Wnt and Frizzled RNA expression in human mesenchymal and embryonic (H7) stem cells

Background

Wnt signals are important for embryonic stem cells renewal, growth and differentiation. Although 19 Wnt, 10 Frizzled genes have been identified in mammals, their expression patterns in stem cells were largely unknown.

Results

We conducted RNA expression profiling for the Wnt ligands, their cellular receptors "Frizzleds" and co-receptors LRP5/6 in human embryonic stem cells (H7), human bone marrow mesenchymal cells, as well as mouse totipotent F9 teratocarcinoma embryonal cells. Except failing to express Wnt2 gene, totipotent F9 cells expressed RNA for all other 18 Wnt genes as well as all 10 members of Frizzled gene family. H7 cells expressed RNA for each of the 19 Wnt genes. In contrast, human mesenchymal cells did not display detectable RNA expression of Wnt1, Wnt8a, Wnt8b, Wnt9b, Wnt10a, and Wnt11. Analysis of Frizzled RNAs in H7 and human mesechymal cells revealed expression of 9 members of the receptor gene family, except Frizzled8. Expression of the Frizzled co-receptor LRP5 and LRP6 genes were detected in all three cell lines. Human H7 and mouse F9 cells express nearly a full complement of both Wnts and Frizzleds genes. The human mesenchymal cells, in contrast, have lost the expression of six Wnt ligands, i.e. Wnt1, 8a, 8b, 9b, 10a and 11.

Conclusion

Puripotent human H7 and mouse F9 embryonal cells express the genes for most of the Wnts and Frizzleds. In contrast, multipotent human mesenchymal cells are deficient in expression of Frizzled-8 and of 6 Wnt genes.     

Mesd encodes an LRP5/6 chaperone essential for specification of mouse embryonic polarity

Specification of embryonic polarity and pattern formation in multicellular organisms requires inductive signals from neighboring cells. One approach toward understanding these interactions is to study mutations that disrupt development. Here, we demonstrate that mesd, a gene identified in the mesoderm development (mesd) deletion interval on mouse chromosome 7, is essential for specification of embryonic polarity and mesoderm induction. MESD functions in the endoplasmic reticulum as a specific chaperone for LRP5 and LRP6, which in conjunction with Frizzled, are coreceptors for canonical WNT signal transduction. Disruption of embryonic polarity and mesoderm differentiation in mesd-deficient embryos likely results from a primary defect in WNT signaling. However, phenotypic differences between mesd-deficient and wnt3(-)(/)(-) embryos suggest that MESD may function on related members of the low-density lipoprotein receptor (LDLR) family, whose members mediate diverse cellular processes ranging from cargo transport to signaling.     

Regulated proteolytic processing of LRP6 results in release of its intracellular domain

Low-density lipoprotein receptor-related protein 6 (LRP6) is a member of low-density lipoprotein receptor (LDLR) family which cooperates with Frizzled receptors to transduce the canonical Wnt signal. As a critical component of the canonical Wnt pathway, LRP6 is essential for appropriate brain development, however, the mechanism by which LRP6 facilitates Wnt canonical signaling has not been fully elucidated. Interestingly, LRP6 which lacks its extracellular domain can constitutively activate TCF/LEF and potentiate the Wnt signal. Further, the free cytosolic tail of LRP6 interacts directly with glycogen synthase kinase (GSK3) and inhibits GSK3's activity in the Wnt canonical pathway which results in increased TCF/LEF activation. However, whether these truncated forms of LRP6 are physiologically relevant is unclear. Recent studies have shown that other members of the LDLR family undergo gamma-secretase dependent regulated intramembrane proteolysis (RIP). Using independent experimental approaches, we show that LRP6 also undergoes RIP. The extracellular domain of LRP6 is shed and released into the surrounding milieu and the cytoplasmic tail is cleaved by gamma-secretase-like activity to release the intracellular domain. Furthermore, protein kinase C, Wnt 3a and Dickkopf-1 modulate this process. These findings suggest a novel mechanism for LRP6 in Wnt signaling: induction of ectodomain shedding of LRP6, followed by the gamma-secretase involved proteolytic releasing its intracellular domain (ICD) which then binds to GSK3 inhibiting its activity and thus activates the canonical Wnt signaling pathway.     

The mechanism of endogenous receptor activation functionally distinguishes prototype canonical and noncanonical Wnts

Wnt glycoproteins are developmentally essential signaling molecules, and lesions afflicting Wnt pathways play important roles in human diseases. Some Wnts signal to the canonical pathway by stabilizing beta-catenin, while others lack this activity. Frizzled serpentine receptors mediate distinct signaling pathways by both classes of Wnts. Here, we tandemly linked noncanonical Wnt5a with the C-terminal half of Dickkopf-2 (Dkk2C), a distinct ligand of the Wnt coreceptor LRP5/6. Whereas Wnt5a, Dkk2C, or both together were incapable of stimulating endogenous canonical signaling, the Wnt5a/Dkk2C chimera efficiently activated this pathway in a manner inhibitable by specific antagonists of either frizzled or LRP receptors. Thus, activation of the canonical pathway requires ligand coupling of an endogenous frizzled/LRP coreceptor complex, rather than Wnt triggering each receptor independently. Moreover, fusion of Wnt5a with Dkk2C unmasked its ability to signal to Dishevelled through multiple frizzleds, indicating that the lack of functional interaction with LRP distinguishes noncanonical Wnt5a from canonical Wnts in mammalian cells. These findings provide a novel mechanism by which the same receptor can be switched between distinct signaling pathways depending on the differential recruitment of a coreceptor by members of the same ligand family.     

A novel set of Wnt-Frizzled fusion proteins identifies receptor components that activate beta -catenin-dependent signaling

Wnt proteins initiate the canonical (beta-catenin-regulated) signaling cascade by binding to seven-transmembrane spanning receptors of the Frizzled (Fz) family together with the coreceptors LRP5 and -6, members of the low density lipoprotein receptor-related protein family (LRP). Several reports have shown physical and functional associations between various Wnt, LRP, and Frizzled molecules; however, the underlying mechanisms for selectivity remain poorly understood. We present data on a novel set of Wnt-Fz fusion constructs that are useful for elucidating mechanisms of Wnt signal transduction specificity in both Xenopus embryos and 293T cells. In 293T cells, coexpression of several Wnt-Fz fusion proteins with LRP6, but not LRP5, significantly activated a Wnt-responsive promoter, Optimized TOPFlash. Interestingly, Wnt proteins from both the Wnt1 and Wnt5A classes, when fused to the same Frizzled, can synergize with LRP6 to activate signaling and induce secondary axes in Xenopus embryos. However, when several Wnt-Fz constructs containing different Frizzled molecules were tested, it was found that all Frizzled molecules are not equivalent in their ability to activate the canonical Wnt pathway in this context. The data suggest that the distinction between the two Wnt classes lies not in intrinsic differences in the molecules but via the Frizzled molecules with which they interact.     

The low density lipoprotein receptor-1, LRP1, interacts with the human frizzled-1 (HFz1) and down-regulates the canonical Wnt signaling pathway

Members of the low density lipoprotein receptor family (LDLR), LRP5/6, were shown to interact with the Frizzled (Fz) receptors and to function as Wnt coreceptors. Here we show that mLRP4T100, a minireceptor of LRP1, another member of the LDLR family, interacts with the human Fz-1 (HFz1), previously shown to serve as a receptor transmitting the canonical Wnt-3a-induced signaling cascade. However, in contrast to LRP5/6, mLRP4T100, as well as the full-length LRP1, did not cooperate with HFz1 in transmitting the Wnt-3a signaling but rather repressed it. mLRP4T100 inhibitory effect was displayed also by endocytosis-defective mLRP4T100 mutants, suggesting that LRP1 repressive effect is not attributable to LRP1-mediated enhanced HFz1 internalization and subsequent degradation. Enforced expression of mLRP4T100 decreased the capacity of HFz1 cysteine-rich domain (CRD) to interact with LRP6, in contrast to HFz1-CRD/Wnt-3a interaction that was not disrupted by overexpressing mLRP4T100. These data suggest that LRP1, by sequestering HFz1, disrupts the receptor/coreceptor complex formation, leading to the repression of the canonical Wnt signaling. Thus, this study implies that the ability to interact with Fz receptors is shared by several members of the LDLR family. However, whereas some members of the LDLR family, such as LRP5/6, interact with Fz and serve as Wnt coreceptors, others negatively regulate Wnt signaling, presumably by sequestering Fz.     

Wnt signaling and heterotrimeric G-proteins: strange bedfellows or a classic romance?

Wnts are secreted ligands with diverse roles in animal development. Wnts bind to cell surface membrane proteins termed Frizzleds. Molecular cloning of members of the Frizzled family revealed hydropathy plots with seven putative, transmembrane-spanning regions, conserved in Frizzleds characterized in mice, humans, flies, and worms. Understanding how Frizzled translates binding of their cognate Wnts into intracellular signals controlling aspects of development has been an elusive goal. Earlier observations gathered from a variety of model systems provided compelling, but indirect, support that the Frizzled receptors may be members of the superfamily of G-protein-coupled receptors that possess seven transmembrane-spanning domains. Search for a linkage between Frizzled and possible downstream heterotrimeric G-proteins has been advanced by the use of bacterial toxins, antisense DNA, and novel chimeric receptor constructs. New data establish that Frizzleds are indeed bona fide G-protein-coupled receptors. Frizzled-1 couples via G-proteins Go and Gq to the canonical beta-catenin-Lef-Tcf pathway. Frizzled-2 couples via Gq and Gt to downstream effectors including calcium mobilization. Frizzleds and G-proteins might once have been considered strange bedfellows, not likely partners in signaling. The new data, consistent with the properties known for virtually all members of the G-protein-coupled receptors, reveal a more classic romance of signaling elements controlling aspects of early development.     

Frizzled-7 turnover at the plasma membrane is regulated by cell density and the Ca(2+) -dependent protease calpain-1

Frizzled are seven-transmembrane domain G-protein coupled receptors involved in cell polarity and Wnt signaling. The mechanisms regulating their turnover at the plasma membrane remain unclear. We have identified a regulated C-terminus cleavage of Frizzled-7 in endothelial cells using ectopic expression of N- and C-termini-tagged Frizzled-7 proteins. This specific cleavage produced a 10 kDa C-terminus fragment that remained associated with intracellular vesicles and was localized within the 3rd intracytoplasmic loop using N-terminal sequencing and targeted mutagenesis. Frizzled-7 mutated forms displaying reduced C-terminus cleavage were also defective for dvl2 translocation at the plasma membrane. PMA, an activator of PKC and endocytosis, but not Wnt13A and Wnt5A, increased the appearance of Frizzled-7 C-terminus-containing vesicles and Frizzled-7 cleavage. Concanavalin-A, an inhibitor of receptor internalization decreased both constitutive and PMA-induced Frizzled-7 cleavage, while inhibition of the endocytic pathway with Delta95-295-Eps15 dominant-negative prevented only PMA-induced Frizzled-7 cleavage. Frizzled-7 C-terminus cleavage was increased with cell density and by the Ca(2+) ionophore ionomycin and was decreased by specific calpain inhibitors, by the expression of DN-calpain-1 and the down-regulation of calpain-1 levels by siRNAs. Altogether, our findings pinpoint calpain-1 as a regulator of Frizzled-7 turnover at the plasma membrane and reveal a link between Frizzled-7 cleavage and its activity.     

β-Catenin-dependent pathway activation by both promiscuous “canonical” WNT3a–, and specific “noncanonical” WNT4– and WNT5a–FZD receptor combinations with strong differences in LRP5 and LRP6 dependency

The WNT/β-catenin signalling cascade is the best-investigated frizzled receptor (FZD) pathway, however, whether and how specific combinations of WNT/FZD and co-receptors LRP5 and LRP6 differentially affect this pathway are not well understood. This is mostly due to the fact that there are 19 WNTs, 10 FZDs and at least two co-receptors. In our attempt to identify the signalling capabilities of specific WNT/FZD/LRP combinations we made use of our previously reported TCF/LEF Gaussia luciferase reporter gene HEK293 cell line (Ring et al., 2011). Generation of WNT/FZD fusion constructs – but not their separate transfection – without or with additional isogenic overexpression of LRP5 and LRP6 in our reporter cells permitted the investigation of specific WNT/FZD/LRP combinations. The canonical WNT3a in fusion to almost all FZDs was able to induce β-catenin-dependent signalling with strong dependency on LRP6 but not LRP5. Interestingly, noncanonical WNT ligands, WNT4 and WNT5a, were also able to act “canonically” but only in fusion with specific FZDs and with selective dependence on LRP5 or LRP6. These data and extension of this experimental setup to the poorly characterized other WNTs should facilitate deeper insight into the complex WNT/FZD signalling system and its function.     

The p75 neurotrophin receptor interacts with multiple MAGE proteins

The p75 neurotrophin receptor has been implicated in diverse aspects of neurotrophin signaling, but the mechanisms by which its effects are mediated are not well understood. Here we identify two MAGE proteins, necdin and MAGE-H1, as interactors for the intracellular domain of p75 and show that the interaction is enhanced by ligand stimulation. PC12 cells transfected with necdin or MAGE-H1 exhibit accelerated differentiation in response to nerve growth factor. Expression of these two MAGE proteins is predominantly cytoplasmic in PC12 cells, and necdin was found to be capable of homodimerization, suggesting that it may act as a cytoplasmic adaptor to recruit a signaling complex to p75. These findings indicate that diverse MAGE family members can interact with the p75 receptor and highlight type II MAGE proteins as a potential family of interactors for signaling proteins containing type II death domains.     

Wnt-4 activates the canonical beta-catenin-mediated Wnt pathway and binds Frizzled-6 CRD: functional implications of Wnt/beta-catenin activity in kidney epithelial cells

The Wnt signaling pathway is central to the development of all animals and to cancer progression, yet largely unknown are the pairings of secreted Wnt ligands to their respective Frizzled transmembrane receptors or, in many cases, the relative contributions of canonical (beta-catenin/LEF/TCF) versus noncanonical Wnt signals. Specifically, in the kidney where Wnt-4 is essential for the mesenchymal to epithelial transition that generates the tissue's collecting tubules, the corresponding Frizzled receptor(s) and downstream signaling mechanism(s) are unclear. In this report, we addressed these issues using Madin-Darby Canine Kidney (MDCK) cells, which are competent to form tubules in vitro. Employing established reporter constructs of canonical Wnt/beta-catenin pathway activity, we have determined that MDCK cells are highly responsive to Wnt-4, -1, and -3A, but not to Wnt-5A and control conditions, precisely reflecting functional findings from Wnt-4 null kidney mesenchyme ex vivo rescue studies. We have confirmed that Wnt-4's canonical signaling activity in MDCK cells is mediated by downstream effectors of the Wnt/beta-catenin pathway using beta-Engrailed and dnTCF-4 constructs that suppress this pathway. We have further found that MDCK cells express the Frizzled-6 receptor and that Wnt-4 forms a biochemical complex with the Frizzled-6 CRD. Since Frizzled-6 did not appear to transduce Wnt-4's canonical signal, data supported recently by Golan et al., there presumably exists another as yet unknown Frizzled receptor(s) mediating Wnt-4 activation of beta-catenin/LEF/TCF. Finally, we report that canonical Wnt/beta-catenin signals cells help maintain cell growth and survival in MDCK cells but do not contribute to standard HGF-induced (nonphysiologic) tubule formation. Our results in combination with work from Xenopus laevis (not shown) lead us to believe that Wnt-4 binds both canonical and noncanonical Frizzled receptors, thereby activating Wnt signaling pathways that may each contribute to kidney tubulogenesis.     

Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6.

BACKGROUND: Dickkopf-1 (Dkk-1) is a head inducer secreted from the vertebrate head organizer and induces anterior development by antagonizing Wnt signaling. Although several families of secreted antagonists have been shown to inhibit Wnt signal transduction by binding to Wnt, the molecular mechanism of Dkk-1 action is unknown. The Wnt family of secreted growth factors initiates signaling via the Frizzled (Fz) receptor and its candidate coreceptor, LDL receptor-related protein 6 (LRP6), presumably through Fz-LRP6 complex formation induced by Wnt. The significance of the Fz-LRP6 complex in signal transduction remains to be established. RESULTS: We report that Dkk-1 is a high-affinity ligand for LRP6 and inhibits Wnt signaling by preventing Fz-LRP6 complex formation induced by Wnt. Dkk-1 binds neither Wnt nor Fz, nor does it affect Wnt-Fz interaction. Dkk-1 function in head induction and Wnt signaling inhibition strictly correlates with its ability to bind LRP6 and to disrupt the Fz-LRP6 association. LRP6 function and Dkk-1 inhibition appear to be specific for the Wnt/Fz beta-catenin pathway. CONCLUSIONS: Our results demonstrate that Dkk-1 is an LRP6 ligand and inhibits Wnt signaling by blocking Wnt-induced Fz-LRP6 complex formation. Our findings thus reveal a novel mechanism for Wnt signal modulation. LRP6 is a Wnt coreceptor that appears to specify Wnt/Fz signaling to the beta-catenin pathway, and Dkk-1, distinct from Wnt binding antagonists, may be a specific inhibitor for Wnt/beta-catenin signaling. Our findings suggest that Wnt-Fz-LRP6 complex formation, but not Wnt-Fz interaction, triggers Wnt/beta-catenin signaling.     

Wnt-3a and Dickkopf-1 stimulate neurite outgrowth in Ewing tumor cells via a Frizzled3- and c-Jun N-terminal kinase-dependent mechanism

Recombinant Wnt-3a stimulated the rapid formation of elongated processes in Ewing sarcoma family tumor (ESFT) cells that were identified as neurites. The processes stained positively for polymerized actin and microtubules as well as synapsin I and growth-associated protein 43. Inhibition of the Wnt receptor, Frizzled3 (Fzd3), with antiserum or by short interfering RNA (siRNA) markedly reduced neurite extension. Knockdown of Dishevelled-2 (Dvl-2) and Dvl-3 also suppressed neurite outgrowth. Surprisingly, disruption of the Wnt/Fzd/lipoprotein receptor-related protein (LRP) complex and the associated beta-catenin signaling by treating cells either with the Wnt antagonist Dickkopf-1 (Dkk1) or LRP5/LRP6 siRNA enhanced neuritogenesis. Neurite outgrowth induced by Dkk1 or with LRP5/LRP6 siRNA was inhibited by secreted Fzd-related protein 1, a Wnt antagonist that binds directly to Wnt. Moreover, Dkk1 stimulation of neurite outgrowth was blocked by Fzd3 siRNA. These results suggested that Dkk1 shifted endogenous Wnt activity from the beta-catenin pathway to Fzd3-mediated, noncanonical signaling that is responsible for neurite formation. In particular, c-Jun amino-terminal kinase (JNK) was important for neurite outgrowth stimulated by both Wnt-3a and Dkk1. Our data demonstrate that Fzd3, Dvl, and JNK activity mediate Wnt-dependent neurite outgrowth and that ESFT cell lines will be useful experimental models for the study of Wnt-dependent neurite extension.     

Arrow (LRP6) and Frizzled2 cooperate to degrade Wingless in Drosophila imaginal discs

Lysosome-mediated ligand degradation is known to shape morphogen gradients and modulate the activity of various signalling pathways. We have investigated the degradation of Wingless, a Drosophila member of the Wnt family of secreted growth factors. We find that one of its signalling receptors, Frizzled2, stimulates Wingless internalization both in wing imaginal discs and cultured cells. However, this is not sufficient for degradation. Indeed, as shown previously, overexpression of Frizzled2 leads to Wingless stabilization in wing imaginal discs. We show that Arrow (the Drosophila homologue of LRP5/6), another receptor involved in signal transduction, abrogates such stabilization. We provide evidence that Arrow stimulates the targeting of Frizzled2-Wingless (but not Dally-like-Wingless) complexes to a degradative compartment. Thus, Frizzled2 alone cannot lead Wingless all the way from the plasma membrane to a degradative compartment. Overall, Frizzled2 achieves ligand capture and internalization, whereas Arrow, and perhaps downstream signalling, are essential for lysosomal targeting.     

Ligand receptor interactions in the Wnt signaling pathway in Drosophila

Secreted Wnt proteins have numerous signaling functions during development, mediated by Frizzled molecules that act as Wnt receptors on the cell surface. In the genome of Drosophila, seven Wnt genes (including wingless; wg), and five frizzled genes have been identified. Relatively little is known about signaling and binding specificities of different Wnt and Frizzled proteins. We have developed an assay to determine the strength of binding between membrane-tethered Wnts and ligand binding domains of the Frizzled receptors. We found a wide spectrum of binding affinities, reflecting known genetic interactions. Most Wnt proteins can bind to multiple Frizzleds and vice versa, suggesting redundancy in vivo. In an extension of these experiments, we tested whether two different subdomains of the Wg protein would by themselves bind to Frizzled and generate a biological response. Whereas these two separate domains are secreted from cells, suggesting that they form independently folded parts of the protein, they were only able to evoke a response when co-transfected, indicating that both are required for function. In addition to the Frizzleds, members of the LRP family (represented by the arrow gene in Drosophila) are also necessary for Wnt signal transduction and have been postulated to act as co-receptors. We have therefore examined whether a soluble form of the Arrow molecule can bind to Wingless and Frizzled, but no interactions were detected.     

Multiplicity of the interactions of Wnt proteins and their receptors

Wnts are secreted proteins that are essential for a wide array of developmental and physiological processes. They signal across the plasma membranes by interacting with serpentine receptors of the Frizzled (Fz) family and members of the low-density-lipoprotein receptor-related protein (LRP) family. Recent advances in the Wnt signaling field have revealed that Wnt-unrelated proteins activate or suppress Wnt signaling by binding to Fzs or LRP5/6 and that atypical receptor tyrosine kinases mediate Wnt signaling independently of Fz and/or function as a Fz co-receptor. This review highlights recent progress in our understanding of the multiplicity of Wnts and their receptors. We discuss how the interaction between the ligands and receptors activate distinct intracellular signaling pathways. We also discuss how intracellular trafficking of Wnt signaling components can regulate the sensitivity of cells to Wnts.     

Wnt signals across the plasma membrane to activate the beta-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP

Wnt-induced signaling via beta-catenin plays crucial roles in animal development and tumorigenesis. Both a seven-transmembrane protein in the Frizzled family and a single transmembrane protein in the LRP family (LDL-receptor-related protein 5/6 or Arrow) are essential for efficiently transducing a signal from Wnt, an extracellular ligand, to an intracellular pathway that stabilizes beta-catenin by interfering with its rate of destruction. However, the molecular mechanism by which these two types of membrane receptors synergize to transmit the Wnt signal is not known. We have used mutant and chimeric forms of Frizzled, LRP and Wnt proteins, small inhibitory RNAs, and assays for beta-catenin-mediated signaling and protein localization in Drosophila S2 cells and mammalian 293 cells to study transmission of a Wnt signal across the plasma membrane. Our findings are consistent with a mechanism by which Wnt protein binds to the extracellular domains of both LRP and Frizzled receptors, forming membrane-associated hetero-oligomers that interact with both Disheveled (via the intracellular portions of Frizzled) and Axin (via the intracellular domain of LRP). This model takes into account several observations reported here: the identification of intracellular residues of Frizzled required for beta-catenin signaling and for recruitment of Dvl to the plasma membrane; evidence that Wnt3A binds to the ectodomains of LRP and Frizzled; and demonstrations that a requirement for Wnt ligand can be abrogated by chimeric receptors that allow formation of Frizzled-LRP hetero-oligomers. In addition, the beta-catenin signaling mediated by ectopic expression of LRP is not dependent on Disheveled or Wnt, but can also be augmented by oligomerization of LRP receptors.