In our study, we have compared the functional activities of two members of the Dickkopf family, Dkk1 and Dkk2. Both Dkks share the ability to inhibit Wnt8 and cooperate with a dominant-negative BMP4 receptor in inducing head structures. In contrast, only Dkk2 synergizes with the Wnt coreceptor LRP6 to activate β-catenin signaling. We show that these distinct activities are due to differences in the N-terminal domains. Analysis of chimeric Dkks shows that the N1 domain inhibits the ability of C1 and C2 to synergize with LRP6 (Fig. and ), whereas the N2 domain does not. Thus, the different properties of N1 and N2 underlie the opposing effects of Dkk1 and Dkk2 on LRP6-dependent axis induction.
We find that Dkk2 is an inhibitor of Wnt8, yet it synergizes with LRP6 in the same signal transduction pathway. This paradoxical finding can be explained by postulating that Dkk2 interferes with the binding of Wnt8 to LRP6, but itself functions as a weak activator of LRP6-mediated signaling (Fig. ). Consistent with this idea, we show that both C-terminal domains of Dkks form a complex with LRP6 (Fig. ). Our conclusions are supported by a recent study in which the C-terminal region of Dkk2 inhibited Wnt3a signaling in cultured cells, but activated a Wnt-responsive promoter when coexpressed with LRP6 (25
). Therefore, depending on the cellular context (i.e., the presence of Wnt ligands or levels of LRP5 or LRP6), Dkk2 can act as an activator or an inhibitor of the β-catenin pathway.
We also observe that C1 can activate LRP-dependent signaling. Unlike Dkk2, there is no evidence that Dkk1 could function as an activator of Wnt signaling, yet our data suggest that it might be the case, especially if there is a mechanism for the release of the C-terminal domain. Although we do not see major proteolytic fragments of Dkk1 in injected embryos (Fig. ), limited evidence for proteolytic processing of Dkks has been reported (24
). Moreover, there is a conserved furin site in the N-terminal regions of Dkks (24
) (Fig. ), suggesting that Dkks may be regulated by proteolysis. A similar situation has been documented for the bipartite zinc finger transcription factor Gli2, which can be activated by the removal of the N-terminal repressor domain (39
). More work is necessary to determine whether Dkk1 activity is indeed regulated in the embryo.
A two-domain structure for the family of Dkk proteins was originally proposed based on the conservation of their amino- and carboxy-terminal cysteine-rich regions, which are separated by variable-length spacer sequences (14
). The functional significance of this two-domain structure was previously unknown. Our experiments demonstrate that individual Dkk domains operate as discrete functional units. We find that the C-terminal regions C1 and C2 are both necessary and sufficient for the ability of Dkks to inhibit Wnt8 signaling and to synergize with LRP6 in axis induction. In contrast, the N-terminal regions have diverged to confer opposing functions to these two highly similar proteins.
Our data suggest that the N1 domain of Dkk1 acts to suppress the ability of the C1 domain to activate LRP6 signaling. The exact mechanism by which this occurs is unclear. One possibility is that the N1 region interacts with the C1 domain, thereby inhibiting its ability to synergize with LRP6. However, we have not been able to detect an association between the N1 and C1 proteins (data not shown). Another possibility is that the N1 region, tethered to LRP6 by the C-terminal Dkk domain, prevents LRP6 from complexing with Frizzled receptors. This idea is supported by the model of Semenov et al. (41
), who have shown that Dkk1 prevents LRP6 from forming complexes with Frizzled and Wnt1. Lack of competition between N1 and Dkk2 (Fig. ) also suggests that the N-terminal domain functions only in the context of a complete Dkk molecule or interacts with non-LRP targets. Similar to N1, the N2 domain may interact with other extracellular modulators of Wnt signaling, such as Frizzled receptors or heparan sulfate proteoglycans. This is likely, since the effects of Dkk2 and C2 on embryonic development appear to be different. Further experiments are required to elucidate the role of other regulators of Dkk/LRP signaling.
Dkk1 and Dkk2 have different expression patterns during development. Xenopus
Dkk1 is expressed in the organizer (14
), a region responsible for specification of head and heart mesoderm. In contrast, the expression of Dkk2 in head mesenchyme, lens, and somites is not detected until organogenesis (48
). It has been proposed that the head- and heart-inducing activities of Dkk1 are due to its activity as a Wnt antagonist (14
). Because the Wnt-inhibitory activity of our various Dkk constructs correlates with their ability to induce head structures (Fig. ), our findings lend support to this hypothesis.
In contrast to the effect of Dkk1, embryos injected with Dkk2 RNA do not develop enlarged head structures (14
; data not shown). This observation indicates that Dkk2 is unlikely to function solely as a Wnt antagonist in vivo. We have shown that Dkk2 may function as an activator or an inhibitor of Wnt signaling, depending on the cell context. Thus, Dkk2 may have opposite activities in different embryonic tissues. To provide more insight into the molecular mechanisms of Dkk2 action during development, a detailed knowledge of the expression levels of endogenous LRP and other proteins interacting with Dkk2 will be required.
Our experiments have investigated regulation of Wnt signaling by two closely related Dkk family members. We have shown that although both proteins inhibit Wnt signaling, they possess an intrinsic ability to activate the Wnt coreceptor LRP6, which is suppressed in the case of Dkk1 by its N-terminal domain. The C-terminal domains of both Dkks associate with LRP6 and appear to modulate LRP6 signaling. Our studies suggest a mechanism for the regulation of Wnt signaling by Dkk1 and Dkk2 and further contribute to our understanding of this signaling pathway.