Figure 1.
Opposing effects of Dkk1 on LRP6 protein level and Wnt/LRP6 signaling.
(A) HEK293 cells were co-transfected with indicated amounts of Dkk1 and Myc-tagged human LRP6 plasmids. Total plasmid levels were balanced with the empty vector. After 48 h, the level of LRP6 was examined by Western blotting using the anti-Myc antibody. (B) HEK293 cells were co-transfected with indicated amounts of LRP6 and Dkk1 plasmids together with 0.1 µg of the TOP-FLASH TCF luciferase construct and 0.1 µg of β-galactosidase-expressing vector. After 48 h, the luciferase activity was determined with normalization to the activity of the β-galactosidase.
Figure 2.
Exogenously added Dkk1 is sufficient to enhance LRP6 protein level.
(A) HEK293 cells in 6 well plates were transiently transfected with 0.8 µg of Myc-tagged human LRP6 plasmid. Twenty four hours following transfection, cells were cultured with Dkk1 CM (+) or vector-transfected control CM (−) for additional 24 h. The level of LRP6 was then examined by Western blotting using the anti-Myc antibody. Similarly, HT1080 cells stably transduced with HA-tagged LRP6 in 6-well plates were cultured with Dkk1 CM or control CM for 24 h, and the level of LRP6 was then examined by Western blotting with the anti-HA antibody. (B) HT1080 cells stably transduced with HA-tagged LRP6 were cultured with Dkk1 CM or control CM for 4, 8, or 16 h. The level of LRP6 was then examined by Western blotting with the anti-HA antibody. (C) HT1080 cells stably transduced with HA-tagged LRP6 in 6 well plates were cultured with recombinant Dkk1 protein (1 µg/ml) or recombinant sFRP1 (1 µg/ml) for 24 h. The level of LRP6 was then examined by Western blotting with the anti-HA antibody. Samples were also probed with anti-actin antibody to verify equal loading.
Figure 3.
HT1080 cells stably transfected with HA-tagged LRP6 were incubated with 5 µg/ml of cycloheximide in the presence of 25% of Dkk1 CM or 25% of control CM for 0, 3, 6 or 9 h. Cells were then harvested, and the level of LRP6 was examined by Western blotting. Samples were also probed with anti-tubulin antibody to verify equal loading. The pixels for each band were measured, normalized and plotted. Data are mean values of three independent experiments with the SE values indicated by error bars. *P<0.05 versus corresponding control value.
Figure 4.
Kremen2 induces LRP6 turnover and Wnt/LRP6 signaling down-regulation.
(A) HEK293 cells were transiently co-transfected with 0.2 µg of Myc-tagged human LRP6 plasmid, and 0.8 µg of human Dkk1, 0.8 µg of mouse Kremen2, or both Dkk1 and Kremen2 plasmids. After 48 h post-transfection, the level of LRP6 was examined by Western blotting with the anti-Myc antibody. Samples were also probed with the anti-actin antibody to verify equal loading. (B) HEK293 cells were transiently co-transfected with 0.2 µg of Myc-tagged human LRP6 plasmid, 0.8 µg of human Dkk1, 0.8 µg of mouse Kremen2, or both Dkk1 and Kremen2 plasmids together with 0.1 µg of the TOP-FLASH TCF luciferase construct, and 0.1 µg of β-galactosidase-expressing vector. After 48 h, the luciferase activity was determined with normalization to the activity of the β-galactosidase.
Figure 5.
Dkk1 blocks Wnt3A-induced LRP6 down-regulation.
(A) HT1080 cells stably transduced with HA-tagged LRP6 in 6 well plates were incubated with 25% of Wnt3A CM, Wnt5A CM or L cell control CM for 24 h. The level of LRP6 was then examined by Western blotting with anti-HA antibody. (B) HT1080 cells stably transduced with HA-tagged LRP6 in 6 well plates were incubated with 0–25% of Wnt3A CM for 24 h. The level of LRP6 was then examined by Western blotting. (C) HT1080 cells stably transfected with HA-tagged LRP6 in 6 well plates were incubated with 25% of Wnt3A CM for 1.5–24 h. The levels of total cellular LRP6, cytosolic free β-catenin and total cellular β-catenin were examined by Western blotting. (D) HT1080 cells stably transduced with HA-tagged LRP6 in 6 well plates were incubated with 25% of L cell control CM, 25% of Wnt3A CM, or 25% of Wnt3A CM with 1 µg/ml of recombinant Dkk1 protein for 24 h. The level of LRP6 was then examined by Western blotting with the anti-HA antibody. Samples were also probed with the anti-actin antibody to verify equal loading.
Figure 6.
LRP6 levels on cell surface and in endosomes after Dkk1 and Wnt3A treatment.
(A) HT1080 cells stably transduced with HA-tagged LRP6 were incubated with 25% of L cell control CM, 25% of Wnt3A CM, 0.5 µg/ml of recombinant Dkk1 protein, or 25% of Wnt3A CM plus 0.5 µg/ml of recombinant Dkk1 protein for 24 h. Cell surface LRP6 was labeled with the anti-HA antibody and detected with goat anti-mouse IgG-FITC by FACS. Each value represents the difference between total and background fluorescence intensities and is the average of triple determinations with the SE indicated by error bar. **P<0.01 compared to L cell control CM-treated cells; ##P<0.01 compared to L cell control CM-treated cells and Wnt3A CM-treated cells. (B) HT1080 cells stably transduced with HA-tagged LRP6 were incubated with control CMs, 25% of Wnt3A CM, 25% of Dkk1 CM, or Wnt3A CM plus Dkk1 CM for 24 h. Levels of LRP6 in endosomes were examined by Western blotting with the anti-HA antibody. Samples were also probed with the anti-Rab5 antibody to verify equal loading and endosome preparation. The pixels for each band were measured, normalized and plotted. Data are mean values of four independent experiments with the SE values indicated by error bars. *P<0.05 compared to L cell control CM-treated cells; #P<0.05 compared to L cell control CM-treated cells and Wnt3A CM plus Dkk1C CM-treated cells.
Figure 7.
Dkk1 stabilizes endogenous LRP6 and blocks Wnt3A-induced LRP6 down-regulation in breast cancer HCC1187 cells.
HCC1187 cells were incubated with 25% of L cell control CM or 25% of Wnt3A CM in the presence or absence of 500 ng/ml of recombinant Dkk1 protein for 24 h. The level of LRP6 was then examined by Western blotting. Samples were also probed with the anti-actin antibody to verify equal loading. The pixels for each band were measured, normalized and plotted. Data are mean values of three independent experiments with the SE values indicated by error bars. * P<0.05 versus control.
Figure 8.
Effects of Filipin III and monodansylcadaverine on LRP6 turnover.
HT1080 cells stably transduced with HA-tagged LRP6 were incubated with 0.5 µg/ml of recombinant Dkk1 protein (A, C) or 25% of Wnt3A CM (B, D), and treated with Filipin III (3 µg/ml) (A, B) or monodansylcadaverine (MDC) (100 µM) for 24 h. The level of LRP6 was then examined by Western blotting with the anti-HA antibody. Samples were also probed with the anti-actin antibody to verify equal loading.
Figure 9.
Model depicting the LRP6 turnover pathways.
The first pathway is mediated by Kremen. In the presence of Kremen, LRP6 antagonist Dkk1 serves as a bridge between LRP6 and Kremen and induces LRP6 internalization and degradation, terminating Wnt signaling. Furthermore, Kremen might directly bind to LRP6 and induce LRP6 degradation. The second pathway is mediated by Wnt/Frizzled. While Wnt binding to its receptors LRP6 and Frizzled (Fz) induces LRP6-Wnt-Fz complex formation, the endocytosis and Wnt/LRP6 signaling decay of this complex occur after Wnt signaling activation. Dkk1 blocks Wnt binding to LRP6 and then inhibits LRP6 turnover mediated by Wnt/Fz.