Elsevier

Gene

Volume 302, Issues 1–2, 2 January 2003, Pages 179-183
Gene

Kremen2 modulates Dickkopf2 activity during Wnt/lRP6 signaling

https://doi.org/10.1016/S0378-1119(02)01106-XGet rights and content

Abstract

Dickkopf1 (Dkk1) is a secreted antagonist of the Wnt/β-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.

Introduction

Wnt signaling plays important roles in many developmental processes and is regulated at multiple levels (Wodarz and Nusse, 1998). Extracellularly, Wnt activity is regulated by several kinds of secreted antagonists, including Dickkopf1 (Dkk1, Glinka et al., 1998), Cerberus (Piccolo et al., 1999), Wnt-interacting factor (WIF, Hsieh et al., 1999) and secreted Frizzled-related proteins (sFRPs, Leyns et al., 1997, Rattner et al., 1997, Wang et al., 1997), of which all except Dkk1 function by direct binding to and inactivating Wnt ligands. Dkk1 acts by binding to and inhibiting the Wnt co-receptor LRP5/6 (Bafico et al., 2001, Mao et al., 2001, Semenov et al., 2001). In an expression screen, we have identified another Dkk1-binding membrane protein, Kremen2 (Krm2) (Mao et al., 2002). We have shown that Dkk1, LRP6 and Krm2 form a ternary complex which triggers internalization and thus clearance of LRP6 from the cell surface. Functionally, Dkk1 and Krm work synergistically in Wnt inhibition. Dkk1 is a member of a multigene family with at least 4 members in human (Krupnik et al., 1999, Monaghan et al., 1999). Among the Dkks tested, Dkk2 can bind to LRP6 and Krm1 and -2 with high affinity similar to Dkk1, while Dkk3 can bind neither LRP6 nor Krm. Despite their sequence homology and their similar binding characteristics, Dkk1 and Dkk2 behave quite different in functional assays. While Dkk1 acts as Wnt inhibitor in all conditions tested, the effect of Dkk2 on Wnt signaling depends on the cellular context. In Xenopus embryos, Dkk2 can weakly activate Wnt/β-catenin signaling when overexpressed and synergistically activates the pathway when co-expressed with certain members of the Frizzled (Fz) family of Wnt receptors. However, in the human 293 fibroblasts cell line, Dkk2 functions as a Wnt inhibitor (Wu et al., 2000). Furthermore, in the presence of overexpressed LRP6, Dkk2C2, which contains only the second cysteine-rich domain (Cys2) of Dkk2, can activate rather than inhibit Wnt signaling in 293 cells (Li et al., 2002).

These complex results suggest that Dkk2 can function either as agonist or antagonist of LRP6, and that its activity might be modulated by an unknown co-factor(s). In this study we show that Krm2 acts as such a co-factor that modulates Dkk2 activity during Wnt/lRP6 signaling: In the presence of Krm2, Dkk2 is converted from a LRP6 agonist to a LRP6 antagonist. Finally we localize both LRP6 and Krm binding sites of Dkk1 to its second cysteine-rich domain.

Section snippets

Constructs

To construct pCS-hdkk4, the hdkk4 open reading frame was amplified from an EST clone (IMAGE Consortium clone 1016173) and inserted into the pCS2+vector (Rupp et al., 1994). To generate flag-Xdkk1-Cys1 (amino acids 24–177), flag-Xdkk1-Cys2 (amino acids 175–260) and flag-mdkk2-Cys2 (amino acids 141–259), the corresponding fragments without signal peptide sequence were amplified by PCR and cloned into pCS-SP-flag, which contains the coding sequence for the mKrm2 signal peptide (Mao et al., 2002)

Dkk-1, -2, -4 but not Dkk3 co-operate with Kremen in Wnt inhibition

We had previously shown that Dkk2 can bind to Krm2, but the functional consequence for Dkk2 activity had not been investigated (Mao et al., 2002). We therefore tested the ability of Dkk1-4 to co-operate with mKrm2 in Wnt inhibition using the Wnt responsive TOP-FLASH luciferase reporter (Korinek et al., 1997). At the limiting doses used, Dkk1, -2 and -4 weakly inhibit Wnt signaling in 293T cells. In contrast, neither Dkk3 nor mKrm2 alone inhibit Wnt signaling (Fig. 1, lanes 2–7). When

Discussion

In this study we confirm that Dkk2 can act either as agonist or antagonist of Wnt/lrp6 signaling and we provide evidence that these two functional activities are regulated by the Dkk receptor Krm2 (Fig. 4D). Consistent with previous observations (Wu et al., 2000, Li et al., 2002), we found that in 293T cells, Dkk2 activates the Wnt signaling pathway when LRP6 is overexpressed. Although LRP6 is expressed endogenously in 293T cells (not shown), Dkk2 alone cannot activate the Wnt/β-catenin

Acknowledgements

We thank Dana Hoppe for technical help, and Dr. W. Wu for the hdkk4 construct and critical reading of the manuscript. We thank Drs. H. Clevers, X. He and J. Nathans for reagents.

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