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???displayArticle.abstract??? Dickkopf1 (Dkk1) is a secreted antagonist of the Wnt/beta-catenin signaling pathway that acts by direct binding to and inhibiting the Wnt co-receptor LRP6. The related Dkk2, however, can function either as LRP6 agonist or antagonist, depending on the cellular context, suggesting that its activity is modulated by unknown co-factors. We have recently identified the transmembrane proteins Kremen1 and -2 as additional Dkk receptors, which bind to both Dkk1 and Dkk2 with high affinity. Here we show that Kremen2 (Krm2) regulates Dkk2 activity during Wnt signaling. In human 293 fibroblasts transfected dkk2 activates LRP6 signaling. However, co-transfection of krm2 blocks the ability of Dkk2 to activate LRP6 and enhances inhibition of Wnt/Frizzled signaling. Krm2 also co-operates with Dkk4 to inhibit Wnt signaling, but not with Dkk3, which has no effect on Wnt signaling. Likewise, in Xenopus embryos, Dkk2 and Krm2 co-operate in Wnt inhibition leading to anteriorized embryos. Finally, we show that interaction with Krm2 is mediated by the second cysteine-rich domain of Dkks. These results suggest that Krm2 can function as a switch that turns Dkk2 from an activator into an inhibitor of Wnt/lRP6 signaling.
Fig. 1.
TOP-FLASH luciferase reporter assays in 293T cells showing that Dkk1, -2 and -4 but not Dkk3 co-operate with mKrm2 in Wnt inhibition. pCS-Xdkk1, mdkk2, mdkk3, hdkk4 and mkrm2 plasmids were used at 1 ng/well; mwnt1, 8 ng/well; mfz8, 1 ng/well.
Fig. 2.
mKrm2 blocks the activity of Dkk2 to activate LRP6. (A) TOP-FLASH luciferase reporter assay in 293T cells. DNA doses used (ng/well): LRP6, 10; all dkk constructs, 3; mkrm2, 3. (B) TOP-FLASH luciferase reporter assays in whole Xenopus embryos harvested at stage 10. mRNA doses injected (pg/blastomere): LRP6, 500; mkrm2, 250; Xdkk1, 250; mdkk2, 250.
Fig. 3.
Dkk2 and mKrm2 interact in Xenopus embryos. All blastomeres of 4-cell stage embryos were injected with the indicated RNAs and scored for phenotypes at stage 27. Representative embryos are shown. (A) Control embryos. (B) mdkk2 (50 pg/blastomere) injected embryos. (C) mkrm2 (50 pg/blastomere) injected embryos. (D) mdkk2/mkrm2 (50/50 pg/blastomere) injected embryos.
Fig. 4.
Both LRP6 and Krm binding sites locate to the Cys2 domain of Dkk1. (A) Schematic representation of the structure of Dkk1 and deletion constructs. SP, signal peptide; L1, L2, linker region 1, 2; Cys1, Cys2, cysteine-rich domain 1, 2. (B) 293T cells were transfected with LRP6 or mkrm2 as indicated, incubated with recombinant XDkk1-AP, AP-XDkk1-Cys1, or AP-XDkk1-Cys2, and bound AP activity was measured colorimetrically at 405 nm. The same AP activity was loaded for all AP-fusion proteins. (C) TOP-FLASH luciferase reporter assay showing the activity of the Dkk1 deletions on Wnt/Fz signaling with or without mKrm2. RLU, relative light units. DNA doses used (ng/well): mwnt1, 8; mfz8, 1; dkk1, 1; flag-Xdkk1-Cys1 and flag-Xdkk1-Cys2, 3; mkrm2, 1. (D) A model showing the functional interactions of Dkk2, LRP6 and Krm. In the absence of Krm, Dkk2 binds to LRP6 and activates the Wnt/lRP6 signaling (a), while in the presence of Krm, Dkk2, LRP6 and Krm form a ternary complex and inhibit the Wnt/lRP6 signaling (b).
