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. 2015 Aug 13;162(4):780-94.
doi: 10.1016/j.cell.2015.07.013.

Alternative Wnt Signaling Activates YAP/TAZ

Hyun Woo Park  1 , Young Chul Kim  2 , Bo Yu  3 , Toshiro Moroishi  1 , Jung-Soon Mo  1 , Steven W Plouffe  1 , Zhipeng Meng  1 , Kimberly C Lin  1 , Fa-Xing Yu  4 , Caroline M Alexander  5 , Cun-Yu Wang  3 , Kun-Liang Guan  6
Affiliations

Alternative Wnt Signaling Activates YAP/TAZ

Hyun Woo Park et al. Cell. .

Abstract

The transcriptional co-activators YAP and TAZ are key regulators of organ size and tissue homeostasis, and their dysregulation contributes to human cancer. Here, we discover YAP/TAZ as bona fide downstream effectors of the alternative Wnt signaling pathway. Wnt5a/b and Wnt3a induce YAP/TAZ activation independent of canonical Wnt/β-catenin signaling. Mechanistically, we delineate the "alternative Wnt-YAP/TAZ signaling axis" that consists of Wnt-FZD/ROR-Gα12/13-Rho GTPases-Lats1/2 to promote YAP/TAZ activation and TEAD-mediated transcription. YAP/TAZ mediate the biological functions of alternative Wnt signaling, including gene expression, osteogenic differentiation, cell migration, and antagonism of Wnt/β-catenin signaling. Together, our work establishes YAP/TAZ as critical mediators of alternative Wnt signaling.

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Figures

Figure 1
Figure 1. Wnt Ligands Activate YAP/TAZ Through Alternative Wnt Signaling
(A) Transgenic expression of Wnt1, but not ΔNβ-catenin, induce YAP/TAZ accumulation in mice. Representative images of YAP/TAZ expression level in Wnt1- and ΔNβ-catenin-expressing hyperplastic mammary glands. Mammary gland tissues from 3 month-old MMTV-Wnt1 and MMTV-ΔNβ-cat transgenic mice were stained with either YAP or TAZ antibodies. Scale bars, 200 µm. (B) Western blot analysis of YAP/TAZ and CTGF in tissues isolated from Wnt1 and ΔNβ-catenin-expressing hyperplastic mammary glands. (C) TAZ accumulation by Wnt3a is insensitive to DKK1. MCF10A cells were serum-starved for 16 hr, and then treated with Wnt3a (100 ng/ml) alone, or in the presence of DKK1 (200 ng/ml). (D) Wnt5a and Wnt5b promote YAP/TAZ accumulation. MCF10A cells were serum-staved and stimulated with either Wnt5a (400 ng/ml) or Wnt5b (400 ng/ml). Upper arrowhead indicates phosphorylated band of Dvl2. (E) YAP/TAZ activation by Wnt ligands is insensitive to RSPO1 and DKK1. ST2 cells were serum-starved and stimulated for 4 hr with Wnt3a or Wnt5a in the presence of RSPO1 (100 ng/ml) or DKK1 (200 ng/ml). Lower arrowhead corresponds to the dephosphorylated, active form of YAP on a phos-tag gel. (F) Differential effects of Wnt ligands and inhibitors on YAP/TAZ and β-catenin. Control and Wnt3a stable L cells were serum-starved then stimulated for 4 hr with indicated recombinant proteins or C3 transferase (2 µg/ml), a RhoA inhibitor. (G and H) Alternative Wnt signaling induces YAP/TAZ nuclear localization. MEFs were serum-starved then pretreated with either DMSO (G) or cycloheximide (CHX, 10 µg/ml) (H) for 1 hr. After Wnt5b stimulation (400 ng/ml) for 4 hr, cells were fixed for immunofluorescence with anti-YAP/TAZ antibody. Scale bar, 20 µm. See also Figure S1.
Figure 2
Figure 2. The Wnt-FZD/ROR-Gα12/13-Rho-Lats1/2 Pathway Activates YAP/TAZ
(A) FZD1 expression activates endogenous YAP/TAZ. HEK293A cells were transfected with FZD1, then serum-starved for 16 hr before harvest. Lower arrowhead in YAP phos-tag blot represents dephosphorylated YAP. Upper arrowhead in Dvl2 blot represents phosphorylated Dvl2. (B) FZD1 expression promotes YAP/TAZ nuclear localization. HEK293A cells were transfected with FZD1. After starvation for 16 hr, cells were fixed for immunofluorescence with anti-YAP/TAZ antibody. Scale bar, 20 µm. (C) ROR1 potentiates FZD-induced YAP/TAZ activation. (D) Knockdown of Gα12/13, but not Gαq/11, suppresses FZD1-induced YAP/TAZ activation. (E and F) Knockout of Gα12/13, but not Gαq/11, blocks Wnt5b-induced YAP/TAZ activation. Experiments in DKO MEFs were performed within 5 days after Cre infection. DKO cells were serum-starved then stimulated with Wnt5b (400 ng/ml). Cre infection efficiency was confirmed by Gα13 and Gαq blots. (G) Rho GTPases mediate FZD1-Gα12/13-induced YAP/TAZ activation. FZD1 was co-transfected with dominant negative (DN) or constitutively active (CA) Rho GTPase mutants in control or Gα12/13 depleted HEK293A cells, respectively. (H) Expression of Rho-GDI inhibits FZD1, LPAR1, and LPA-induced YAP/TAZ activation. (I) Lats1/2 are required for YAP/TAZ activation by FZD1. HEK293A cells with deletion of Sav1, Mst1/2, or Lats1/2 were transfected with FZD1 then serum-starved for 16 hr before harvest. (J) Lats1/2 are required for Wnt3a–induced TAZ activation. WT and Lats1/2 DKO MEFs were serum-starved then stimulated with Wnt3a (100 ng/ml). (K) Inhibition of Lats1 kinase activity by FZD1. In vitro kinase assay was performed with immunoprecipitated Lats1 and recombinant YAP protein. (L) Components of the alternative Wnt-YAP/TAZ signaling axis. See also Figure S2.
Figure 3
Figure 3. Wnt3a Activates YAP/TAZ Independent of LRP5/6
(A) Wnt3a activates YAP/TAZ in LRP5/6 DKO cells. LRP5/6 DKO MEFs were starved for 16 hr then stimulated with Wnt3a (100 ng/ml). LRP5/6 deletion was confirmed by western blot. (B) LRP5/6 are required for Wnt3a–induced expression of β-catenin target gene Axin2, but not YAP/TAZ target gene Ctgf. Wnt3a–induced expression of Axin2 and Ctgf mRNA was measured by qRT-PCR in control and LRP5/6 DKO MEFs for 0, 2, 4 hr. (C and D) Experiments similar to panels A and B were performed in 3T3-L1 cells. Data are presented as mean ± SEM. See also Figure S3.
Figure 4
Figure 4. YAP/TAZ Mediate Alternative Wnt Signaling in Osteogenesis and Cell Migration
(A) Transgenic expression of Wnt4 induces YAP/TAZ activation. Representative images of YAP/TAZ expression levels in femur sections from 2-month-old WT and OB-Wnt4 mice. Scale bar, 50 µm. (B and C) ALP staining (B) and activity (C) in MSC after treatment with normal culture media (control), osteoinduction media (OI), and Wnt4 (100ng/ml) for 3 days. Depletion of YAP/TAZ by siRNA impairs Wnt4-induced ALP activity. (D) ARS staining in MSCs after treatment with OI and Wnt4 (100ng/ml) for 7 days. Depletion of YAP/TAZ by siRNA impairs Wnt4-induced calcium deposition in MSCs. *P < 0.05, **P < 0.01 (Student’s t-test), n.s.: P > 0.05 by one-way ANOVA with Tukey’s post-hoc test. (E) YAP/TAZ mediate FZD1-induced cell migration. Migration of HEK293A cells transfected with indicated constructs were assessed by transwell cell migration assays. Data are presented as mean ± SEM. (F) Depletion of TEAD blocks Wnt5b-induced wound closure. A scratch was made across a confluent monolayer of stable TEAD knockdown MCF10A cells, and Wnt5b-induced wound closure (400 ng/ml) was monitored for 12 hr. See also Figure S4.
Figure 5
Figure 5. YAP/TAZ Induce Secreted Inhibitors of Wnt/β-Catenin Signaling
(A and B) Inhibition of Wnt/β-catenin signaling by nuclear YAP/TAZ. Stable MCF10A–YAP-5SA (A) and TAZ-4SA (B) cells were serum-starved, then stimulated with Wnt3a (100 ng/ml). (C) Impaired Wnt/β-catenin signaling in Lats1/2 DKO cells. Axin2 mRNA level was measured in Wnt3a (100 ng/ml) stimulated Lats1 KO and Lats1/2 DKO MEFs. Data are presented as mean ± SEM. *P < 0.05 (Student’s t-test). (D) Inhibition of Wnt/β-catenin signaling by TAZ-4SA conditioned media. HEK293A cells were pretreated for 2 hr with conditioned media (CM) from control and TAZ-4SA-expressing MCF10A cells then stimulated with Wnt3a (100 ng/ml). In lanes 4–6, DKK1 (200 ng/ml) was added in control-CM. (E) RNA-seq data in YAP-overexpressing (YAP-OE) MCF10A cells. The fold-induction of each gene is ranked among 22,341 genes. (F-I) Induction of YAP target genes among the Wnt ligands (F), DKK family (G), BMP ligands (H), and IGFBP family (I). Fold-change by YAP is indicated above each bar graph. Ligands without corresponding number were undetectable or below threshold in RNA-seq analysis. (J) Inhibition of β-catenin/TCF target genes by YAP. (K) Induction of secreted Wnt inhibitors in YAP transgenic mouse liver. qRT-PCR analysis in the liver of YAP transgenic (YAP Tg) and control (WT) mice fed with 0.2 mg/ml doxycycline for 2 weeks. n = 3 mice per group; Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 (Student’s t-test). See also Figure S5.
Figure 6
Figure 6. WNT5A/B Are YAP/TAZ-TEAD-Mediated Wnt Target Genes
(A) TEAD induce Wnt5a/b and inhibit Wnt/β-catenin signaling. Stable MCF10A-4SA and 4SA/S51A cells were serum-starved for 16 hr then stimulated with Wnt3a (100 ng/ml). (B) WNT5A promoter region contains two TEAD binding sequences (TBS; GGAATG). Arrows underline primers used to amplify the region with each TBS. (C) TEAD1 binds WNT5A promoter. ChIP experiment using anti-TEAD1 antibody was performed in TAZ-4SA-MCF10A cells. CTGF, positive control; GAPDH and WNT5A–CR (coding region), negative controls. Data are presented as mean ± SEM. *IgG versus anti-TEAD1 antibody, P < 0.05 (Student’s t-test). (D) Activation of YAP/TAZ-Wnt5a/b axis by FZD1/ROR1. Stable MCF10A–FZD1/ROR1 cells were stimulated with Wnt3a (100 ng/ml). Note that Wnt3a induces Wnt5a/b accumulation in control cells (lanes 1–3). (E) VP suppresses FZD1/ROR1-induced WNT5A gene expression. TEAD target genes, WNT5A and CTGF, and TCF target gene MYC mRNA were measured in MCF10A–FZD1/ROR1 (F+R) cells after VP (7 mM) treatment for 0, 2, 4 hr. Data are presented as mean ± SEM. *Compared to no VP treatment, P < 0.05 (Student’s t-test). (F and G) TEAD mediates Wnt3a–induced WNT5A and WNT5B gene expression. Wnt3a (100 ng/ml) treatment for 0, 2, 4 hr induced mRNA expression of WNT5A (F) and WNT5B (G) in MCF10A and 3T3-L1 cells, respectively. In TEAD knockdown cells, Wnt3a–induced WNT5A/B expression was impaired. Data are presented as mean ± SEM. *Control versus shTEAD at 4 hr, P < 0.05 (Student’s t-test). (H) Effect of oncogenic PIK3CA mutations on YAP/TAZ activation. PIK3CA-E545K and H1047R knockin MCF10A were starved for 16 hr then harvested. (I) VP (7 µM) suppresses PIK3CA-H1047R–induced WNT5A and CTGF gene expression. Data are presented as mean ± SEM. *Compared to no VP treatment, P < 0.05 (Student’s t-test). (J) VP (7 µM) induces β-catenin/TCF target genes in PIK3CA-H1047R knockin cells. See also Figure S6.
Figure 7
Figure 7. Alternative Wnt-YAP/TAZ-TEAD Axis Antagonizes Wnt/β-Catenin Signaling in Adipogenesis
(A and B) Wnt5b promotes YAP/TAZ accumulation (A), and nuclear translocation (B) in 3T3-L1 preadipocytes. Scale bar, 20 µm (B). (C and D) Differential regulation of YAP/TAZ-TEAD and β-catenin/TCF target genes by VP (7 µM) (C) and TEAD depletion (D). (E) TEAD knockdown potentiates Wnt3a–induced Wnt/β-catenin signaling. Axin2 mRNA was measured after Wnt3a (100 ng/ml) stimulation for 0, 2, 4 hr in serum-starved control and shTEAD 3T3-L1 cells. Data are presented as mean ± SEM. (F) TEAD knockdown impairs Wnt5b-induced inhibition of Wnt/β-catenin signaling. Note that left y-axis is for control, and right y-axis is for shTEAD 3T3-L1 cells. Data are presented as mean ± SEM. (G) Wnt5a antagonizes Wnt/β-catenin signaling in adipogenesis. Control and stable 3T3-L1-Wnt5a cells were treated with Wnt3a (0, 50, 100 ng/ml) after induction of adipogenesis. Experiment was terminated within 6 days after ADM treatment to compare panels 2 and 6. Stable expression of Wnt5a derepressed Wnt3a–induced adipogenesis inhibition. (H) TAZ and TEAD mediate the antagonistic effect of Wnt5a on Wnt3a in adipogenesis. In control cells, pretreatment of Wnt5a (400 ng/ml) impaired the effect of Wnt3a (100 ng/ml) in adipogenesis. In contrast, Wnt5a did not antagonize the effect of Wnt3a in TAZ or TEAD-knockdown 3T3-L1 cells. See also Figure S7.
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