Wnt signaling is implicated in many developmental decisions, including stem cell control, as well as in cancer. There are relatively few target genes known of the Wnt pathway.
We have identified target genes of Wnt signaling using microarray technology and human embryonic carcinoma cells stimulated with active Wnt protein. The ~50 genes upregulated early after Wnt addition include the previously known Wnt targets Cyclin D1, MYC, ID2 and βTRCP. The newly identified targets, which include MSX1, MSX2, Nucleophosmin, Follistatin, TLE/Groucho, Ubc4/5E2, CBP/P300, Frizzled and REST/NRSF, have important implications for understanding the roles of Wnts in development and cancer. The protein synthesis inhibitor cycloheximide blocks induction by Wnt, consistent with a requirement for newly synthesized β-catenin protein prior to target gene activation. The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin. Several of the target genes have a cooperative response to a combination of Wnt and BMP.
Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.
There is currently great interest in using extracellular signaling proteins to influence the gene expression program and differentiation of embryonic cells, in particular embryonic stem cells. Of the factors that contribute to specifying cell fate during development, Wnt proteins are among the most attractive candidates to use in such in vitro experiments. Wnt proteins control numerous aspects of development, ranging from stem cell control to differentiation and cell polarity [1,2]. It has been problematic however to test directly whether Wnt proteins can be used as reagents in cell culture, because working with soluble Wnt proteins is difficult and few cell lines are known to respond to Wnt proteins. Wnt signal transduction proceeds through a complex series of protein interactions, initiated by binding of the Wnt protein to cell surface receptors (Frizzled and LRP [3-5]) which generates a signal to downstream components. A key event in signaling is the regulation of the GSK3 protein kinase and its substrate β-catenin. In the absence of a Wnt signal, GSK3 phosphorylates β-catenin, which then becomes targeted for degradation . The binding of Wnt to its receptors initiates a cascade of events that inhibit GSK3 and ultimately prevent degradation of β-catenin. Together with the DNA binding protein TCF, β-catenin activates expression of Wnt target genes. In this work, we have identified Wnt target genes using micro-array technology.
Identifying Wnt targets on microarrays
We tested human teratocarcinoma cells (NCCIT) cells for a Wnt response. Despite the cancerous origin of these cells, they share many properties with embryonic stem cells . These cells express several members of the Frizzled family (including FZD7), one of the receptors for Wnt (data not shown; Figure 2A). To stimulate the NCCIT cells with Wnt, we used tissue culture medium containing active Wnt-3A protein produced by mouse L cells (Wnt-3A CM ), in comparison to control conditioned medium (CCM). In initial experiments, we found that Wnt-3A protein elevates the levels of β-catenin 5–10 fold (Figure 4); and that a transiently transfected TCF reporter construct is activated 3–4 fold (not shown).
Figure 1. Microarray cluster analysis demonstrating increased expression of Wnt-3A responsive genes, including MSX1, MSX2, ID2, Versican, NPM, Frizzled-7, TLE/Groucho, Cyclin D1, and MYC. NCCIT cells were exposed to control conditioned medium (CCM) and Wnt-3A CM  for 4 hours. Shown here are 5 separate experiments. Note also that MSX 2 is included on this particular array in duplicate which provides an internal check on the precision of the measurements. Additional array experiments led to the identification of Follistatin and REST/NRSF as elevated genes (which were confirmed in Figure 2A). Brightest red corresponds to ratio of = 2:1 and dimmer red corresponds to ratio of 2:1. The full data set will be available at the Wnt homepage http://www.stanford.edu/~rnusse/wntwindow.html webcite and the Stanford Microarray database http://genome-www5.stanford.edu/MicroArray/SMD/ webcite
Figure 2. A) Confirmation of microarray data using Northern analysis. Northern blot analysis using MSX2, ID2, REST/NRSF, FZD7 (Frizzled7) and Follistatin cDNA probes. NCCIT cells were treated with CCM (-) or Wnt-3A CM (+) for the designated number of hours. 1 ug mRNA was loaded into each lane. GAPDH is shown as a sample loading control. B) MSX1 induction by Wnt-3A CM is dependent on the presence of soluble Wnt-3A protein. C = CCM. Wnt = Wnt-3A CM. Wnt-depleted = Wnt-3A CM. Cells were incubated with the various media for 4 hours.
Figure 3. Time course of Wnt induced gene expression and cooperation with BMP. NCCIT cells were exposed to Wnt-3A CM, BMP-4 (10 ng/ml final concentration) or the combination of Wnt-3A and BMP-4 for the specified number of hours. Shown here are MSX1, MSX2 and ID2 demonstrating that Wnt-3A induced effects are not seen until 2 hours (which corresponds to β-catenin accumulation). BMP-4 mediated induction of MSX1, MSX2 and ID2 occurs rapidly, as early as 30 minutes and stays relatively constant. Cooperative effects of Wnt-3A and BMP-4 are not evident until approximately 2 hours. The height of the bars presents the ratio induced/non-induced, quantified by phosphoimager analysis of Northern blots and normalized to GAPDH expression measured in the same experiment. Each bar represents average values obtained from 3–5 experiments for each mRNA tested.
Figure 4. A) Western blot demonstrating cycloheximide effects of Wnt-3A mediated accumulation of β-catenin. NCCIT cells were stimulated for 4 hours with CCM (C) or Wnt-3A CM (Wnt) in the presence (20 microgram/ml) or absence (0) of cycloheximide. Note that β-catenin does not accumulate in response to Wnt-3A CM when cycloheximide is present. B) Northern analysis demonstrating that MSX2 induction by Wnt-3A is abolished in the presence of cycloheximide. Cells were stimulated for 4 hours with CCM (C), BMP-4 or Wnt-3A CM (Wnt) in the presence (20 microgram/ml media) or absence of cycloheximide. As expected, BMP-4 mediated induction of MSX2 is not affected by cycloheximide which contrasts with the inhibition of Wnt-3A mediated induction of MSX2 in the presence of cycloheximide. GAPDH was used to verify equal loading. The inhibitory effect was also seen for MSX1, ID2, Follistatin, Versican and Cyclin D1 (not shown).
To identify target genes, we applied Wnt-3A CM or CCM to the NCCIT cells and isolated RNA at several time points. We performed differential hybridization to microarray slides containing approximately 23,000 spots of human cDNAs, using RNA from CCM-exposed cells as a reference. Figure 1 shows a cluster analysis of the hybridization results. We found approximately 50 genes that were upregulated between 2 and 10 fold by Wnt-3A CM whereas a few genes were repressed, i.e. expressed at lower levels in the Wnt-3A-treated cells. The latter group consisted mostly of ESTs of unknown genes and has yet to be characterized.
We verified Wnt-induced expression of several genes with Northern blots (Figure 2). In all cases tested, we confirmed the increase observed in the microarray; in some cases we found even higher differences in expression. For example, MSX2, ID2, REST/NRSF, FZD7 and Follistatin were confirmed by Northern analysis to be significantly elevated by Wnt-3A CM after two hours (Figure 2A).
None of these genes was induced by the CCM, suggesting that they are Wnt-specific. It remained possible however that the observed changes in gene expression were influenced by other differences between Wnt-3A CM and CCM. For example, Wnt-3A could have induced the expression of another secreted factor in the mouse L cells which would then be present only in the Wnt-3A CM. We addressed this possibility by specifically removing active Wnt-3A from the conditioned medium, using a combination of the ligand binding domain of the Drosophila Wnt/Wingless receptor Frizzled-2 fused to Alkaline Phosphatase [3,9] and Wnt-3A specific antibodies. This procedure specifically depleted active Wnt-3A from the medium. As expected, neither β-catenin levels nor a TCF reporter gene were elevated by the depleted CM (not shown). The depleted medium also failed to induce MSX1, MSX2, ID2 and CyclinD1 expression as assayed by Northern blots (Figure 2B shows loss of MSX1) and in a DNA microarray analysis with CCM as a reference (not shown). Similarly, adding Dickkopf protein, an inhibitor of Wnt signaling that binds to the co-receptor LRP , to the medium abrogated the transcriptional response (data not shown). We conclude that the transcriptional response to Wnt-3A CM is indeed controlled by the Wnt-3A protein rather than by other factors.
Cooperativity between Wnt-3A and BMP
Several of the Wnt target genes had been identified previously as targets of BMP signaling . We were therefore interested in comparing the effects of BMP and Wnt-3A and examining their combined effects. As has been reported before for other cells , MSX1, MSX2 and ID2 are elevated by BMP-4 in NCCIT cells (Figure 3). When Wnt-3A and BMP-4 were combined, gene expression was increased to yet higher levels. MSX1 expression was markedly elevated by the combination of BMP-4 and Wnt-3A. Similar but less dramatic effects were found for ID2 and MSX2 (Figure 3). Hence, BMP-4 and Wnt-3A appear to have an additive or perhaps synergistic effect on target gene expression. However, when the time courses of Wnt- and BMP-induced gene expression were compared, it appeared that BMP-4 acts as soon as at 30 minutes, but that Wnt takes 2 hours to have an effect (Figure 3). Hence, Wnt-induced changes in gene expression are slower than BMP, even on the same target genes and in the same cell type.
Wnt signaling is blocked by cycloheximide
The delay in transcriptional response to soluble Wnt raised the concern that the genes we identified might not be primary targets of Wnt but dependent on the prior induction of other genes. Such concerns are usually addressed by blocking protein synthesis in cells because the expression of primary target genes, or so-called immediate early genes, should not be sensitive whereas secondary targets should be blocked. This approach is not readily applicable to the Wnt response because the critical step in signal transduction is the block in β-catenin phosphorylation and degradation, leading to the accumulation of newly-synthesized β-catenin. Whether Wnt target gene activation is cycloheximide sensitive has never been tested however. When we incubated cells with cycloheximide, the accumulation of β-catenin by Wnt-3A was blocked; and Wnt targets, like MSX2, were not elevated (Figure 4). The inhibitory effect was also seen for MSX1, ID2, Follistatin, Versican and Cyclin D1 (not shown). In contrast, cycloheximide did not block induction of target genes by BMP (Figure 4), the mechanism of which does not involve protein accumulation . These findings underscore the importance of β-catenin protein elevation in the response to Wnt. This unique aspect of Wnt signaling may explain its relatively slow effects on target genes – protein synthesis is slower than protein phosphorylation or other signal transduction events.
The requirement for protein synthesis in Wnt signaling means that we cannot use cycloheximide sensitivity as a criterion to discriminate between primary and secondary targets.
The Follistatin promoter is dependent on a TCF binding site
In the Wnt target gene promoters that are functionally mapped (MSX1, MSX2, ID2, ID3, Follistatin, REST/NRSF, Versican) we invariably found TCF binding sites. Moreover, we found that a luciferase reporter gene, placed under the control of the Follistatin promoter, was activated by Wnt-3A protein added to transiently transfected NCCIT cells (Figure 5). Mutating the single TCF binding site on the Follistatin promoter eliminated the response. These data suggest that Follistatin is activated directly through the Wnt signal transduction pathway and not by another pathway. Because the kinetics of Follistatin activation by Wnt are similar to that of the other target genes (Figure 2A), we suggest that there are no "earlier" targets, i.e. all identified targets are direct.
Figure 5. Response of a reporter containing the Follistatin promoter to Wnt-3A protein in NCCIT cells . The Follistatin promoter  contains one putative TCF binding site (CTTTGAT). This promoter linked to luciferase was transfected in NCCIT cells, which were then exposed to Wnt-3A CM and CCM for 8 hours. This assay showed a significant increase in activity when cells were stimulated directly with Wnt-3A CM or co-transfected with activated β-catenin (not shown). This effect was abrogated by co-transfection of dominant-negative TCF-4 or axin (not shown). When the TCF site within the Follistatin promoter was mutated into CATCGAT, the Wnt-3A response was abolished.