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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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Related in: MedlinePlus

Wnt-5a suppressed canonical Wnt signaling activity. β-Catenin/TCF transcriptional activity was indicated by TOPFLASH luciferase activity. FOPFLASH, which contains mutant LEF/TCF binding sites, was used as a negative control for pathway specificity. Luciferase activity was measured 48 h after transfection and the results are shown as relative luciferase activity. The histograms are the average ± SD from three independent transfections. (A) Wnt-3a activated the reporter activity and such activation was gradually inhibited by higher doses of Wnt-3a. A low dose of Wnt-5a (67 ng) down-regulated the reporter activity induced by different doses of Wnt-3a (10 ng–1 μg) in 293 cells. The reporter activity activated by a low dose of Wnt-3a (33 ng) was inhibited by Wnt-5a in a dose dependent manner (10–167 ng). The mutant reporter FOPFLASH did not respond to Wnt-3a or Wnt-5a. (B) Wnt-5a down-regulated the reporter activity induced by β-catenin in 293 cells. (C) 293 cells were transfected with β-catenin and GFP or Wnt-5a where indicated. Wnt-5a expression led to a decrease in β-catenin protein levels.
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fig1: Wnt-5a suppressed canonical Wnt signaling activity. β-Catenin/TCF transcriptional activity was indicated by TOPFLASH luciferase activity. FOPFLASH, which contains mutant LEF/TCF binding sites, was used as a negative control for pathway specificity. Luciferase activity was measured 48 h after transfection and the results are shown as relative luciferase activity. The histograms are the average ± SD from three independent transfections. (A) Wnt-3a activated the reporter activity and such activation was gradually inhibited by higher doses of Wnt-3a. A low dose of Wnt-5a (67 ng) down-regulated the reporter activity induced by different doses of Wnt-3a (10 ng–1 μg) in 293 cells. The reporter activity activated by a low dose of Wnt-3a (33 ng) was inhibited by Wnt-5a in a dose dependent manner (10–167 ng). The mutant reporter FOPFLASH did not respond to Wnt-3a or Wnt-5a. (B) Wnt-5a down-regulated the reporter activity induced by β-catenin in 293 cells. (C) 293 cells were transfected with β-catenin and GFP or Wnt-5a where indicated. Wnt-5a expression led to a decrease in β-catenin protein levels.

Mentions: To understand the mechanism by which Wnt-5a inhibits the canonical Wnt signaling, we used a TCF reporter (TOPFLASH; Korinek et al., 1997) as a readout for the canonical Wnt pathway activity in a variety of cell lines. In immortalized human embryonic kidney cells (HEK 293 cells), Wnt-3a expression led to up-regulation of the canonical Wnt activity as indicated by the increased luciferase activity (Fig. 1 A). When Wnt-5a was cotransfected with Wnt-3a, we found that the luciferase activity was largely reduced. Interestingly, although lower levels of Wnt-3a expression (10 and 33 ng) activated TOPFLASH in a dose-dependent manner, higher levels of Wnt-3a (167 ng–1 μg) resulted in gradually reduced stimulation of TOPFLASH activity (Fig. 1 A). However, inhibition of the canonical Wnt signaling by Wnt-5a was not simply a result of higher Wnt expression. This is because Wnt-5a did not stimulate TOPFLASH when expressed at both low and high levels (not depicted), and a very low level of Wnt-5a expression (10 ng) inhibited TOPFLASH activity stimulated by a low level of Wnt-3a expression (33 ng; Fig. 1 A).


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 suppressed canonical Wnt signaling activity. β-Catenin/TCF transcriptional activity was indicated by TOPFLASH luciferase activity. FOPFLASH, which contains mutant LEF/TCF binding sites, was used as a negative control for pathway specificity. Luciferase activity was measured 48 h after transfection and the results are shown as relative luciferase activity. The histograms are the average ± SD from three independent transfections. (A) Wnt-3a activated the reporter activity and such activation was gradually inhibited by higher doses of Wnt-3a. A low dose of Wnt-5a (67 ng) down-regulated the reporter activity induced by different doses of Wnt-3a (10 ng–1 μg) in 293 cells. The reporter activity activated by a low dose of Wnt-3a (33 ng) was inhibited by Wnt-5a in a dose dependent manner (10–167 ng). The mutant reporter FOPFLASH did not respond to Wnt-3a or Wnt-5a. (B) Wnt-5a down-regulated the reporter activity induced by β-catenin in 293 cells. (C) 293 cells were transfected with β-catenin and GFP or Wnt-5a where indicated. Wnt-5a expression led to a decrease in β-catenin protein levels.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Wnt-5a suppressed canonical Wnt signaling activity. β-Catenin/TCF transcriptional activity was indicated by TOPFLASH luciferase activity. FOPFLASH, which contains mutant LEF/TCF binding sites, was used as a negative control for pathway specificity. Luciferase activity was measured 48 h after transfection and the results are shown as relative luciferase activity. The histograms are the average ± SD from three independent transfections. (A) Wnt-3a activated the reporter activity and such activation was gradually inhibited by higher doses of Wnt-3a. A low dose of Wnt-5a (67 ng) down-regulated the reporter activity induced by different doses of Wnt-3a (10 ng–1 μg) in 293 cells. The reporter activity activated by a low dose of Wnt-3a (33 ng) was inhibited by Wnt-5a in a dose dependent manner (10–167 ng). The mutant reporter FOPFLASH did not respond to Wnt-3a or Wnt-5a. (B) Wnt-5a down-regulated the reporter activity induced by β-catenin in 293 cells. (C) 293 cells were transfected with β-catenin and GFP or Wnt-5a where indicated. Wnt-5a expression led to a decrease in β-catenin protein levels.
Mentions: To understand the mechanism by which Wnt-5a inhibits the canonical Wnt signaling, we used a TCF reporter (TOPFLASH; Korinek et al., 1997) as a readout for the canonical Wnt pathway activity in a variety of cell lines. In immortalized human embryonic kidney cells (HEK 293 cells), Wnt-3a expression led to up-regulation of the canonical Wnt activity as indicated by the increased luciferase activity (Fig. 1 A). When Wnt-5a was cotransfected with Wnt-3a, we found that the luciferase activity was largely reduced. Interestingly, although lower levels of Wnt-3a expression (10 and 33 ng) activated TOPFLASH in a dose-dependent manner, higher levels of Wnt-3a (167 ng–1 μg) resulted in gradually reduced stimulation of TOPFLASH activity (Fig. 1 A). However, inhibition of the canonical Wnt signaling by Wnt-5a was not simply a result of higher Wnt expression. This is because Wnt-5a did not stimulate TOPFLASH when expressed at both low and high levels (not depicted), and a very low level of Wnt-5a expression (10 ng) inhibited TOPFLASH activity stimulated by a low level of Wnt-3a expression (33 ng; Fig. 1 A).

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