Although it is widely accepted that the canonical Wnt/β-catenin signaling axis is initiated by Wnt-mediated Fz and LRP5/6 heterodimerization 
, key molecular details involved in canonical Wnt signaling propagation remain elusive, such as the details of how Dvl activates the dismantling of the destruction complex. Bilic et al. demonstrated that Dvl plays an integral role in clustering and phosphorylation of LRP6 and formation of the LRP6-signalosome. By utilizing real-time confocal microscopy they demonstrated that LRP6 aggregates in signaling foci within 15 min of Wnt stimulation, suggesting that oligomerization of LRP6 is a prerequisite for Wnt signaling propagation. The necessity for LRP6 clustering to promote Wnt signaling propagation provoked us to design an inducible Wnt system by marrying the LRP5/6 signaling domain with CID technology. In a previous study, Liu et al demonstrated that wild-type LRP6 also forms spontaneous, Wnt-independent dimers; however, these dimerized LRP6 subunits were inactive. Moreover, forced dimerization of the cytoplasmic LRP6 domain inhibited its activity 
, although, we also observed constitutive dimerization (or oligomerization) of LRP5c (data not shown). In contrast to that study, our results show that forced dimerization of LRP5c either at the plasma membrane or within the cytosol is sufficient to induce β-catenin stabilization (). Idiosyncrasies of the bulkier DNA gyrase B (30 kD)/coumermycin CID system used by Liu et al, relative to FKBP12 (12 kD)-based dimerization, could account for these disparate results. Consistent with our results, a separate study showed that dimerization of a membrane-associated LRP6 intracellular domain can stimulate signaling 
. In addition, it has been reported that Frizzled receptors can also dimerize following Wnt binding, contributing to Wnt/β-catenin induction 
. Taken together, rather than inhibiting LRP5/6 signaling, these data support the conclusion that oligomerization of LRP5 and other Wnt-associated proteins can potentiate Wnt signaling.
Our study also reveals novel plasma membrane-distal aspects of LRP5 function. It has been generally accepted that plasma membrane localization of LRP5 precedes phosphorylation by membrane-associated CK1α, which is required for Axin sequestration and β-catenin stabilization 
. However, using non-localized signaling domains and membrane-permeable ligands, we unexpectedly discovered that dimerization of cytosolic LRP5 is sufficient for localization to Dvl-containing punctate structures, Axin-binding and canonical Wnt signaling. This indicates a link between LRP5 oligomerization and recruitment to intra-cytoplasmic compartments that contain the Dvl/Axin complex. It is also tempting to speculate that a similar mechanism can explain the requirement for intracellular trafficking to an endosomal protein complex by the Wingless (Wnt homologue) signaling complex in Drosophila melanogaster
that includes Arrow (LRP5 homologue) and Dishevelled 
Amino-terminal deletion and site-directed mutagenesis analysis of the cytoplasmic signaling domain of LRP5 revealed that a previously unidentified region of LRP5 (1516MFYSSNIPATVRPYRPY1531), devoid of consensus CK1 phosphorylation sites, is essential for β-catenin induction, and intra-cytoplasmic localization. Nonetheless, we consistently observed that CK1 activity is critical for LRP5 function. Since dimerization of iLRP5 within intra-cytoplasmic compartments appears functionally intact, it is likely that cytoplasmic CK1 isoforms, associated with Dvl/Axin complexes, are able to induce LRP5 phosphorylation and activation, comparable to membrane-bound CK1. However, it remains to be tested what additional factor(s) target the newly identified signaling domain required for iLRP5 signaling and whether additional factor(s) are also critical for endogenous LRP5 function.
In addition to helping elucidate Wnt signaling mechanisms, our initial in vitro
data suggested that iLRP5 could also be utilized for induction of Wnt signaling in vivo
, justifying development of the Ubi-Cat mouse model with widespread inducible β-catenin signaling. Ubi-Cat mice should permit analysis of Wnt signaling induction in multiple tissues, consistent with qRT-PCR-based detection of iLRP5 in multiple tissues. The role of Wnt signaling and the pro-neoplastic consequence of Wnt deregulation in mammary and prostate tissues have been well documented 
. Therefore, we initially selected these two tissue types to examine more carefully the utility of targeted iLRP5 signaling.
In order to demonstrate the cell autonomous activity of iLRP5, we selected prostate B/SCs as our initial working model. Activation of Wnt signaling in prostate progenitors results in the induction of known canonical Wnt target genes, expansion of p63+
cell population, enlargement of prostaspheres and development of prostate hyperplasia 
. Similarly, activation of iLRP5 in LSCs promoted upregulation of known canonical Wnt target genes, such as cyclin D1, c-myc and survivin. Prostasphere assays indicated that iLRP5 activation results in the enlargement of prostaspheres and expansion of the p63+
cell population. Moreover, prostate reconstitution assays using Ubi-Cat LSCs revealed that activation of iLRP5 results in expansion of p63+
cells and increased cyclin D1 staining. We also observed increased hyperplasia in iLRP5-induced grafts. In vivo
activation of iLRP5 by administering AP20187 to male Ubi-Cat mice resulted in the progression of prostate tumor development from hyperplasia to adenocarcinoma. Altogether, our in vitro
and in vivo
data involving manipulation of Ubi-Cat LSCs and intact prostate, mirrors that of previous reports on the effect of Wnt signaling in prostate progenitors and tumorigenesis 
The prostate is a highly organized branched organ that relies on coordinated communication between multiple signaling pathways, such as Wnt, Notch, fibroblast growth factor (FGF) and transforming growth factor β (TGF-β), for proper development. Multiple studies have described the presence of activated canonical Wnt signaling and its importance in prostate architecture organization and cell specification during prostate development and also androgen-dependent regeneration 
. UGSM-derived tissue provides a suitable niche for glandular development due to its strong inductive characteristic 
, and hence UGSM cells are regularly used for prostate reconstitution assays. Hence, we also tested the effect of iLRP5-mediated Wnt pathway activation within adult prostate stroma and its influence on wildtype LSC cells during prostate reconstitution. Surprisingly, induction of the iLRP5 switch in Ubi-Cat stroma enhanced prostate tissue regeneration when compared to uninduced stromal tissue. This observation emphasizes the importance of Wnt signaling regulation during prostate development. It is likely that iLRP5/Wnt signaling activation induces development-associated signaling pathways that are know to act in a paracrine fashion, such as Notch, FGF and TGF-β, in order to enhance prostate regeneration. Future studies will more closely examine the possible existence of such cross-regulations upon iLRP5 activation.
In this manuscript, we have provided two examples of Ubi-Cat-derived tissues that can be manipulated using a single dimerizing ligand. In addition to the effects of iLRP5 signaling in prostate epithelial and stromal tissue, we also investigated the outcome of iLRP5 induction within mammary cells. Mammary cell transplants into the mammary fat pad also indicated the cell autonomous effects of iLRP5 in mammary epithelial cells during development and homeostasis. In this case, induction of iLRP5 resulted in the formation mammary tumors, demonstrating that induction of unaltered β-catenin is sufficient to drive mammary tumor progression. This result further demonstrates the potency and efficacy of the iLRP5 switch as an inducer of the Wnt signaling pathway. Future studies will investigate the role of canonical Wnt signaling in other tissues in both reconstitution transplant models and in intact Ubi-Cat mice. Additional animal models based on tissue-specific regulatory elements or conditional, tissue-specific iLRP5 expression should further broaden the utility of this technology.
Overall, this study has demonstrated the generation of a novel inducible Wnt signaling switch by manipulating the Wnt pathway co-receptor LRP5. We observed that membrane recruitment of iLRP5 via CID leads to activation of the Wnt signaling pathway. Oligomerization of LRP5 is also demonstrated to be a potent inducer of Wnt pathway induction, independent of membrane recruitment. This observation led us to generate an inducible Wnt switch (iLRP5) using only the cytoplasmic domain of LRP5. The activation of iLRP5 is achieved via CID technology, in which iLRP5-mediated Wnt signaling induction is dependent on exposure to synthetic homodimerizer drugs, like AP20187.
Next, we investigated the molecular and biochemical events that follow upon iLRP5 activation, leading to Wnt signaling activation. We were able to demonstrate that iLRP5 interacts with Dvl; however, this interaction is not critical for the downstream activity of iLRP5. Instead, the iLRP5 switch has the ability to induce Wnt signaling by sequestering the Wnt inhibitory molecule, Axin. The iLRP5/Axin interaction appears sufficient to activate the Wnt pathway, even in the absences of full-length Dvl, suggesting that iLRP5 does not require Dvl activity or membrane localization. We also showed that iLRP5 phosphorylation via CK1 is required for proper iLRP5 activity. Finally, we generated the Ubi-Cat transgenic mouse model. In two distinct organ systems, Ubi-Cat mice demonstrated their utility as a practical tool for investigating the effect of Wnt signaling induction in vitro and in vivo.