CD44 functions in Wnt signaling by regulating LRP6 localization and activation

Abstract

Wnt reception at the membrane is complex and not fully understood. CD44 is a major Wnt target gene in the intestine and is essential for Wnt-induced tumor progression in colorectal cancer. Here we show that CD44 acts as a positive regulator of the Wnt receptor complex. Downregulation of CD44 expression decreases, whereas CD44 overexpression increases Wnt activity in a concentration-dependent manner. Epistasis experiments place CD44 function at the level of the Wnt receptor LRP6. Mechanistically, CD44 physically associates with LRP6 upon Wnt treatment and modulates LRP6 membrane localization. Moreover, CD44 regulates Wnt signaling in the developing brain of Xenopus laevis embryos as shown by a decreased expression of Wnt targets tcf-4 and en-2 in CD44 morphants.

Figure 1

Expression pattern of CD44 isoforms. All CD44 isoforms are encoded by one single gene present on chromosome 11 in humans and on chromosome 2 in mice. The CD44 gene is composed of 20 exons designated as constant and variable exons. Exons 1–5 and 16–20 are constant exons that encode the N-terminal and C-terminal regions of CD44, respectively. Exon 19 is, however, spliced out in most CD44 isoforms. Ten variant exons (v1–v10) encoded by exons 6–15 can be alternatively spliced and included in various combinations in the stem region, thereby giving rise to many CD44 variant isoforms. In human cells, exon v1 (exon 6) encodes a stop codon and is not expressed. The smallest CD44 isoform, CD44s, does not contain any variant exon in the stem region and is virtually ubiquitously expressed. In contrast, the expression of CD44 variant isoforms is restricted to specific tissues

Figure 2

CD44 is a positive regulator of Wnt/β-catenin signaling. (a) HEK293 cells transfected with control siRNA or siRNA against all CD44 isoforms were transfected with TOPFlash and control TK-Renilla vectors. After stimulation with Wnt3a-CM or Co-CM (20 h), cells were lysed and subjected to luciferase measurements or, in parallel, to western blotting (WB) analysis. Insert: siRNA-transfected HEK293 cells were transfected with FOPFlash and control TK-Renilla vectors and 48 h later subjected to luciferase measurements. (b) HEK293 cells were transfected with TOPFlash and control TK-Renilla vectors together with increasing amounts of hCD44s cDNAs. After stimulation with Wnt3a-CM or Co-CM (20 h), cells were lysed and subjected to luciferase measurements or, in parallel, to WB analysis. Insert: HEK293 cells were transfected with an empty vector or hCD44s (75 ng) together with FOPFlash and control TK-Renilla vectors and 48 h later subjected to luciferase measurements. (c) HeLa cells were transfected with control siRNA or siRNA against all CD44 isoforms and treated either with Co-CM or Wnt3a-CM for 3 h. Cells were subjected to WB analysis 72 h after siRNA transfection. The numbers indicate the fold intensity values of the ABC bands normalized to the actin bands. (d) HeLa cells were transfected with control-siRNA or siRNA against all CD44 isoforms. Forty-eight hours after siRNA transfection, cells were treated with Co-CM or Wnt3a-CM for 24 h. Subsequently, cells were subjected to immunofluorescence staining using antibodies against β-catenin (red). The nucleus was stained with DAPI (4,6-diamidino-2-phenylindole; blue). Afterwards, cells were analyzed by confocal microscopy (left panel: β-catenin only; right panel: overlay; scale bar=10 μm), and the percentage of cells with nuclear β-catenin was evaluated (diagram). Data represent mean±S.D. of all confocal microscopy experiments (four independent experiments; n>25 cells per experiment were evaluated). Statistical significance was analyzed using the Student's t-test (*P<0.05). The knockdown of CD44 was confirmed by WB analysis. (e) HEK293 cells were transfected with TOPFlash and control TK-Renilla vectors together with 75 ng of rCD44v4-v7, rCD44v6 or rCD44s cDNAs. After stimulation with Wnt3a-CM or Co-CM (20 h), cells were lysed and subjected to luciferase measurements or, in parallel, to WB analysis. Reporter gene assays and WB analysis are described in ‘Materials and Methods'. All TOPFlash data represent mean±S.D. from at least four independent experiments performed in triplicates. Statistical significance was analyzed using the Student's t-test (*P<0.05, ***P<0.005)

Figure 3

CD44 requires the ICD and its binding to F-actin via Ezrin to regulate Wnt signaling. (a) HEK293 cells were transfected with TOPFlash and control TK-Renilla vectors together with an empty vector or cDNAs for rCD44v4-v7 or rCD44v4-v7ΔCyt (75 ng). After stimulation with Wnt3a-CM or Co-CM (20 h), cells were lysed and subjected to luciferase measurements or, in parallel, to WB analysis. (b) HEK293 cells were transfected with TOPFlash and control TK-Renilla vectors together with empty vector or cDNAs for GST-tagged CD44-Cyt (CD44 ICD, 75 ng) or GST-tagged CD44-CytΔEzBD (CD44 ICD mutated in the Ezrin binding domain, 75 ng). After stimulation with Wnt3a-CM or Co-CM (20 h), cells were lysed and subjected to luciferase measurements or, in parallel, to WB analysis. (c) HeLa cells were transfected either with empty vector or cDNAs for CD44-Cyt or CD44-CytΔEzBD, treated with Wnt3a-CM for 3 h and subjected to WB analysis. (d) HEK293 cells transfected with control siRNA or siRNA against Ezrin were transfected with TOPFlash and control TK-Renilla vectors. After stimulation with Wnt3a-CM or Co-CM (20 h), cells were lysed and subjected to luciferase measurements or, in parallel, to WB analysis. (e) HEK293 cells were transfected with TOPFlash reporter and control Renilla luciferase vectors together with an empty vector or a cDNA corresponding to a DN V-SVG-tagged mutant of Ezrin (DN-Ezrin, 20 ng) mutated in the F-actin binding domain. After stimulation with Wnt3a-CM or Co-CM (20 h), cells were lysed and subjected to luciferase measurements or, in parallel, to WB analysis. Reporter gene assays and WB analysis are described in ‘Materials and Methods'. All data represent mean±S.D. from at least four independent experiments performed in triplicates. Statistical significance was analyzed using the Student's t-test (*P<0.05; n.s.=not significant)

Figure 4

CD44 regulates Wnt signaling at the level of the membrane. (a) HEK293 cells were transfected with control siRNA or siRNA against all CD44 isoforms. Twenty hours later, cells were transfected with TOPFlash and control TK-Renilla vectors. The reporter was activated either by treatment with Wnt3a-CM or by co-transfection of cDNAs for LRP6 (20 ng), Dsh (20 ng) or constitutive ABC (3 ng). Forty hours after DNA transfection, cells were lysed and subjected to luciferase measurements or, in parallel, to WB analysis. (b) HEK293 cells were transfected with TOPFlash and control TK-Renilla vectors together with empty vector or a cDNA for rCD44s (75 ng). The reporter was activated either by treatment with Wnt3a-CM or by co-transfection of LRP6 (20 ng), Dsh-1 (20 ng) or constitutive ABC (3 ng). Forty hours after transfection, cells were analyzed for luciferase activity. Forty hours after transfection, cells were lysed and subjected to luciferase measurements or, in parallel, to WB analysis. Reporter gene assays and WB analysis are described in ‘Materials and Methods'. All data represent mean±S.D. from at least four independent experiments performed in triplicates. Statistical significance was analyzed using the Student's t-test (*P<0.05; n.s.=not significant)

Figure 5

CD44 interacts with LRP6 and is required for LRP6 activation. (a) HeLa cells were transfected with cDNAs for rCD44v4-v7 or rCD44v4-v7ΔCyt together with a FLAG-tagged LRP6 construct. Twenty-four hours after transfection, cells were treated either with Co-CM or Wnt3a-CM for 30 min, lysed and proteins were immunoprecipitated with an antibody against rCD44. Immunoprecipitates were subjected to WB analysis using the FLAG and rCD44 antibodies. (b) HeLa cells were treated for the indicated time points with Wnt3a-CM, lysed and proteins were immunoprecipitated with antibodies against CD44. Immunoprecipitates were subjected to WB analysis with an antibody against LRP6 and CD44. The numbers indicate the fold increase of co-immunoprecipitated LRP6 normalized to the immunoprecipitated CD44. (c) HeLa cells were transfected with cDNAs for DN-Ezrin together with a FLAG-tagged LRP6 construct. Twenty-four hours after transfection, cells were treated either with Co-CM or Wnt3a-CM for 30 min, lysed and proteins were immunoprecipitated with an antibody against hCD44. Immunoprecipitates were subjected to WB analysis using the FLAG and hCD44 antibodies. (d) HeLa cells were transfected with control siRNA or siRNA against all CD44 isoforms and treated either with Co-CM or Wnt3a-CM for 2 h. Cells were subjected to WB analysis 72 h after siRNA transfection (asterisks=mature LRP6 band of untreated (red) or Wnt3a treated (green) cells). The numbers indicate the fold intensity values of the p-LRP6 bands normalized to the bands of total LRP6

Figure 6

Membrane targeting of LRP6 is dependent on CD44 and its binding to F-actin via Ezrin. (a) HeLa cells transfected either with control siRNA or siRNA against all CD44 isoforms were transfected with LRP6-GFP or Ror2-mCherry constructs, and the cellular localization of LRP6-GFP/Ror-mCherry was determined using a Leica SP5 fluorescence microscope with a 63X objective (scale bar=10 μm). Where indicated, in addition to hCD44 siRNA, rCD44s was co-transfected. Quantification is shown in the histogram. (b) HeLa cells transfected either with control siRNA or siRNA against all CD44 isoforms were transfected with a construct for LRP6-GFP and counterstained with an ER marker. Where indicated, cells were co-transfected with Mesd cDNA. Localization of LRP6-GFP was detected as in panel (a). In addition, the percentage of cells with colocalized LRP6-GFP and ER marker was evaluated. (c) HeLa cells transfected either with an empty vector or constructs for CD44 ICD (CD44-Cyt), the CD44 ICD mutated in the Ezrin binding domain (CD44-CytΔEzBD) or DN-Ezrin together with a construct for LRP6-GFP. LRP6-GFP was detected as in panel (a). Data represent mean±S.D. of all confocal microscopy experiments (at least four independent experiments, n>30 cells per condition were evaluated, scale bar=10 μm). Statistical significance was analyzed using the Student's t-test (*P<0.05; n.s.=not significant)

Figure 7

CD44 is required for Wnt target gene expression in Xenopus laevis. (a) HEK293 cells were transfected with TOPFlash and control TK-Renilla vectors together with 75 ng of cDNAs for hCD44s, rCD44s or xCD44s. After stimulation with Wnt3a-CM or Co-CM (20 h), cells were lysed and subjected to luciferase measurements. Data represent mean±S.D. from at least four independent experiments performed in triplicates. Statistical significance was analyzed using the Student's t-test (*P< 0.05). (b) In situ hybridization for the canonical Wnt target genes tcf-4 and en-2 in 32-stage X. laevis embryos. Embryos were injected in the right blastomere at two-cell stage with Co-MO, xCD44-MO alone, xCD44-MO together with cDNA for hCD44s expression vectors or xCD44-MO together with β-catenin expression vectors. FITC-labeled Dextran was co-injected to assess the side of injection. (c) Quantification of experiments described in panel (b). Data represent mean±S.D. from at least four independent experiments; n>75 per condition were evaluated; statistical significance was analyzed using the Student's t-test (*P<0.05)