Due to early overexpression studies in
embryos, the Wnts were grouped into various classes based on their canonical Wnt signaling ability without regard for the cellular context in which they were overexpressed. Receptor expression, however, clearly plays a role in determining signaling output; when coexpressed with the appropriate Fz receptor, the prototypical “noncanonical” Wnt,
Wnt5a, can signal in a canonical fashion to induce the formation of a secondary axis [
]. Whereas some have argued that the lack of functional interaction with LRP distinguishes Wnt5a from so-called canonical Wnts in mammalian cells [
], previous studies and our present work confirm that Wnt signaling output is not intrinsically related to the Wnt protein itself but rather due to a combination of factors including receptor availability [
Several controversies exist in the literature today regarding the mechanisms by which Wnt proteins signal. Much of the conflicting data, however, can be attributed to the variety of cellular and organismal systems studied coupled with the previous lack of soluble, active Wnt proteins. In the case of Wnt5a, for example, one report has shown that overexpression of Wnt5a inhibits canonical signaling by promoting the degradation of β-catenin protein [
]. By contrast, we found that, in accordance with other groups, Wnt5a protein treatment has no affect on β-catenin protein levels but rather inhibits canonical Wnt signaling at the level of TCF transcription [
]. Although our data are in agreement with Wnt5a overexpression studies by Ishitani et al. [
] with respect to the inhibition occurring downstream of β-catenin protein stabilization, we found that Wnt5a does not stimulate Ca
flux and that Wnt5a-mediated inhibition is not sensitive to pertussis toxin treatment. These data effectively eliminate G protein–mediated activation of calcium signaling as the primary mechanism of Wnt5a-mediated inhibition.
Fz receptor signaling capabilities have also often been debated. Previous studies pertaining to Fz4′s role in the disease familial exudative vitreoretinopathy have suggested that the Fz4 receptor does not mediate Wnt/β-catenin signaling but rather elicits intracellular calcium flux that subsequently activates downstream calcium effector molecules [
]. By contrast, we show here that in multiple cell lines, Fz4 allows Wnt5a to specifically
TCF/Lef transcription in the presence of LRP5 and that mFz4 expression does not enhance calcium flux (
C). The true signaling capabilities of the Fz4 receptor may have been previously overlooked due to the use of an inappropriate Wnt ligand in the analysis, Wnt1 as opposed to Wnt5a. Additionally, as calcium signaling capabilities were attributed to Fz4 in the absence of exogenous ligand stimulation, it remains to be seen whether Wnt ligands are required for Fz-mediated Ca
We observe that when mFz4 is overexpressed alone, Wnt5a protein treatment is sufficient to induce β-catenin protein stabilization but not STF reporter activation. It is possible that Wnt5a does not activate a transcriptional response when mFz4 is expressed alone in 293 cells due to the endogenous expression of mRor2. In addition, coreceptor expression and heterodimerization following ligand stimulation have previously been shown to enhance ligand binding and signal transduction in other systems [
]. As Wnt5a-induced β-catenin stabilization in cells expressing mFz4 is less robust than Wnt3a-induced stabilization, overexpression of both mFz4 and LRP5 may be necessary for full potentiation of the canonical signal resulting in transcriptional activation. Furthermore, previous reports have suggested that the accumulation of β-catenin and Wnt signal transduction are separable events by showing that β-catenin protein levels alone do not dictate signal output but rather the phosphorylation state of β-catenin [
]. Coexpression of LRP5 may be necessary for the dephosphorylation, and hence full activation, of β-catenin resulting in optimal signal transduction.
The disparities between previous reports and our current observations may be due to the fact that in this study, the effects of Wnt5a could be monitored immediately following Wnt5a protein addition as opposed to several hours or cell divisions following cellular expression, thereby allowing us to separate early and late effects of Wnt5a treatment. Activation of downstream transcriptional targets of Wnt5a, such as
may contribute to the overall Wnt5a-mediated inhibition of Wnt/β-catenin signaling subsequent to the initial signaling events that we observed [
]. Additionally, in the proposed Wnt/Ca
signaling model, Wnt5a-stimulated Ca
flux leading to NLK activation is required for transcriptional inhibition [
]. However, Wnt1 has recently been shown to inhibit β-catenin–TCF–mediated transcription via activation of NLK, although Wnt1 has never been shown to induce Ca
]. Thus, Wnt5a may exert its inhibitory effects through the activation of NLK in a similar Ca
-independent manner. Further experimentation into the mechanism by which Wnt5a inhibits canonical signaling is therefore necessary and will be addressed in subsequent reports.
In this study, we show that one Wnt ligand can function in two discreet pathways based on receptor availability (
). Data in the literature suggest that this may also apply to other Wnts. For example, Wnt1 is known to activate canonical Wnt signaling through β-catenin and TCF and act as an oncogene [
]. However, using overexpression in various cell lines, Smit et al. presented evidence that Wnt1 inhibits TCF activity [
]. Another example is Wnt11, which has been implicated in the noncanonical convergence-extension pathway in zebrafish [
]. Recent work, by contrast, demonstrates that Wnt11 is the long-sought ligand that activates the β-catenin pathway in the early
embryo, showing that one Wnt can activate two different pathways, possibly also by activating different receptors [
Model. Wnt5a Can Activate or Inhibit Canonical Wnt Signaling Depending on Receptor Context
Although the ability of one Wnt ligand to function in two distinct pathways based on receptor context is novel for the Wnt field, we note that in an entirely different system, opposing effects brought about by a single ligand have also been explained by the use of different receptor classes. This example is formed by the Netrins, ligands that can either attract or repel axons depending on whether they interact with the DCC (deleted in colorectal carcinoma) or UNC5 (UNCoordinated family member 5) families of receptors [
]. Thus, ligands engaging multiple receptors to effect different signaling outcomes is not unprecedented in nature.
With as many as 19 mammalian homologs known, the question of whether all Wnt family members have evolved to signal in the same fashion is an important one. In this report, we address controversies in the literature regarding how one Wnt family member, Wnt5a, functions. Through quantitative kinetic analyses, we provide evidence for the first time that Wnt5a protein can directly inhibit canonical Wnt/β-catenin signaling and that the single-pass transmembrane receptor Ror2 is required to mediate this activity. Although Wnt5a can inhibit canonical Wnt signaling when Ror2 is expressed at detectable levels, Fz 4 and LRP5 coreceptor expression allows Wnt5a to signal in a canonical fashion. This ability of Wnt5a to toggle between two considerably diverse forms of signaling is particularly intriguing when one considers that Wnt5a has been classified as both a tumor suppressor and an oncogene in various cell types [
]. While previously somewhat paradoxical, it is now clear that in the former role, Wnt5a expression may inhibit uncontrolled Wnt/β-catenin signaling in the presence of Ror2, whereas in the latter, Wnt5a could promote canonical Wnt signaling when Fz4 and LRP5 are expressed. Future study into specific receptor-ligand interactions will thus contribute to understanding the complexities of Wnt signaling.