnferrin, EGF, and insulin, and that clathrin-dependent internalization of these receptors is inhibited in cells expressing the ENTH domain of Epsin (Nakashima et al, 1999; Itoh et al, 2001). As expected, the Wnt5a-dependent activation of Rac was inhibited in CHO cells expressing the ENTH domain (Figure 3D). These results suggest that the clathrin-dependent internalization is required for Wnt5a-dependent activation of Rac. Figure 3.Clathrin-dependent internalization of Fz2 is required for Rac activation. (A) HeLaS3 cells transfected with control or clathrin siRNA were treated with 50 ng/ml Wnt5a for 1 h, and then the cells were subjected to the Rac activation assay. The results shown are means±s.e. from four independent experiments. (B) HeLaS3 cells pretreated with MDC or chlorpromazine were incubated with Wnt5a for 1 h. (C) HeLaS3 cells transfected with the indicated siRNAs were treated with Wnt5a for 1 h. (D) CHO cells or CHO cells expressing Myc-ENTH (CHO/Myc-ENTH) were treated with Wnt5a for 1 h. (E) HEK293T cells expressing HA-Dvl2 or β-arrestin2-GFP were subjected to the Rac activation assay. (F) After HeLaS3 cells expressing FLAG-Fz2 with HA-Dvl2 or β-arrestin2-GFP were kept for 30 min at 4°C and further incubated with anti-FLAG antibody for 1 h at 4°C, the cells were incubated for 20 min at 37°C without Wnt5a stimulation. In the merged images, FLAG-Fz2 is shown in red and HA-Dvl2 or β-arrestin2-GFP is in green. Colocalization of FLAG-Fz2 and HA-Dvl2 appears as yellow. Top panel, confocal images; bottom panel, quantification of internalized FLAG-Fz2 after incubation for 20 min at 37°C. The results shown are means of four independent experiments. Scale bar, 5 μm. Download figure Download PowerPoint Consistent with the results from mouse embryonic fibroblasts (MEFs) (Bryja et al, 2008), the overexpression of β-arrestin2 in HEK293T cells activated Rac to the same extent as with Dvl2 (Figure 3E). Whether the activation of Rac by Dvl2 or β-arrestin2 is related with the internalization of Fz2 without Wnt5a stimulation was examined. After HeLas3 cells expressing Fz2 was kept at 4°C, internalization was initiated by the shift to 37°C. When Fz2 was expressed alone, Fz2 was localized to the cell surface membrane after 20-min incubation at 37°C (Figure 3F). When Fz2 and Dvl2 were coexpressed, Dvl2 was observed at the cell surface membrane at 4°C, and the expression of Dvl2 induced the internalization of Fz2 at 37°C (Figure 3F). Moreover, the internalized Fz2 was colocalized with Dvl2 (Figure 3F). The expression of β-arrestin2 also induced the internalization of Fz2 without Wnt5a stimulation although β-arrestin2 was distributed throughout the cytoplasm (Figure 3F). Dvl2- and β-arrestin2-induced internalization of Fz2 was observed even in the presence of secreted Fz-related protein 2 (sFRP2), which binds to Wnt proteins and blocks Wnt signalling (Kawano and Kypta, 2003) (Supplementary Figure S7A). It is notable that Dvl2-induced Rac activation was observed in Fz2 knockdown cells, whereas β-arrestin2-induced Rac activation was lost by knockdown of Fz2 (Supplementary Figure S7B). Taken together, these results suggest that the clathrin-dependent internalization of Fz2 has a function in Wnt5a-dependent activation of Rac, and it is likely that overexpressed Dvl2 by itself has another way to activate Rac. Wnt5a inhibits Wnt3a-dependent accumulation of β-catenin One of the earliest observations about Wnt5a was that it has an ability to inhibit the β-catenin pathway. As the effects of Wnt5a were analysed using artificial reporter gene assays in earlier studies (Ishitani et al, 2003; Mikels and Nusse, 2006), this study investigated the inhibitory mechanism of the β-catenin pathway by Wnt5a by measuring target gene expression, β-catenin stability, and the phosphorylation of LRP6 at endogenous levels. Among target genes such as c-Myc, cyclinD1, Dkk1, and Axin2 in the β-catenin pathway, Wnt3a increased Axin2 mRNA levels greatly in HeLaS3, NIH3T3, and C3H10T1/2 cells (Figure 4A). Wnt5a did indeed suppress Wnt3a-dependent increases in Axin2 mRNA levels in these cells, and it also inhibited the Wnt3a-dependent β-catenin accumulation (Figure 4A and B). Furthermore, the inhibition of β-catenin accumulation by Wnt5a was observed in MKN-1, CHO, and L cells (Figure 4B), suggesting that Wnt5a is able to suppress the β-catenin pathway by inhibiting the accumulation of β-catenin. Figure 4.Wnt5a inhibits Wnt3a-dependent accumulation of β-catenin. (A) Top panel, HeLaS3 cells were treated with 50 ng/ml Wnt3a in the presence of the indicated concentrations of Wnt5a for 12 h. Total RNA was extracted from these cells, and semi-quantitative RT–PCR analyses for Axin2 mRNA expression were performed. Middle and bottom panels, NIH3T3 or C3H10T1/2 cells were treated with 20 ng/ml Wnt3a and/or 100 ng/ml Wnt5a for 12 h. The results are expressed as fold increase compared with that without Wnt3a stimulation, and indicate means±s.e. from three independent experiments. (B) Top panel, HeLaS3 cells were treated with 50 ng/ml Wnt3a in the presence of the indicated concentrations of Wnt5a for 3 h. The lysates were probed with anti-β-catenin and anti-β-actin antibodies. β-Actin was used as a loading control. Bottom panel, NIH3T3, C3H10T1/2, MKN-1, CHO, and L cells were treated with 50 ng/ml Wnt3a with or without 300 ng/ml Wnt5a for 3 h. (C) After stimulation with 20 ng/ml Wnt3a for 3 h, HeLaS3 cells were washed with PBS, and then treated with 100 ng/ml Wnt5a for the indicated periods of time. (D) HeLaS3 cells were treated with 20 ng/ml Wnt3a, 10 mM LiCl, or 10 μM lactacystin for 3 h with or without 100 ng/ml Wnt5a. Download figure Download PowerPoint Wnt5a has been reported to induce the expression of Siah2, which functions as an E3 ubiquitin ligase to degrade β-catenin, in HEK293 cells (Topol et al, 2003). However, in our hand Wnt5a did not increase Siah1 or Siah2 mRNA levels in HEK293 or HeLaS3 cells (data not shown). Although Axin2 is also known to degrade β-catenin and acts as a negative regulator of the β-catenin signalling (Yamamoto et al, 1998; Lustig et al, 2002), Wnt5a alone did not increase Axin2 mRNA levels in contrast to Wnt3a (Figure 4A). After HeLaS3 cells were treated with Wnt3a for 3 h, under conditions where β-catenin accumulated maximally, Wnt3a was removed from the culture medium. The β-catenin levels reduced gradually in a time-dependent manner, but the addition of Wnt5a did not affect the β-catenin levels (Figure 4C). Furthermore, the accumulation of β-catenin following LiCl treatment, which acts as a GSK-3 inhibitor, or lactacystin, which functions as a proteasome inhibitor, was not suppressed by Wnt5a (Figure 4D). These results suggest that Wnt5a does not induce the degradation of β-catenin but is involved in the regulation of β-catenin stabilization upstream of β-catenin. Wnt5a inhibits Wnt3a-dependent phosphorylation of LRP6 Although Wnt5a suppressed the increases in Axin2 mRNA levels induced by overexpression of Wnt3a, it did not affect the increases due to LRP6ΔN, a constitutively active form of LRP6, or Dvl2 in HeLaS3 cells (Figure 5A). Wnt3a induced the phosphorylation of LRP6 at Ser1490 and Thr1479 (Davidson et al, 2005; Zeng et al, 2005), and Wnt5a inhibited it in HeLaS3 cells (Figure 5B). These findings were confirmed in NIH3T3, L, and C3H10T1/2 cells (Figure 5C), suggesting that Wnt5a affects the β-catenin pathway at the receptor level. Figure 5.Wnt5a inhibits Wnt3a-dependent phosphorylation of LRP6. (A) HeLaS3 cells expressing Wnt3a, LRP6ΔN-GFP, or FLAG-Dvl2 were treated with the indicated concentrations of Wnt5a, and semi-quantitative RT–PCR analyses for Axin2 mRNA expression were performed. The results are expressed as fold increase as compared with the Axin2 mRNA expression level in control cells, and indicate means±s.e. from three independent experiments. (B) HeLaS3 cells were treated with 50 or 100 ng/ml Wnt3a in the presence of the indicated concentrations of Wnt5a for 1 h. The lysates were probed with the indicated antibodies. S1490P, anti-phospho-S1490 LRP6 antibody; T1479P, anti-phospho-T1479 LRP6 antibody. Clathrin was used as a loading control. (C) NIH3T3, L, and C3H10T1/2 cells were treated with 100 ng/ml Wnt3a in the presence of the indicated concentrations of Wnt5a for 1 h. (D) Top panels, lysates of MEFs isolated from wild-type embryos (Wnt5a+/+) or Wnt5a knockout embryos (Wnt5a−/−) were probed with anti-Wnt5a/b and anti-clathrin antibodies. Clathrin was used as a loading control. Bottom panels, Wnt5a+/+ MEFs or Wnt5a−/− MEFs were treated with 50 ng/ml Wnt3a for 9 h (left panel) or with 10 mM LiCl for 9 h (right panel), and semi-quantitative RT–PCR analyses of Axin2 mRNA expression were performed. The results are expressed as fold increases as compared with the Axin2 mRNA expression levels in Wnt5a+/+ MEFs without stimulation, and indicate means±s.e. from three independent experiments. *P<0.05. (E) Wnt5a+/+ MEFs or Wnt5a−/− MEFs were treated with the indicated concentrations of Wnt3a for 1 h. The lysates were probed with indicated antibodies. The results shown are representative of four different pairs of Wnt5a−/− and the littermate Wnt5a+/+ mice. The signals of S1490P were quantified using NIH Image. The results are expressed as arbitrary units as compared with the signal intensity from Wnt5a+/+ MEFs with 100 ng/ml Wnt3a stimulation and are shown as means±s.e. from four independent experiments. *P<0.05. (F) Wnt5a+/+ and Wnt5a−/− embryos were stained with X-gal to detect BATlacZ reporter activity at E10.5. Embryos are shown in anterior view. In the telencephalon, β-galactosidase activity of Wnt5a−/− embryo was stronger than that of Wnt5a+/+ (arrowheads). The staining in otic vesicles (arrows) and other regions showed similar staining intensity between Wnt5a+/+ and Wnt5a−/− embryos. Download figure Download PowerPoint To further examine the loss of function of Wnt5a, MEFs were prepared from Wnt5a knockout (Wnt5a−/−) mice (Figure 5D). Wnt3a-dependent increases in Axin2 mRNA levels were enhanced in Wnt5a−/− MEFs as compared with wild-type (Wnt5a+/+) MEFs (Figure 5D). However, increases in the levels of Axin2 mRNA following LiCl treatment were similar between Wnt5a−/− and Wnt5a+/+ MEFs (Figure 5D). Furthermore, the Wnt3a-dependent phosphorylation of LRP6 at Ser1490 was enhanced in Wnt5a−/− MEFs (Figure 5E). These results support the findings that Wnt5a acts on the receptor to inhibit the β-catenin pathway. The in vivo activity of the β-catenin pathway was assessed directly in the Wnt5a−/− embryos using BATlacZ transgenic mice, in which LacZ expression is under the control of eight Tcf/Lef-binding sites (Maretto et al, 2003; Nakaya et al, 2005). It has been reported that the Wnt5a mRNA is expressed in the fronted nasal process, telencephalon, diencephalons, mesencephalon, limb buds, genital primordial, and tailbud at 9.5–12.5 d.p.c (Takada et al, 1994; Yamaguchi et al, 1999). BATlacZ expression was clearly detected in the telencephalon, diencephalons, limb buds, and tailbud in Wnt5a+/+ embryos at 10.5 d.p.c., which was consistent with the earlier observation (Maretto et al, 2003). Ectopic LacZ staining was enhanced on the dorsal side of the telencephalon of Wnt5a−/−/BATlacZ mice (Figure 5F, arrowheads), whereas otic vesicles (Figure 5F, arrows) and other regions including the bronchial arches, limb buds, and somites (data not shown) were stained at similar levels between Wnt5a+/+ and Wnt5a−/− embryos. Therefore, these observations suggest that the activity of the β-catenin pathway was enhanced at least in the telencephalon in vivo by removing Wnt5a. Wnt5a competes with Wnt3a for binding to Fz2 It has been reported that overexpression of Ror2 mediates Wnt5a-dependent inhibition of the β-catenin pathway in L cells (Mikels and Nusse, 2006). However, knockdown of Ror2 and/or Ror1 in HeLaS3 cells did not affect the Wnt5a-induced inhibition of β-catenin accumulation and Axin2 mRNA expression (Figure 6A). It has been proposed that Wnt3a induces the phosphorylation of LRP6 by CK1γ in a Dvl-dependent manner, and the recruitment of Axin further enhances the phosphorylation of the PPPSP motifs by GSK-3 (Bilic et al, 2007; Zeng et al, 2008). The present results also raised the possibility that Wnt5a inhibits CK1γ phosphorylation of LRP6. However, Wnt5a could not affect the phosphorylation of LRP6, which was induced by overexpression of CK1γ (Figure 6B).