Both Axin and its homolog Axin2/Conductin/Axil are believed to act as scaffold proteins, which bind several components of the canonical Wnt signal transduction pathway and promote the phosphorylation of β-catenin by GSK-3 and its consequent degradation. Thus, both Axin and Axin2 appear to serve as negative regulators of the signaling pathway, and consistent with this role, both have been shown to act as tumor suppressors in humans. Axin, which is expressed ubiquitously, is believed to act as a constitutive modulator of the Wnt pathway and is thus a key component of the mechanism that prevents spontaneous signal transduction in the absence of a Wnt signal. Here, we show that Axin2 plays a complementary role, being transcriptionally induced following the reception of a Wnt/β-catenin signal. This property of Axin2 may create a negative feedback loop to silence the signaling pathway following transduction of the Wnt signal (Fig. ).
FIG. 8. A model for the role of Axin2 in Wnt signal transduction. Upon transduction of a Wnt signal, transcription of the Axin2 gene is induced via the β-catenin/Tcf pathway. Our point mutation analysis, as well as data from cotransfection of DN-Tcf, (more ...)
The evidence that first led us to test this hypothesis was the apparent overlap between several sites of Axin2
gene expression during embryogenesis and organogenesis. For example, the expression of Axin2
in the primitive streak and the dorsal neural tube (Fig. ) was reminiscent of the expression patterns of Wnt-1
, and Wnt-3a
). Another notable site of Axin2
expression was the intervillus epithelium of the fetal gut, a region that gives rise to the crypts in which the gut stem cells arise and proliferate. The proliferation of these stem cells is known to be regulated by the Wnt/β-catenin/Tcf4 pathway, and mutations in APC
), as well as Axin2
), predispose the cells to oncogenic transformation leading to colon cancer. Therefore, the strong expression of Axin2 in these cells might be explained if the Axin2
gene were itself a target of this signaling pathway. It is also important to point out that the overlap between Axin2
expression is only partial: there are many sites where members of the Wnt pathway (including those believed to activate the canonical Wnt pathway) are expressed where Axin2
is apparently not expressed. Conversely, it is not clear that every site of Axin2
expression corresponds to a site where the Wnt/β-catenin/Tcf pathway is active. Indeed, in the adult mouse as well as the adult human, Axin2
mRNA is found in most if not all tissues (T. Zhang and F. Costantini, unpublished data), and it seems unlikely that all of these adult expression sites are maintained by Wnt signaling. Therefore, the Wnt pathway appears to be only one of perhaps several mechanisms by which transcription of Axin2
can be activated and/or maintained.
In the mouse mammary gland cell line C57MG, we found that the level of endogenous Axin2
mRNA was strongly induced following expression of the chimeric Wnt1/5. Similarly, in rat fetal gut endoderm cocultured with cells expressing Wnt-1, the endogenous rat Axin2
mRNA was strongly induced. These experiments clearly showed that the expression of Axin2
can be induced by Wnts both in cell lines and in fetal tissue. Further support for this conclusion comes from recent findings of Lustig et al. (25a
), who have observed that expression of Conductin (Axin2) is highly elevated in several tumors and tumor cell lines that are induced by β-catenin/Wnt signaling.
While these findings did not distinguish between direct and indirect induction, several observations strongly argue for a direct effect. First, the time required for induction of Axin2 expression following treatment of 293T cells with LiCl (Fig. ) is within the range observed for several other Wnt/β-catenin target genes whose promoters contain Tcf/LEF sites (2 to 8 h [J. Willert and R. Nusse, personal communication]). Second, when fused to a luciferase reporter, a 5.6-kb mouse Axin2 DNA fragment including all eight Tcf/LEF sites was sufficient to mediate Wnt- or β-catenin-inducible expression of luciferase in 293T cells. This indicates that the induction of Axin2 is mediated at the transcriptional level. Furthermore, this induction could be largely inhibited by cotransfection of a dominant-negative mutant form of Tcf-4E, showing that the transcriptional activation is mediated by Tcf/LEF factors. Third, introduction of a single point mutation into each of the eight Tcf/LEF sites, in various combinations, reduced the degree of induction by β-catenin, confirming that it is mediated, at least in part, through a subset of the Tcf/LEF consensus sites. While the failure of these mutations to eliminate entirely the induction by β-catenin suggests that part of the induction might be indirect, it is also possible that the single point mutations were insufficient to eliminate the function of the Tcf/LEF motifs. For this reason, we examined the effects of a triple mutation in site T2 in the Axin2 promoter using reporter and protein binding assays. While a 398-bp promoter fragment including site T2 mediated 2.3-fold induction of the reporter, mutation of site T2 eliminated most of the induction. Fourth, in an EMSA, the ability of an oligonucleotide including site T2 to bind proteins from 293T cell extracts was largely eliminated by the same mutation. Together, these results strongly suggest that at least some of the consensus Tcf/LEF sites in the promoter and first intron of the Axin2 gene are responsible for direct transcriptional regulation by Tcf/LEF factors.
The ability of the Axin2 promoter and first intron to direct tissue-specific expression of a d2EGFP reporter was tested in transgenic mice, and the transgene was found to largely recapitulate the expression of endogenous Axin2 during embryogenesis and organogenesis. This suggests that the Tcf/LEF sites and the surrounding conserved sequences that we have identified play a role in the in vivo expression pattern of Axin2, although the role of specific sequences for tissue-specific expression remains to be tested. The transgenic mice that we have produced will also be useful for studies of the regulation of Axin2, for example, crossing the mice with those with mutations in various Wnt pathway components.
The most important implication of our studies is that Axin2 appears to participate in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal. Such negative feedback loops are critical for the precise control of signaling during development and have been identified in many of the well-characterized signaling pathways (7
). There are several other examples of mechanisms that could provide additional negative feedback loops in the Wnt pathway. First, it has been shown that Tcf1
is a target gene for Tcf4 in epithelial cells and that the most abundant Tcf1 isoforms lack a β-catenin interaction domain, so that Tcf1 might serve as a feedback repressor of β-catenin/Tcf4 target genes (33
). Second, β-TrCP, which targets the ubiquitination and degradation of β-catenin, is itself induced (through a posttranscriptional mechanism) by β-catenin/Tcf signaling, causing accelerated degradation of β- catenin (39
). Third, the protein naked cuticle, which is induced by wingless in Drosophila melanogaster
, acts directly through Dsh to limit wingless activity (34
). Therefore, the induction of Axin2
appears to be one of several mechanisms for feedback regulation of β-catenin upon activation of the Wnt pathway.