Increasingly complex: new players enter the Wnt signaling network

Wnt proteins can activate different intracellular signaling cascades in various organisms by interacting with receptors of the Frizzled family. The first identified Wnt signaling pathway, the Wnt/beta-catenin pathway, has been studied in much detail and is highly conserved among species. As to non-canonical Wnt pathways, the current situation is more nebulous partly because the intracellular mediators of this pathway are not yet fully understood and, in some cases, even identified. However, there are increasing data that prove the existence of non-canonical Wnt signaling and demonstrate its involvement in different developmental processes. In vertebrates, Wnt-11 and Wnt-5A can activate the Wnt/JNK pathway, which resembles the planar cell polarity pathway in Drosophila. The Wnt/Ca(2+)-pathway has only been described in Xenopus and zebrafish so far and it is unclear whether it also exists in other organisms. Two recent papers provide us with new insight into non-canonical Wnt signaling by (1) presenting a new intracellular mediator of non-canonical signaling in Xenopus1 and (2) implicating the existence of an additional non-canonical Wnt signaling pathway in flies.     

Non-canonical Wnt signaling in Xenopus: regulation of axis formation and gastrulation

Wnt proteins form a family of secreted glycoproteins that are involved in different developmental processes such as differentiation, proliferation, cell migration and cell polarity. To exert its function, Wnt proteins activate different intracellular signaling cascades. Whereas the canonical, Wnt/beta-catenin pathway is well characterized, less is known about the function of non-canonical Wnt pathways in vertebrates. I here summarize recent findings implicating important roles for Wnt/Ca(2+) and Wnt/JNK signaling during different aspects of early Xenopus laevis development, namely axis formation and gastrulation movements.     

The roles of canonical and non-canonical Wnt signaling in human de-differentiated articular chondrocytes

Osteoarthritis is the most prevalent form of arthritis in the world and it is becoming a major public health problem. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to de-differentiation. The involvement of signaling pathways, such as the Wnt pathway, during cartilage pathology has been reported. Wnt signaling regulates critical biological processes. Wnt signals are transduced through at least three intracellular signaling pathways including the canonical Wnt/β-catenin pathway, the Wnt/Ca2 + pathway and the Wnt/planar cell polarity pathway. We investigated the involvement of the Wnt canonical and non-canonical pathways in human articular chondrocyte de-differentiation in vitro. Human articular chondrocytes were cultured through four passages with no treatment, or with sFRP3 treatment, an inhibitor of Wnt pathways, or with DKK1 treatment, an inhibitor of the canonical pathway. Chondrocyte-secreted markers and Wnt pathway components were analyzed using western blotting and qPCR. Inhibition of the Wnt pathway showed that the canonical Wnt signaling probably is responsible for inhibition of collagen II expression, activation of metalloproteinase 13 expression and regulation of Wnt7a and c-jun expression during chondrocyte de-differentiation in vitro. Our results also suggest that expressions of eNOS, Wnt5a and cyclinE1 are regulated by non-canonical Wnt signaling.     

Wnt5a induces homodimerization and activation of Ror2 receptor tyrosine kinase

Wnts are secreted glycoproteins that control vital biological processes, including embryogenesis, organogenesis and tumorigenesis. Wnts are classified into several subfamilies depending on the signaling pathways they activate, with the canonical subfamily activating the Wnt/beta-catenin pathway and the non-canonical subfamily activating a variety of other pathways, including the Wnt/calcium signaling and the small GTPase/c-Jun NH2-terminal kinase pathway. Wnts bind to a membrane receptor Frizzled and a co-receptor, the low-density lipoprotein receptor related protein. More recently, both canonical and non-canonical Wnts were shown to bind the Ror2 receptor tyrosine kinase. Ror2 is an orphan receptor that plays crucial roles in skeletal morphogenesis and promotes osteoblast differentiation and bone formation. Here we examine the effects of a canonical Wnt3a and a non-canonical Wnt5a on the signaling of the Ror2 receptor. We demonstrate that even though both Wnt5a and Wnt3a bound Ror2, only Wnt5a induced Ror2 homo-dimerization and tyrosine phosphorylation in U2OS human osteoblastic cells. Furthermore, Wnt5a treatment also resulted in increased phosphorylation of the Ror2 substrate, 14-3-3beta scaffold protein, indicating that Wnt5a binding causes activation of the Ror2 signaling cascade. Functionally, Wnt5a recapitulated the Ror2 activation phenotype, enhancing bone formation in the mouse calvarial bone explant cultures and potentiating osteoblastic differentiation of human mesenchymal stem cells. The effect of Wnt5a on osteoblastic differentiation was largely abolished upon Ror2 down-regulation. Thus we show that Wnt5a activates the classical receptor tyrosine kinase signaling cascade through the Ror2 receptor in cells of osteoblastic origin.     

Role of glypican 4 in the regulation of convergent extension movements during gastrulation in Xenopus laevis

Coordinated morphogenetic cell movements during gastrulation are crucial for establishing embryonic axes in animals. Most recently, the non-canonical Wnt signaling cascade (PCP pathway) has been shown to regulate convergent extension movements in Xenopus and zebrafish. Heparan sulfate proteoglycans (HSPGs) are known as modulators of intercellular signaling, and are required for gastrulation movements in vertebrates. However, the function of HSPGs is poorly understood. We analyze the function of Xenopus glypican 4 (Xgly4), which is a member of membrane-associated HSPG family. In situ hybridization revealed that Xgly4 is expressed in the dorsal mesoderm and ectoderm during gastrulation. Reducing the levels of Xgly4 inhibits cell-membrane accumulation of Dishevelled (Dsh), which is a transducer of the Wnt signaling cascade, and thereby disturbs cell movements during gastrulation. Rescue analysis with different Dsh mutants and Wnt11 demonstrated that Xgly4 functions in the non-canonical Wnt/PCP pathway, but not in the canonical Wnt/beta-catenin pathway, to regulate gastrulation movements. We also provide evidence that the Xgly4 protein physically binds Wnt ligands. Therefore, our results suggest that Xgly4 functions as positive regulator in non-canonical Wnt/PCP signaling during gastrulation.     

β-Arrestin Interacts with the Beta/Gamma Subunits of Trimeric G-Proteins and Dishevelled in the Wnt/Ca2+ Pathway in Xenopus Gastrulation

β-Catenin independent, non-canonical Wnt signaling pathways play a major role in the regulation of morphogenetic movements in vertebrates. The term non-canonical Wnt signaling comprises multiple, intracellularly divergent, Wnt-activated and β-Catenin independent signaling cascades including the Wnt/Planar Cell Polarity and the Wnt/Ca²⁺ cascades. Wnt/Planar Cell Polarity and Wnt/Ca²⁺ pathways share common effector proteins, including the Wnt ligand, Frizzled receptors and Dishevelled, with each other and with additional branches of Wnt signaling. Along with the aforementioned proteins, β-Arrestin has been identified as an essential effector protein in the Wnt/β-Catenin and the Wnt/Planar Cell Polarity pathway. Our results demonstrate that β-Arrestin is required in the Wnt/Ca²⁺ signaling cascade upstream of Protein Kinase C (PKC) and Ca²⁺/Calmodulin-dependent Protein Kinase II (CamKII). We have further characterized the role of β-Arrestin in this branch of non-canonical Wnt signaling by knock-down and rescue experiments in Xenopus embryo explants and analyzed protein-protein interactions in 293T cells. Functional interaction of β-Arrestin, the β subunit of trimeric G-proteins and Dishevelled is required to induce PKC activation and membrane translocation. In Xenopus gastrulation, β-Arrestin function in Wnt/Ca²⁺ signaling is essential for convergent extension movements. We further show that β-Arrestin physically interacts with the β subunit of trimeric G-proteins and Dishevelled, and that the interaction between β-Arrestin and Dishevelled is promoted by the beta/gamma subunits of trimeric G-proteins, indicating the formation of a multiprotein signaling complex.     

Wnt signal transduction pathways

The Wnt signaling pathway is an ancient and evolutionarily conserved pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. The Wnts are secreted glycoproteins and comprise a large family of nineteen proteins in humans hinting to a daunting complexity of signaling regulation, function and biological output. To date major signaling branches downstream of the Fz receptor have been identified including a canonical or Wnt/β-catenin dependent pathway and the non-canonical or β-catenin-independent pathway which can be further divided into the Planar Cell Polarity and the Wnt/Ca²⁺ pathways, and these branches are being actively dissected at the molecular and biochemical levels. In this review, we will summarize the most recent advances in our understanding of these Wnt signaling pathways and the role of these pathways in regulating key events during embryonic patterning and morphogenesis.Key words: Wnt, frizzled, dishevelled, canonical, non-canonical, β-catenin, Planar Cell Polarity     

Nuclear factor of activated T cells (NFAT) proteins repress canonical Wnt signaling via its interaction with Dishevelled (Dvl) protein and participate in regulating neural progenitor cell proliferation and differentiation

The Ca(2+) signaling pathway appears to regulate the processes of the early development through its antagonism of canonical Wnt/β-catenin signaling pathway. However, the underlying mechanism is still poorly understood. Here, we show that nuclear factor of activated T cells (NFAT), a component of Ca(2+) signaling, interacts directly with Dishevelled (Dvl) in a Ca(2+)-dependent manner. A dominant negative form of NFAT rescued the inhibition of the Wnt/β-catenin pathway triggered by the Ca(2+) signal. NFAT functioned downstream of β-catenin without interfering with its stability, but influencing the interaction of β-catenin with Dvl by its competitively binding to Dvl. Furthermore, we demonstrate that NFAT is a regulator in the proliferation and differentiation of neural progenitor cells by modulating canonical Wnt/β-catenin signaling pathway in the neural tube of chick embryo. Our findings suggest that NFAT negatively regulates canonical Wnt/β-catenin signaling by binding to Dvl, thereby participating in vertebrate neurogenesis.     

Dapper-1 is essential for Wnt5a induced cardiomyocyte hypertrophy by regulating the Wnt/PCP pathway

The Wnt signaling pathway was identified as crucial mediator of cardiomyocyte hypertrophy. In this study we found that activation of non-canonical Wnt signaling by Wnt5a stimulates protein synthesis and enlargement of cardiomyocyte surface area. These hypertrophic features were inhibited in Dapper-1 (Dpr1) depleted cells. On the molecular level, we observed inhibition of the non-canonical Wnt/planar-cell-polarity (PCP) pathway denoted by reduction of c-jun-n-terminal-kinase (JNK) phosphorylation. Upstream of JNK, increased protein levels of the Wnt/PCP trans-membrane receptor van-Gogh-like-2 (Vangl2) were observed along with an enrichment of Vangl2 in perinuclear located vesicles. The findings suggest that Dpr1 is essential for execution of the Wnt/PCP pathway and regulation of the Vangl2/JNK axis. Depletion of Dpr1 inhibits non-canonical Wnt signaling induced cardiomyocyte hypertrophy by blocking Wnt/PCP signaling.     

Both Canonical and Non-Canonical Wnt Signaling Independently Promote Stem Cell Growth in Mammospheres

The characterization of mammary stem cells, and signals that regulate their behavior, is of central importance in understanding developmental changes in the mammary gland and possibly for targeting stem-like cells in breast cancer. The canonical Wnt/β-catenin pathway is a signaling mechanism associated with maintenance of self-renewing stem cells in many tissues, including mammary epithelium, and can be oncogenic when deregulated. Wnt1 and Wnt3a are examples of ligands that activate the canonical pathway. Other Wnt ligands, such as Wnt5a, typically signal via non-canonical, β-catenin-independent, pathways that in some cases can antagonize canonical signaling. Since the role of non-canonical Wnt signaling in stem cell regulation is not well characterized, we set out to investigate this using mammosphere formation assays that reflect and quantify stem cell properties. Ex vivo mammosphere cultures were established from both wild-type and Wnt1 transgenic mice and were analyzed in response to manipulation of both canonical and non-canonical Wnt signaling. An increased level of mammosphere formation was observed in cultures derived from MMTV-Wnt1 versus wild-type animals, and this was blocked by treatment with Dkk1, a selective inhibitor of canonical Wnt signaling. Consistent with this, we found that a single dose of recombinant Wnt3a was sufficient to increase mammosphere formation in wild-type cultures. Surprisingly, we found that Wnt5a also increased mammosphere formation in these assays. We confirmed that this was not caused by an increase in canonical Wnt/β-catenin signaling but was instead mediated by non-canonical Wnt signals requiring the receptor tyrosine kinase Ror2 and activity of the Jun N-terminal kinase, JNK. We conclude that both canonical and non-canonical Wnt signals have positive effects promoting stem cell activity in mammosphere assays and that they do so via independent signaling mechanisms.     

Galphaq negatively regulates the Wnt-beta-catenin pathway and dorsal embryonic Xenopus laevis development

The non-canonical Wnt/Ca2+ signaling pathway has been implicated in the regulation of axis formation and gastrulation movements during early Xenopus laevis embryo development, by antagonizing the canonical Wnt/beta-catenin dorsalizing pathway and specifying ventral cell fate. However, the molecular mechanisms involved in this antagonist crosstalk are not known. Since Galphaq is the main regulator of Ca2+ signaling in vertebrates and from this perspective probably involved in the events elicited by the non-canonical Wnt/Ca2+ pathway, we decided to study the effect of wild-type Xenopus Gq (xGalphaq) in dorso-ventral axis embryo patterning. Overexpression of xGalphaq or its endogenous activation at the dorsal animal region of Xenopus embryo both induced a strong ventralized phenotype and inhibited the expression of dorsal-specific mesoderm markers goosecoid and chordin. Dorsal expression of an xGalphaq dominant-negative mutant reverted the xGalphaq-induced ventralized phenotype. Finally, we observed that the Wnt8-induced secondary axis formation is reverted by endogenous xGalphaq activation, indicating that it is negatively regulating the Wnt/beta-catenin pathway.     

Wnt signal transduction pathways

The Wnt signaling pathway is an ancient and evolutionarily conserved pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. The Wnts are secreted glycoproteins and comprise a large family of nineteen proteins in humans hinting to a daunting complexity of signaling regulation, function and biological output. To date major signaling branches downstream of the Fz receptor have been identified including a canonical or Wnt/β-catenin dependent pathway and the non-canonical or β-catenin-independent pathway which can be further divided into the Planar Cell Polarity and the Wnt/Ca²⁺ pathways, and these branches are being actively dissected at the molecular and biochemical levels. In this review, we will summarize the most recent advances in our understanding of these Wnt signaling pathways and the role of these pathways in regulating key events during embryonic patterning and morphogenesis.     

Regulation of convergent extension in Xenopus by Wnt5a and Frizzled-8 is independent of the canonical Wnt pathway

The Wnt signaling pathway is increasingly recognized as a highly branched signaling network. Experimental uncoupling of the different branches of this pathway has proven difficult, as many single components are shared downstream by multiple, distinct pathways. In this report, we demonstrate that the upstream Wnt antagonists Xwnt5a and Nxfz-8, which inhibit normal morphogenetic movements during Xenopus gastrulation, act independently of the canonical Wnt signaling pathway. This finding is important, as it highlights the promiscuity of upstream Wnt signaling components and further establishes an important role for non-canonical Wnt signaling in Xenopus morphogenesis.     

The endocannabinoid anandamide inhibits cholangiocarcinoma growth via activation of the noncanonical Wnt signaling pathway

Cholangiocarcinomas are cancers that have poor prognosis and limited treatment options. The noncanonical Wnt pathway is mediated predominantly by Wnt 5a, which activates a Ca(2+)-dependent pathway involving protein kinase C, or a Ca(2+)-independent pathway involving the orphan receptor Ror2 and subsequent activation of Jun NH(2)-terminal kinase (JNK). This pathway is associated with growth-suppressing effects in numerous cell types. We have shown that anandamide decreases cholangiocarcinoma growth in vitro. Therefore, we determined the effects of anandamide on cholangiocarcinoma tumor growth in vivo using a xenograft model and evaluated the effects of anandamide on the noncanonical Wnt signaling pathways. Chronic administration of anandamide decreased tumor growth and was associated with increased Wnt 5a expression in vitro and in vivo. Treatment of cholangiocarcinoma cells with recombinant Wnt 5a decreased cell proliferation in vitro. Neither anandamide nor Wnt 5a affected intracellular calcium release, but both increased the JNK phosphorylation. Stable knockdown of Wnt 5a or Ror2 expression in cholangiocarcinoma cells abolished the effects of anandamide on cell proliferation and JNK activation. Modulation of the endocannabinoid system may be important in cholangiocarcinoma treatment. The antiproliferative actions of the noncanonical Wnt signaling pathway warrants further investigation to dissect the mechanism by which this may occur.     

Ror2 modulates the canonical Wnt signaling in lung epithelial cells through cooperation with Fzd2

Background  

Wnt signaling is mediated through 1) the beta-catenin dependent canonical pathway and, 2) the beta-catenin independent pathways. Multiple receptors, including Fzds, Lrps, Ror2 and Ryk, are involved in Wnt signaling. Ror2 is a single-span transmembrane receptor-tyrosine kinase (RTK). The functions of Ror2 in mediating the non-canonical Wnt signaling have been well established. The role of Ror2 in canonical Wnt signaling is not fully understood.     

Live Imaging of Xwnt5A-ROR2 Complexes

Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/β-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner.     

Wnt4 enhances murine hematopoietic progenitor cell expansion through a planar cell polarity-like pathway

Background

While the role of canonical (β-catenin-mediated) Wnt signaling in hematolymphopoiesis has been studied extensively, little is known of the potential importance of non-canonical Wnt signals in hematopoietic cells. Wnt4 is one of the Wnt proteins that can elicit non-canonical pathways. We have previously shown that retroviral overexpression of Wnt4 by hematopoietic cells increased thymic cellularity as well as the frequency of early thymic progenitors and bone marrow hematopoietic progenitor cells (HPCs). However, the molecular pathways responsible for its effect in HPCs are not known.

Methodology/Principal Findings

Here we report that Wnt4 stimulation resulted in the activation of the small GTPase Rac1 as well as Jnk kinases in an HPC cell line. Jnk activity was necessary, while β-catenin was dispensable, for the Wnt4-mediated expansion of primary fetal liver HPCs in culture. Furthermore, Jnk2-deficient and Wnt4 hemizygous mice presented lower numbers of HPCs in their bone marrow, and Jnk2-deficient HPCs showed increased rates of apoptosis. Wnt4 also improved HPC activity in a competitive reconstitution model in a cell-autonomous, Jnk2-dependent manner. Lastly, we identified Fz6 as a receptor for Wnt4 in immature HPCs and showed that the absence of Wnt4 led to a decreased expression of four polarity complex genes.

Conclusions/Significance

Our results establish a functional role for non-canonical Wnt signaling in hematopoiesis through a pathway involving Wnt4, Fz6, Rac1 and Jnk kinases.     

Stromelysin-1 and mesothelin are differentially regulated by Wnt-5a and Wnt-1 in C57mg mouse mammary epithelial cells

Background  

The Wnt signal transduction pathway is important in a wide variety of developmental processes as well as in the genesis of human cancer. Vertebrate Wnt pathways can be functionally separated into two classes, the canonical Wnt/beta-catenin pathway and the non-canonical Wnt/Ca²⁺ pathway. Supporting differences in Wnt signaling, gain of function of Wnt-1 in C57mg mouse mammary epithelial cells leads to their morphological transformation while loss of function of Wnt-5a leads to the same transformation. Many downstream target genes of the Wnt/beta-catenin pathway have been identified. In contrast, little is known about the Wnt/Ca²⁺ pathway and whether it regulates gene expression.