The pseudoreceptor BMP and activin membrane-bound inhibitor positively modulates Wnt/beta-catenin signaling

Abstract

The canonical Wnt/beta-catenin pathway plays a pivotal role in regulating embryogenesis and tumorigenesis by promoting cell proliferation. BAMBI (BMP and activin membrane-bound inhibitor) has previously been shown to negatively regulate the signaling activity of transforming growth factor-beta, activin, and BMP and was identified as a target of beta-catenin in colorectal and hepatocellular tumor cells. In this study, we provide evidence that BAMBI can promote the transcriptional activity of Wnt/beta-catenin signaling. Overexpression of BAMBI enhances the expression of Wnt-responsive reporters, whereas knockdown of endogenous BAMBI attenuates them. Accordingly, BAMBI also promotes the nuclear translocation of beta-catenin. BAMBI interacts with Wnt receptor Frizzled5, coreceptor LRP6, and Dishevelled2 and increases the interaction between Frizzled5 and Dishevelled2. Finally we show that BAMBI promotes the expression of c-myc and cyclin D1 and increases Wnt-promoted cell cycle progression. Altogether, our data indicate that BAMBI can function as a positive regulator of the Wnt/beta-catenin pathway to promote cell proliferation.

FIGURE 1.

BAMBI enhances the expression of the Wnt-responsive reporters. In A: Left panel, BAMBI increases the expression of the Wnt-responsive LEF-luciferase. HEK293T cells were transfected with LEF-luciferase (0.5 μg) along with Wnt-1 (0.2 μg) or BAMBI (0.2 μg) and at 48 h post-transfection, cells were harvested for luciferase measurement. Right panel, BAMBI enhances the Wnt-1-activated expression of LEF-luciferase in a dose-dependent manner. Increasing amount of BAMBI plasmids (0.1, 0.2, to 0.4 μg) were transfected into HEK293T cells with LEF-luciferase (0.5 μg) and Wnt-1 (0.1 μg), and reporter assay was performed at 48 h post-transfection. B, BAMBI increases the Wnt-1-induced expression of Topflash. Topflash (left panel) or Fopflash (right panel) (0.5 μg each) was transfected into HEK293T cells along with Wnt-1 (0.2 μg) or BAMBI (0.2 μg), and reporter assay was performed at 48 h post-transfection. For transfection, the total amount of DNA was adjusted with empty plasmid. The pRL-tk Renilla reporter (20 ng) was co-transfected to normalize transfection efficiency. Each experiment was performed in triplicate, and the data represent the mean ± S.D. after normalized to Renilla activity. The asterisks indicate a statistically significant difference (**, p < 0.01).

FIGURE 2.

Knockdown of endogenous BAMBI expression impairs Wnt signaling. A, BAMBI siRNA effectively suppresses BAMBI expression. HEK293T cells were transfected with indicated constructs, and at 40 h post-transfection, the cells were harvested for immunoblotting. Tubulin was used as a loading control. B and C, knockdown of endogenous BAMBI expression impairs the Wnt-induced expression of LEF-luciferase. LEF-luciferase was cotransfected into HEK293T (B) or Hep3B (C) cells with indicated plasmids (0.3 μg) together with or without Wnt-1 (0.2 μg). Then luciferase activity was determined as in Fig. 1. The asterisks indicate a statistically significant difference (**, p < 0.01). D, BAMBI enhances the nuclear accumulation of β-catenin. HEK293T cells were transfected with the constructs as indicated. At 40 h post-transfection, the cells were harvested, and anti-β-catenin immunoblotting was performed with the nuclear lysates. Lamin was used as a nuclear marker. Si-NS, nonspecific siRNA; si-BAMBI, siRNA specific for BAMBI.

FIGURE 3.

BAMBI interacts with Fzd5 and LRP6. A, BAMBI interacts with Fzd5 at the endogenous protein levels. HEK293T cell lysates were subject to immunoprecipitation with control rabbit IgG or anti-BAMBI antibody followed by anti-Fzd5 immunoblotting (upper panel). Protein expression was confirmed by immunoblotting (middle and lower panels). C, rabbit control IgG. B and C, BAMBI associates with Fzd5 when overexpressed. HEK293T cells were transfected with indicated HA-tagged Fzd5 and myc-tagged BAMBI. At 40 h post-transfection, the cell lysates were harvested for anti-myc (B) or anti-HA (C) immunoprecipitation and anti-HA (B) or anti-myc (C) immunoblotting (top panel). Protein expression was confirmed by immunoblotting of 10% of the total lysate (middle and lower panels). IgG-L, light chain IgG; IgG-H, heavy chain IgG. D, BAMBI interacts with LRP6. Protein interaction was examined as in B. E, Fzd5 binds to both the extracellular (BAMBI-N) and intracellular (BAMBI-C) domains of BAMBI, whereas LRP6 associates only with BAMBI-C. HEK293 cells were transfected with the constructs (5 μg each) as indicated. Protein interaction was examined as in B. F, BAMBI-C, but not BAMBI-N, has the ability to promote Wnt signaling. Reporter assay in HEK293T cells was performed as in Fig. 1. In all the experiments shown here, the total amount of plasmid DNA was adjusted with pCMV5 empty plasmid.

FIGURE 4.

BAMBI associates with Dishevelled. A, BAMBI interacts with Dvl2. HEK293T cells were transfected with FLAG-tagged Dvl2 and myc-tagged BAMBI. B, BAMBI associates with Dvl2 at endogenous levels. HEK293T cell lysates were subject to immunoprecipitation with control IgG or anti-Dvl2 antibody followed by anti-BAMBI immunoblotting. C, BAMBI binds to three isoforms of Dvl. HEK293T cells were transfected with indicated constructs. D, BAMBI interacts with Dvl2 via its intracellular domain. E, both the PDZ and DEP domains of Dvl2 can interact with BAMBI. In all of the immunoprecipitation assays (except B), 5 μg of DNA for each construct was transfected and the total amount of plasmid DNA was adjusted with pCMV5 empty vector. At 40 h post-transfection, the cells were harvested and subjected to immunoprecipitation and then immunoblotting analysis (upper panel). Protein expression was confirmed by immunoblotting of total cell lysates (middle and lower panels).

FIGURE 5.

BAMBI enhances the interaction between Fzd5 and Dvl2. A, BAMBI increases the interaction between Fzd5 and Dvl2. Extracts from HEK293T cells ectopically expressing the constructs (5 μg for each) as indicated were subjected to anti-FLAG immunoprecipitation and anti-myc immunoblotting (top panel). Protein expression was confirmed by immunoblotting of 10% of the total lysate (middle and lower panels). IgG-H, heavy chain IgG. B and C, BAMBI synergizes with Dvl2 to increase the expression of LEF-luciferase in HEK293T (B) and Hep3B (C) cells. Cells were transfected with LEF-luciferase together with Dvl2 (0.2 μg) and BAMBI (0.3 μg). Reporter assay was performed as in Fig. 1. The asterisks indicate a statistically significant difference (*, p < 0.05; **, p < 0.01).

FIGURE 6.

BAMBI enhances the cell proliferation-promoting effect of Wnt1. A, BAMBI increases the expression of a cyclin D1 promoter-driven reporter. HEK293T cells were co-transfected with a cyclin D1 luciferase reporter as well as indicated constructs, and the luciferase activity was measured at 48 h post-transfection. B, BAMBI increases the expression of a c-myc promoter-driven reporter. The asterisks indicate a statistically significant difference (**, p < 0.01). C, knockdown of endogenous BAMBI reduces the expression of Wnt target genes in HEK293T cells. HEK293T cells were transfected with indicated plasmids (3 μg for each), and the total amount of DNA plasmid was adjusted with corresponding empty vector. At 48 h post-transfection, the cells were harvested for RT-PCR analysis. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as a control. D, BAMBI promotes cell growth. HEK293T cells were transfected with control vector or BAMBI together with or without Wnt-1 plasmid (3 μg for each). At 48 h post-transfection, the cells were harvested for fluorescence-activated cell sorting analysis.