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

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.

Figure 1.

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.

Figure 2.

Wnt-5a promoted β-catenin degradation through a mechanism independent of GSK-3 and β-TrCP. (A) 24 h after transfection, 293 cells were treated with 40 mM LiCl for 16 h before cells were harvested. LiCl treatment up-regulated TOPFLASH reporter activity. Wnt-5a suppressed the effect of LiCl. (B) Cells were transfected with the indicated plasmids and harvested 48 h later for luciferase assay. Δβ-TrCP up-regulated the TOPFLASH reporter activity and this effect was suppressed by Wnt-5a. (C) 293 cells were transfected with the indicated plasmids and treated with LiCl as in A. LiCl treatment or Δβ-TrCP expression stabilized the endogenous β-catenin and Wnt-5a inhibited this effect. (D) Wnt-5a promoted the degradation of two mutant stabilized forms of β-catenin in 293 cells. Cells were transfected with the indicated plasmids and 48 h after transfection, cells were harvested for western analysis.

Figure 3.

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.

Figure 4.

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.

Figure 5.

Canonical Wnt signaling was increased in the Wnt-5a ^−/− limb buds. Gene expression in embryos was examined by whole mount in situ hybridization. (A) At 12.5 dpc, the expression of Sox9, which marks early chondrogenesis, was not detected in the distal-most limb bud of the Wnt-5a ^{− / −} limb (arrow). (B) Ectopic LacZ staining (arrow) was detected in the Wnt-5a ^{− / −} /TOPGAL distal limb at 12.5 dpc. (C) The β-catenin protein level was increased in the distal Wnt-5a ^{− / −} limb at 11.5 dpc. (D) Sfrp-2 expressing cells (red), but not control cells (blue), induced ectopic expression of ColII (arrows), a marker for chondrocyte differentiation in both wild-type and Wnt-5a ^{− / −} limbs.

Figure 6.

Wnt-5a decreased β-catenin activity and protein stability in the SW48 cells but not in SW480 cells. (A) Wnt-5a expression inhibits TOPFLASH activity in SW48 cells. (B) Wnt-5a expression reduced the protein level of β-catenin in SW48 cells. Both ΔSiah1 and ΔSiah2 blocked the activity of Wnt-5a in reducing β-catenin protein. (C) Wnt-5a also induces Siah2 expression in SW48 cells. (D) Wnt-5a expression in SW480 cells did not lead to the inhibition of β-catenin activity and protein degradation. Expression of APC inhibited β-catenin activity and promoted β-catenin degradation.