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Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation.

Topol L, Jiang X, Choi H, Garrett-Beal L, Carolan PJ, Yang Y - J. Cell Biol. (2003)

Bottom Line: Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems.We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin.This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent.

View Article: PubMed Central - PubMed

Affiliation: Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Wnts are secreted signaling molecules that can transduce their signals through several different pathways. Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems. We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin. This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent. Furthermore, we provide evidence that Wnt-5a also acts in vivo to promote beta-catenin degradation in regulating mammalian limb development and possibly in suppressing tumor formation.

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Activation of NF-AT and CaMKII is not required for Wnt-5a–induced β-catenin degradation. Cyclosporin A (CsA) inhibited NF-AT reporter activity. Luciferase activity was measured 40 h after transfection. (B) Activated calcineurin (pCN) or CaMKII (ca CaMKII) inhibited the TOPFLASH activity stimulated by the mutant β-catenin (S37A). Inhibiting calcineurin–NF-AT pathway by CsA or inhibiting CaMKII by KN93 did not block Wnt-5a–induced inhibition of TOPFLASH activity in 293 cells. 24 h after transfection with indicated plasmids, CsA or KN93 were added. After 16 h of incubation with the inhibitors, cells were lysed for luciferase assay. (C) 293 cells were transfected with c-Jun and indicated plasmids. JNK activation was detected by anti–phospho-c-Jun (Ser 63) antibodies. Dishevelled, but not Wnt-5a, activated JNK. (D) CHO cells were transfected with mutant β-catenin (S37A) and Wnt-5a or GFP where indicated, and then treated with CsA or KN93 to inhibit calcineurin or CaMKII, respectively. CsA or KN93 treatment or expression of a dominant negative CaMKII (dn CaMKII) appeared to stabilize the mutant β-catenin, but neither treatment inhibited Wnt-5a–induced β-catenin degradation.
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fig3: Activation of NF-AT and CaMKII is not required for Wnt-5a–induced β-catenin degradation. Cyclosporin A (CsA) inhibited NF-AT reporter activity. Luciferase activity was measured 40 h after transfection. (B) Activated calcineurin (pCN) or CaMKII (ca CaMKII) inhibited the TOPFLASH activity stimulated by the mutant β-catenin (S37A). Inhibiting calcineurin–NF-AT pathway by CsA or inhibiting CaMKII by KN93 did not block Wnt-5a–induced inhibition of TOPFLASH activity in 293 cells. 24 h after transfection with indicated plasmids, CsA or KN93 were added. After 16 h of incubation with the inhibitors, cells were lysed for luciferase assay. (C) 293 cells were transfected with c-Jun and indicated plasmids. JNK activation was detected by anti–phospho-c-Jun (Ser 63) antibodies. Dishevelled, but not Wnt-5a, activated JNK. (D) CHO cells were transfected with mutant β-catenin (S37A) and Wnt-5a or GFP where indicated, and then treated with CsA or KN93 to inhibit calcineurin or CaMKII, respectively. CsA or KN93 treatment or expression of a dominant negative CaMKII (dn CaMKII) appeared to stabilize the mutant β-catenin, but neither treatment inhibited Wnt-5a–induced β-catenin degradation.

Mentions: Wnt-5a signaling may activate PKC and intracellular Ca2+ mobilization to trigger a series of downstream effects including activation of NF-AT and CaMKII (Kuhl et al., 2000a; Saneyoshi et al., 2002). During early Xenopus development, Wnt-5a activates calcineurin, which leads to NF-AT nuclear localization and increased β-catenin degradation (Saneyoshi et al., 2002). To address how Wnt-5a transduces its signal in mammalian cells, we checked whether Wnt-5a activates NF-AT transcriptional activity and whether such activation is required for Wnt-5a–induced β-catenin degradation. We found that NF-AT transcriptional activity was only weakly activated by Wnt-5a (∼50%), whereas it was strongly up-regulated by activated calcineurin (∼300%; Fig. 3 A). In addition, we found that although activated calcineurin was able to inhibit the TOPFLASH activity up-regulated by β-catenin S37A, Wnt-5a had an additive effect in its presence (Fig. 3 B). Moreover, specific inhibition of calcineurin by Cyclosporin A (CsA; Crabtree and Olson, 2002) did not block the inhibitory effect of Wnt-5a on β-catenin activity and protein stability (Fig. 3, B and D), although it strongly inhibited NF-AT transcriptional activity (Fig. 3 A) and up-regulated β-catenin protein level in the absence of Wnt-5a (Fig. 3 D). These data indicate that the calcineurin–NF-AT pathway is not the major one mediating the effect of Wnt-5a on β-catenin degradation in 293 cells. Next, we also examined in 293 cells whether Wnt-5a activated PKC, CaMKII or JNK, all of which have been implicated in transducing the noncanonical Wnt-5a signal (Kuhl et al., 2000a; Yamanaka et al., 2002). Consistent with what has been shown before, we found that Wnt-5a signaling led to the activation of PKC and CaMKII in 293 cells (unpublished data). However, no significant JNK activation was detected (Fig. 3 C). In addition, we found that, in contrast to Wnt-5a signaling, PKC activation led to stabilization of β-catenin as shown previously (Chen et al., 2000; unpublished data). As an activated form of CaMKII was able to inhibit TOPFLASH activity (Fig. 3 B), we tested whether CaMKII activation is required for Wnt-5a–induced β-catenin degradation. We blocked CaMKII activity with a dominant negative CaMKII (Kuhl et al., 2000a) or KN93 (Rich and Schulman, 1998). We found that blocking CaMKII activity did not rescue the TOPFLASH activity or β-catenin protein stability inhibited by Wnt-5a (Fig. 3, B and D), indicating that CaMKII is not required in the Wnt-5a signaling pathway for β-catenin degradation. Interestingly, in the absence of Wnt-5a, inhibition of CaMKII by a dominant negative CaMKII or KN93 also resulted in the up-regulation of β-catenin protein level (Fig. 3 D). Together, our results indicate that activation of NF-AT or CaMKII may play a role in promoting β-catenin degradation. However, inhibition of canonical Wnt activity by Wnt-5a can be mediated by pathways other than the activation of PKC, NF-AT, and CaMKII in mammalian cells.


Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation.

Topol L, Jiang X, Choi H, Garrett-Beal L, Carolan PJ, Yang Y - J. Cell Biol. (2003)

Activation of NF-AT and CaMKII is not required for Wnt-5a–induced β-catenin degradation. Cyclosporin A (CsA) inhibited NF-AT reporter activity. Luciferase activity was measured 40 h after transfection. (B) Activated calcineurin (pCN) or CaMKII (ca CaMKII) inhibited the TOPFLASH activity stimulated by the mutant β-catenin (S37A). Inhibiting calcineurin–NF-AT pathway by CsA or inhibiting CaMKII by KN93 did not block Wnt-5a–induced inhibition of TOPFLASH activity in 293 cells. 24 h after transfection with indicated plasmids, CsA or KN93 were added. After 16 h of incubation with the inhibitors, cells were lysed for luciferase assay. (C) 293 cells were transfected with c-Jun and indicated plasmids. JNK activation was detected by anti–phospho-c-Jun (Ser 63) antibodies. Dishevelled, but not Wnt-5a, activated JNK. (D) CHO cells were transfected with mutant β-catenin (S37A) and Wnt-5a or GFP where indicated, and then treated with CsA or KN93 to inhibit calcineurin or CaMKII, respectively. CsA or KN93 treatment or expression of a dominant negative CaMKII (dn CaMKII) appeared to stabilize the mutant β-catenin, but neither treatment inhibited Wnt-5a–induced β-catenin degradation.
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Related In: Results  -  Collection

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fig3: Activation of NF-AT and CaMKII is not required for Wnt-5a–induced β-catenin degradation. Cyclosporin A (CsA) inhibited NF-AT reporter activity. Luciferase activity was measured 40 h after transfection. (B) Activated calcineurin (pCN) or CaMKII (ca CaMKII) inhibited the TOPFLASH activity stimulated by the mutant β-catenin (S37A). Inhibiting calcineurin–NF-AT pathway by CsA or inhibiting CaMKII by KN93 did not block Wnt-5a–induced inhibition of TOPFLASH activity in 293 cells. 24 h after transfection with indicated plasmids, CsA or KN93 were added. After 16 h of incubation with the inhibitors, cells were lysed for luciferase assay. (C) 293 cells were transfected with c-Jun and indicated plasmids. JNK activation was detected by anti–phospho-c-Jun (Ser 63) antibodies. Dishevelled, but not Wnt-5a, activated JNK. (D) CHO cells were transfected with mutant β-catenin (S37A) and Wnt-5a or GFP where indicated, and then treated with CsA or KN93 to inhibit calcineurin or CaMKII, respectively. CsA or KN93 treatment or expression of a dominant negative CaMKII (dn CaMKII) appeared to stabilize the mutant β-catenin, but neither treatment inhibited Wnt-5a–induced β-catenin degradation.
Mentions: Wnt-5a signaling may activate PKC and intracellular Ca2+ mobilization to trigger a series of downstream effects including activation of NF-AT and CaMKII (Kuhl et al., 2000a; Saneyoshi et al., 2002). During early Xenopus development, Wnt-5a activates calcineurin, which leads to NF-AT nuclear localization and increased β-catenin degradation (Saneyoshi et al., 2002). To address how Wnt-5a transduces its signal in mammalian cells, we checked whether Wnt-5a activates NF-AT transcriptional activity and whether such activation is required for Wnt-5a–induced β-catenin degradation. We found that NF-AT transcriptional activity was only weakly activated by Wnt-5a (∼50%), whereas it was strongly up-regulated by activated calcineurin (∼300%; Fig. 3 A). In addition, we found that although activated calcineurin was able to inhibit the TOPFLASH activity up-regulated by β-catenin S37A, Wnt-5a had an additive effect in its presence (Fig. 3 B). Moreover, specific inhibition of calcineurin by Cyclosporin A (CsA; Crabtree and Olson, 2002) did not block the inhibitory effect of Wnt-5a on β-catenin activity and protein stability (Fig. 3, B and D), although it strongly inhibited NF-AT transcriptional activity (Fig. 3 A) and up-regulated β-catenin protein level in the absence of Wnt-5a (Fig. 3 D). These data indicate that the calcineurin–NF-AT pathway is not the major one mediating the effect of Wnt-5a on β-catenin degradation in 293 cells. Next, we also examined in 293 cells whether Wnt-5a activated PKC, CaMKII or JNK, all of which have been implicated in transducing the noncanonical Wnt-5a signal (Kuhl et al., 2000a; Yamanaka et al., 2002). Consistent with what has been shown before, we found that Wnt-5a signaling led to the activation of PKC and CaMKII in 293 cells (unpublished data). However, no significant JNK activation was detected (Fig. 3 C). In addition, we found that, in contrast to Wnt-5a signaling, PKC activation led to stabilization of β-catenin as shown previously (Chen et al., 2000; unpublished data). As an activated form of CaMKII was able to inhibit TOPFLASH activity (Fig. 3 B), we tested whether CaMKII activation is required for Wnt-5a–induced β-catenin degradation. We blocked CaMKII activity with a dominant negative CaMKII (Kuhl et al., 2000a) or KN93 (Rich and Schulman, 1998). We found that blocking CaMKII activity did not rescue the TOPFLASH activity or β-catenin protein stability inhibited by Wnt-5a (Fig. 3, B and D), indicating that CaMKII is not required in the Wnt-5a signaling pathway for β-catenin degradation. Interestingly, in the absence of Wnt-5a, inhibition of CaMKII by a dominant negative CaMKII or KN93 also resulted in the up-regulation of β-catenin protein level (Fig. 3 D). Together, our results indicate that activation of NF-AT or CaMKII may play a role in promoting β-catenin degradation. However, inhibition of canonical Wnt activity by Wnt-5a can be mediated by pathways other than the activation of PKC, NF-AT, and CaMKII in mammalian cells.

Bottom Line: Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems.We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin.This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent.

View Article: PubMed Central - PubMed

Affiliation: Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Wnts are secreted signaling molecules that can transduce their signals through several different pathways. Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems. We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin. This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent. Furthermore, we provide evidence that Wnt-5a also acts in vivo to promote beta-catenin degradation in regulating mammalian limb development and possibly in suppressing tumor formation.

Show MeSH
Related in: MedlinePlus