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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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Wnt-5a promoted β-catenin degradation through a mechanism that requires Siah2 and APC. (A) β-catenin degradation in response to Wnt-5a was inhibited by the proteasome inhibitor epoxomicin. 293 cells were transfected with indicated plasmids. 38 h after transfection, cells were treated with 100 nM epoxomicin for 8 h before they were harvested. (B) A dominant negative Siah2 (ΔSiah2) blocked Wnt-5a mediated inhibition of TOPFLASH activity activated by exogenous β-catenin. ΔSiah1 or ΔSiah2 alone also up-regulated TOPFLASH activity and this was not inhibited by Wnt-5a. FOPFLASH responded to ΔSiah1 or ΔSiah2 very weakly. (C) ΔSiah2 blocked the degradation of both wild-type and mutant β-catenin promoted by Wnt-5a. (D) ΔAPC blocked Wnt-5a–induced inhibition of β-catenin activity and degradation. ΔAPC alone also up-regulated TOPFLASH, which was hardly inhibited by Wnt-5a. FOPFLASH did not respond to ΔAPC. (E) RT-PCR was performed to detect Siah2 transcript. Expression of Siah2 was induced by Wnt-5a and p53 in 293 cells. (F) Wnt-5a did not activate p53 transcriptional activity.
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fig4: Wnt-5a promoted β-catenin degradation through a mechanism that requires Siah2 and APC. (A) β-catenin degradation in response to Wnt-5a was inhibited by the proteasome inhibitor epoxomicin. 293 cells were transfected with indicated plasmids. 38 h after transfection, cells were treated with 100 nM epoxomicin for 8 h before they were harvested. (B) A dominant negative Siah2 (ΔSiah2) blocked Wnt-5a mediated inhibition of TOPFLASH activity activated by exogenous β-catenin. ΔSiah1 or ΔSiah2 alone also up-regulated TOPFLASH activity and this was not inhibited by Wnt-5a. FOPFLASH responded to ΔSiah1 or ΔSiah2 very weakly. (C) ΔSiah2 blocked the degradation of both wild-type and mutant β-catenin promoted by Wnt-5a. (D) ΔAPC blocked Wnt-5a–induced inhibition of β-catenin activity and degradation. ΔAPC alone also up-regulated TOPFLASH, which was hardly inhibited by Wnt-5a. FOPFLASH did not respond to ΔAPC. (E) RT-PCR was performed to detect Siah2 transcript. Expression of Siah2 was induced by Wnt-5a and p53 in 293 cells. (F) Wnt-5a did not activate p53 transcriptional activity.

Mentions: To test whether Wnt-5a–induced degradation of β-catenin S37A is also mediated by proteasome, we used epoxomicin to block proteasome-mediated protein degradation (Meng et al., 1999). We found that β-catenin S37A degradation induced by Wnt-5a was substantially inhibited when the cells were treated with Epoxomicin (Fig. 4 A). As the Siah–APC–Ebi E3 ubiquitin ligase complex has been implicated in GSK-3–independent β-catenin degradation (Liu et al., 2001; Matsuzawa and Reed, 2001), we then examined whether Siah activity is required for Wnt-5a–induced β-catenin degradation. We found that both Siah1 and 2 exhibit activities similar to Wnt-5a in inhibiting canonical Wnt activities in 293 cells (unpublished data). Moreover, a dominant negative Siah2 (ΔSiah2) was able to rescue the β-catenin activity that had been suppressed by Wnt-5a (Fig. 4 B). In addition, degradation of wild-type and an activated form of β-catenin by Wnt-5a signaling were also suppressed by ΔSiah1 and ΔSiah2 (Fig. 4 C and not depicted). Furthermore, we found that, as a result of deceased degradation of the endogenous β-catenin, both ΔSiah1 and ΔSiah2 up-regulated the TOPFLASH activity and this was not inhibited by Wnt-5a (Fig. 4 B). As it has been shown that Siah-dependent β-catenin degradation requires APC activity (Liu et al., 2001; Matsuzawa and Reed, 2001), we next examined whether a dominant negative form of APC (ΔAPC), which has been shown to sequester Siah proteins, could also block β-catenin degradation induced by Wnt-5a signaling. We found that indeed both the activity and protein levels of β-catenin were rescued by ΔAPC (Fig. 4 D). In addition, Wnt-5a hardly inhibited the TOPFLASH activity up-regulated by ΔAPC when there was no exogenous β-catenin (Fig. 4 D). All these results demonstrate that Wnt-5a–induced β-catenin degradation requires functional Siah and APC.


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)

Wnt-5a promoted β-catenin degradation through a mechanism that requires Siah2 and APC. (A) β-catenin degradation in response to Wnt-5a was inhibited by the proteasome inhibitor epoxomicin. 293 cells were transfected with indicated plasmids. 38 h after transfection, cells were treated with 100 nM epoxomicin for 8 h before they were harvested. (B) A dominant negative Siah2 (ΔSiah2) blocked Wnt-5a mediated inhibition of TOPFLASH activity activated by exogenous β-catenin. ΔSiah1 or ΔSiah2 alone also up-regulated TOPFLASH activity and this was not inhibited by Wnt-5a. FOPFLASH responded to ΔSiah1 or ΔSiah2 very weakly. (C) ΔSiah2 blocked the degradation of both wild-type and mutant β-catenin promoted by Wnt-5a. (D) ΔAPC blocked Wnt-5a–induced inhibition of β-catenin activity and degradation. ΔAPC alone also up-regulated TOPFLASH, which was hardly inhibited by Wnt-5a. FOPFLASH did not respond to ΔAPC. (E) RT-PCR was performed to detect Siah2 transcript. Expression of Siah2 was induced by Wnt-5a and p53 in 293 cells. (F) Wnt-5a did not activate p53 transcriptional activity.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172823&req=5

fig4: Wnt-5a promoted β-catenin degradation through a mechanism that requires Siah2 and APC. (A) β-catenin degradation in response to Wnt-5a was inhibited by the proteasome inhibitor epoxomicin. 293 cells were transfected with indicated plasmids. 38 h after transfection, cells were treated with 100 nM epoxomicin for 8 h before they were harvested. (B) A dominant negative Siah2 (ΔSiah2) blocked Wnt-5a mediated inhibition of TOPFLASH activity activated by exogenous β-catenin. ΔSiah1 or ΔSiah2 alone also up-regulated TOPFLASH activity and this was not inhibited by Wnt-5a. FOPFLASH responded to ΔSiah1 or ΔSiah2 very weakly. (C) ΔSiah2 blocked the degradation of both wild-type and mutant β-catenin promoted by Wnt-5a. (D) ΔAPC blocked Wnt-5a–induced inhibition of β-catenin activity and degradation. ΔAPC alone also up-regulated TOPFLASH, which was hardly inhibited by Wnt-5a. FOPFLASH did not respond to ΔAPC. (E) RT-PCR was performed to detect Siah2 transcript. Expression of Siah2 was induced by Wnt-5a and p53 in 293 cells. (F) Wnt-5a did not activate p53 transcriptional activity.
Mentions: To test whether Wnt-5a–induced degradation of β-catenin S37A is also mediated by proteasome, we used epoxomicin to block proteasome-mediated protein degradation (Meng et al., 1999). We found that β-catenin S37A degradation induced by Wnt-5a was substantially inhibited when the cells were treated with Epoxomicin (Fig. 4 A). As the Siah–APC–Ebi E3 ubiquitin ligase complex has been implicated in GSK-3–independent β-catenin degradation (Liu et al., 2001; Matsuzawa and Reed, 2001), we then examined whether Siah activity is required for Wnt-5a–induced β-catenin degradation. We found that both Siah1 and 2 exhibit activities similar to Wnt-5a in inhibiting canonical Wnt activities in 293 cells (unpublished data). Moreover, a dominant negative Siah2 (ΔSiah2) was able to rescue the β-catenin activity that had been suppressed by Wnt-5a (Fig. 4 B). In addition, degradation of wild-type and an activated form of β-catenin by Wnt-5a signaling were also suppressed by ΔSiah1 and ΔSiah2 (Fig. 4 C and not depicted). Furthermore, we found that, as a result of deceased degradation of the endogenous β-catenin, both ΔSiah1 and ΔSiah2 up-regulated the TOPFLASH activity and this was not inhibited by Wnt-5a (Fig. 4 B). As it has been shown that Siah-dependent β-catenin degradation requires APC activity (Liu et al., 2001; Matsuzawa and Reed, 2001), we next examined whether a dominant negative form of APC (ΔAPC), which has been shown to sequester Siah proteins, could also block β-catenin degradation induced by Wnt-5a signaling. We found that indeed both the activity and protein levels of β-catenin were rescued by ΔAPC (Fig. 4 D). In addition, Wnt-5a hardly inhibited the TOPFLASH activity up-regulated by ΔAPC when there was no exogenous β-catenin (Fig. 4 D). All these results demonstrate that Wnt-5a–induced β-catenin degradation requires functional Siah and APC.

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