Wnt Signaling through the Ror Receptor in the Nervous System

The receptor tyrosine kinase-like orphan receptor (Ror) proteins are conserved tyrosine kinase receptors that play roles in a variety of cellular processes that pattern tissues and organs during vertebrate and invertebrate development. Ror signaling is required for skeleton and neuronal development and modulates cell migration, cell polarity, and convergent extension. Ror has also been implicated in two human skeletal disorders, brachydactyly type B and Robinow syndrome. Rors are widely expressed during metazoan development including domains in the nervous system. Here, we review recent progress in understanding the roles of the Ror receptors in neuronal migration, axonal pruning, axon guidance, and synaptic plasticity. The processes by which Ror signaling execute these diverse roles are still largely unknown, but they likely converge on cytoskeletal remodeling. In multiple species, Rors have been shown to act as Wnt receptors signaling via novel non-canonical Wnt pathways mediated in some tissues by the adapter protein disheveled and the non-receptor tyrosine kinase Src. Rors can either activate or repress Wnt target expression depending on the cellular context and can also modulate signal transduction by sequestering Wnt ligands away from their signaling receptors. Future challenges include the identification of signaling components of the Ror pathways and bettering our understanding of the roles of these pleiotropic receptors in patterning the nervous system.     

Structure-based Discovery of Novel Small Molecule Wnt Signaling Inhibitors by Targeting the Cysteine-rich Domain of Frizzled

Frizzled is the earliest discovered glycosylated Wnt protein receptor and is critical for the initiation of Wnt signaling. Antagonizing Frizzled is effective in inhibiting the growth of multiple tumor types. The extracellular N terminus of Frizzled contains a conserved cysteine-rich domain that directly interacts with Wnt ligands. Structure-based virtual screening and cell-based assays were used to identify five small molecules that can inhibit canonical Wnt signaling and have low IC₅₀ values in the micromolar range. NMR experiments confirmed that these compounds specifically bind to the Wnt binding site on the Frizzled8 cysteine-rich domain with submicromolar dissociation constants. Our study confirms the feasibility of targeting the Frizzled cysteine-rich domain as an effective way of regulating canonical Wnt signaling. These small molecules can be further optimized into more potent therapeutic agents for regulating abnormal Wnt signaling by targeting Frizzled.     

The autophagy-related kinase UNC-51 and its binding partner UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5 in Caenorhabditis elegans

UNC-51 and UNC-14 are required for the axon guidance of many neurons in Caenorhabditis elegans. UNC-51 is a serine/threonine kinase homologous to yeast Atg1, which is required for autophagy. The binding partner of UNC-51, UNC-14, contains a RUN domain that is predicted to play an important role in multiple Ras-like GTPase signaling pathways. How these molecules function in axon guidance is largely unknown. Here we observed that, in unc-51 and unc-14 mutants, UNC-5, the receptor for axon-guidance protein Netrin/UNC-6, abnormally localized in neuronal cell bodies. By contrast, the localization of many other proteins required for axon guidance was undisturbed. Moreover, UNC-5 localization was normal in animals with mutations in the genes for axon guidance proteins, several motor proteins, vesicle components and autophagy-related proteins. We also found that unc-5 and unc-6 interacted genetically with unc-51 and unc-14 to affect axon guidance, and that UNC-5 co-localized with UNC-51 and UNC-14 in neurons. These results suggest that UNC-51 and UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5, and that UNC-5 uses a unique mechanism for its localization; the functionality of UNC-5 is probably regulated by this localization.     

Extracellular Matrix Regulates UNC-6 (Netrin) Axon Guidance by Controlling the Direction of Intracellular UNC-40 (DCC) Outgrowth Activity

How extracellular molecules influence the direction of axon guidance is poorly understood. The HSN axon of Caenorhabditis elegans is guided towards a ventral source of secreted UNC-6 (netrin). The axon’s outgrowth response to UNC-6 is mediated by the UNC-40 (DCC) receptor. We have proposed that in response to the UNC-6 molecule the direction of UNC-40-mediated axon outgrowth is stochastically determined. The direction of guidance is controlled by asymmetric cues, including the gradient of UNC-6, that regulate the probability that UNC-40-mediated axon outgrowth is directed on average, over time, in a specific direction. Here we provide genetic evidence that a specialized extracellular matrix, which lies ventral to the HSN cell body, regulates the probability that UNC-40-mediated axon outgrowth will be directed ventrally towards the matrix. We show that mutations that disrupt the function of proteins associated with this matrix, UNC-52 (perlecan), UNC-112 (kindlin), VAB-19 (Kank), and UNC-97 (PINCH), decrease the probability of UNC-40-mediated axon outgrowth in the ventral direction, while increasing the probability of outgrowth in the anterior and posterior directions. Other results suggest that INA-1 (α integrin) and MIG-15 (NIK kinase) signaling mediate the response in HSN. Although the AVM axon also migrates through this matrix, the mutations have little effect on the direction of AVM axon outgrowth, indicating that responses to the matrix are cell-specific. Together, these results suggest that an extracellular matrix can regulate the direction of UNC-6 guidance by increasing the probability that UNC-40-mediated axon outgrowth activity will be oriented in a specific direction.     

Lignin Induces ES Cells to Differentiate into Neuroectodermal Cells through Mediation of the Wnt Signaling Pathway

Embryonic stem cells (ES cells) are characterized by their pluripotency and infinite proliferation potential. Ever since ES cells were first established in 1981, there have been a growing number of studies aimed at clinical applications of ES cells. In recent years, various types of differentiation inducement systems using ES cells have been established. Further studies have been conducted to utilize differentiation inducement systems in the field of regenerative medicine. For cellular treatments using stem cells including ES cells, differentiation induction should be performed in a sufficient manner to obtain the intended cell lineages. Lignin is a high-molecular amorphous material that forms plants together with cellulose and hemicelluloses, in which phenylpropane fundamental units are complexly condensed. Lignin derivatives have been shown to have several bioactive functions. In spite of these findings, few studies have focused on the effects of lignin on stem cells. Our study aimed to develop a novel technology using lignin to effectively induce ES cells to differentiate into neuroectodermal cells including ocular cells and neural cells. Since lignin can be produced at a relatively low cost in large volumes, its utilization is expected for more convenient differentiation induction technologies and in the field of regenerative medicine in the future.     

The Wnt Coreceptor Ryk Regulates Wnt/Planar Cell Polarity by Modulating the Degradation of the Core Planar Cell Polarity Component Vangl2

The Wnt signaling pathways control many critical developmental and adult physiological processes. In vertebrates, one fundamentally important function of Wnts is to provide directional information by regulating the evolutionarily conserved planar cell polarity (PCP) pathway during embryonic morphogenesis. However, despite the critical roles of Wnts and PCP in vertebrate development and disease, little is known about the molecular mechanisms underlying Wnt regulation of PCP. Here, we have found that the receptor-like tyrosine kinase (Ryk), a Wnt5a-binding protein required in axon guidance, regulates PCP signaling. We show that Ryk interacts with Vangl2 genetically and biochemically, and such interaction is potentiated by Wnt5a. Loss of Ryk in a Vangl2^+/− background results in classic PCP defects, including open neural tube, misalignment of sensory hair cells in the inner ear, and shortened long bones in the limbs. Complete loss of both Ryk and Vangl2 results in more severe phenotypes that resemble the Wnt5a^−/− mutant in many aspects such as shortened anterior-posterior body axis, limb, and frontonasal process. Our data identify the Wnt5a-binding protein Ryk as a general regulator of the mammalian Wnt/PCP signaling pathway. We show that Ryk transduces Wnt5a signaling by forming a complex with Vangl2 and that Ryk regulates PCP by at least in part promoting Vangl2 stability. As human mutations in WNT5A and VANGL2 are found to cause Robinow syndrome and neural tube defects, respectively, our results further suggest that human mutations in RYK may also be involved in these diseases.     

SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells

In mammals, the receptor of the neuropeptide gonadotropin-releasing hormone (GnRHR) is unique among the G protein-coupled receptor (GPCR) family because it lacks the carboxyl-terminal tail involved in GPCR desensitization. Therefore, mechanisms involved in the regulation of GnRHR signaling are currently poorly known. Here, using immunoprecipitation and GST pull-down experiments, we demonstrated that SET interacts with GnRHR and targets the first and third intracellular loops. We delineated, by site-directed mutagenesis, SET binding sites to the basic amino acids ⁶⁶KRKK⁶⁹ and ²⁴⁶RK²⁴⁷, located next to sequences required for receptor signaling. The impact of SET on GnRHR signaling was assessed by decreasing endogenous expression of SET with siRNA in gonadotrope cells. Using cAMP and calcium biosensors in gonadotrope living cells, we showed that SET knockdown specifically decreases GnRHR-mediated mobilization of intracellular cAMP, whereas it increases its intracellular calcium signaling. This suggests that SET influences signal transfer between GnRHR and G proteins to enhance GnRHR signaling to cAMP. Accordingly, complexing endogenous SET by introduction of the first intracellular loop of GnRHR in αT3-1 cells significantly reduced GnRHR activation of the cAMP pathway. Furthermore, decreasing SET expression prevented cAMP-mediated GnRH stimulation of Gnrhr promoter activity, highlighting a role of SET in gonadotropin-releasing hormone regulation of gene expression. In conclusion, we identified SET as the first direct interacting partner of mammalian GnRHR and showed that SET contributes to a switch of GnRHR signaling toward the cAMP pathway.     

Netrin stimulates tyrosine phosphorylation of the UNC-5 family of netrin receptors and induces Shp2 binding to the RCM cytodomain

Caenorhabditis elegans UNC-5 and its mammalian homologues such as RCM are receptors for the secreted axon guidance cue UNC-6/netrin and are required to mediate the repulsive effects of UNC-6/netrin on growth cones. We find that C. elegans UNC-5 and mouse RCM are phosphorylated on tyrosine in vivo. C. elegans UNC-5 tyrosine phosphorylation is reduced in unc-6 null mutants, and RCM tyrosine phosphorylation is induced by netrin-1 in transfected HEK-293 cells, demonstrating that phosphorylation of UNC-5 proteins is enhanced by UNC-6/netrin stimulation in both worms and mammalian cells. An activated Src tyrosine kinase induces phosphorylation of RCM at multiple cytoplasmic tyrosine residues creating potential binding sites for cytoplasmic signaling proteins. Indeed, the NH2-terminal SH2 domain of the Shp2 tyrosine phosphatase bound specifically to a Tyr(568) RCM phosphopeptide. Furthermore, Shp2 associated with RCM in a netrin-dependent manner in transfected cells, and co-immunoprecipitated with RCM from an embryonic mouse brain lysate. A Y568F mutant RCM receptor failed to bind Shp2 and was more highly phosphorylated on tyrosine than the wild type receptor. These results suggest that netrin-stimulated phosphorylation of RCM Tyr(568) recruits Shp2 to the cell membrane where it can potentially modify RCM phosphorylation and function.     

Post-translational Glycoprotein Modifications Regulate Colon Cancer Stem Cells and Colon Adenoma Progression in Apcmin/+ Mice through Altered Wnt Receptor Signaling

Deletion of GnT-V (MGAT5), which synthesizes N-glycans with β(1,6)-branched glycans, reduced the compartment of cancer stem cells (CSC) in the her-2 mouse model of breast cancer, leading to delay of tumor onset. Because GnT-V levels are also commonly up-regulated in colon cancer, we investigated their regulation of colon CSC and adenoma development. Anchorage-independent cell growth and tumor formation induced by injection of colon tumor cells into NOD/SCID mice were positively associated with GnT-V levels, indicating regulation of proliferation and tumorigenicity. Using Apc^min/+ mice with different GnT-V backgrounds, knock-out of GnT-V had no significant effect on the number of adenoma/mouse, but adenoma size was significantly reduced and accompanied increased survival of Apc^min/+ mice with GnT-V deletion (p < 0.01), suggesting an inhibition in the progression of colon adenoma caused by deletion of GnT-V. Decreased expression levels of GnT-V down-regulated the population of colon (intestine) CSC, affecting their ability for self-renewal and tumorigenicity in NOD/SCID mice. Furthermore, altered nuclear translocation of β-catenin and expression of Wnt target genes were positively associated with expression levels of GnT-V, indicating the regulation of canonical Wnt/β-catenin signaling. By overexpressing the Wnt receptor, FZD-7, in colon cancer cells, we found that FZD-7 receptors expressed N-linked β(1,6) branching, indicating that FZD-7 can be modified by GnT-V. The aberrant Wnt signaling observed after modulating GnT-V levels is likely to result from altered N-linked β(1,6) branching on FZD-7, thereby affecting Wnt signaling, the compartment of CSC, and tumor progression.     

The Chemokine CCL5 Regulates Glucose Uptake and AMP Kinase Signaling in Activated T Cells to Facilitate Chemotaxis

Recruitment of effector T cells to sites of infection or inflammation is essential for an effective adaptive immune response. The chemokine CCL5 (RANTES) activates its cognate receptor, CCR5, to initiate cellular functions, including chemotaxis. In earlier studies, we reported that CCL5-induced CCR5 signaling activates the mTOR/4E-BP1 pathway to directly modulate mRNA translation. Specifically, CCL5-mediated mTOR activation contributes to T cell chemotaxis by initiating the synthesis of chemotaxis-related proteins. Up-regulation of chemotaxis-related proteins may prime T cells for efficient migration. It is now clear that mTOR is also a central regulator of nutrient sensing and glycolysis. Herein we describe a role for CCL5-mediated glucose uptake and ATP accumulation to meet the energy demands of chemotaxis in activated T cells. We provide evidence that CCL5 is able to induce glucose uptake in an mTOR-dependent manner. CCL5 treatment of ex vivo activated human CD3(+) T cells also induced the activation of the nutrient-sensing kinase AMPK and downstream substrates ACC-1, PFKFB-2, and GSK-3β. Using 2-deoxy-d-glucose, an inhibitor of glucose uptake, and compound C, an inhibitor of AMPK, experimental data are presented that demonstrate that CCL5-mediated T cell chemotaxis is dependent on glucose, as these inhibitors inhibit CCL5-mediated chemotaxis in a dose-dependent manner. Altogether, these findings suggest that both glycolysis and AMPK signaling are required for efficient T cell migration in response to CCL5. These studies extend the role of CCL5 mediated CCR5 signaling beyond lymphocyte chemotaxis and demonstrate a role for chemokines in promoting glucose uptake and ATP production to match energy demands of migration.     

Apela Regulates Fluid Homeostasis by Binding to the APJ Receptor to Activate Gi Signaling

Apela (APJ early endogenous ligand, also known as elabela or toddler) is a recently discovered peptide hormone. Based on genetic studies in zebrafish, apela was found to be important for endoderm differentiation and heart development during embryogenesis. Although common phenotypes of apela and APJ-null zebrafish during embryonic development suggested that apela interacts with the APJ receptor, kinetics of apela binding to APJ and intracellular signaling pathways for apela remain unknown. The role of apela in adults is also uncertain. Using a chimeric apela ligand, we showed direct binding of apela to APJ with high affinity (K_d = 0.51 nm) and the ability of apelin, the known peptide ligand for APJ, to compete for apela binding. Apela, similar to apelin, acts through the inhibitory G protein pathway by inhibiting forskolin-stimulated cAMP production and by inducing ERK1/2 phosphorylation. In adult rats, apela is expressed exclusively in the kidney, unlike the wide tissue distribution of apelin. In vivo studies demonstrated the ability of apela to regulate fluid homeostasis by increasing diuresis and water intake. Dose-response studies further indicated that apela induces 2- and 5-fold higher maximal responses than apelin in ERK1/2 phosphorylation and diuresis/water intake, respectively. After designing an apela antagonist, we further demonstrated the role of endogenous ligand(s) in regulating APJ-mediated fluid homeostasis. Our results identified apela as a potent peptide hormone capable of regulating fluid homeostasis in adult kidney through coupling to the APJ-mediated G_i signaling pathway.     

The Roles of Multiple UNC-40 (DCC) Receptor-Mediated Signals in Determining Neuronal Asymmetry Induced by the UNC-6 (Netrin) Ligand

The polarization of post-mitotic neurons is poorly understood. Preexisting spatially asymmetric cues, distributed within the neuron or as extracellular gradients, could be required for neurons to polarize. Alternatively, neurons might have the intrinsic ability to polarize without any preestablished asymmetric cues. In Caenorhabditis elegans, the UNC-40 (DCC) receptor mediates responses to the extracellular UNC-6 (netrin) guidance cue. For the HSN neuron, an UNC-6 ventral-dorsal gradient asymmetrically localizes UNC-40 to the ventral HSN surface. There an axon forms, which is ventrally directed by UNC-6. In the absence of UNC-6, UNC-40 is equally distributed and the HSN axon travels anteriorly in response to other cues. However, we find that a single amino acid change in the UNC-40 ectodomain causes randomly oriented asymmetric UNC-40 localization and a wandering axon phenotype. With UNC-6, there is normal UNC-40 localization and axon migration. A single UNC-6 amino acid substitution enhances the mutant phenotypes, whereas UNC-6 second-site amino acid substitutions suppress the phenotypes. We propose that UNC-40 mediates multiple signals to polarize and orient asymmetry. One signal triggers the intrinsic ability of HSN to polarize and causes randomly oriented asymmetry. Concurrently, another signal biases the orientation of the asymmetry relative to the UNC-6 gradient. The UNC-40 ectodomain mutation activates the polarization signal, whereas different forms of the UNC-6 ligand produce UNC-40 conformational changes that allow or prohibit the orientation signal.A major challenge for developmental neuroscience has been to understand how axons are able to detect and follow molecular gradients of different extracellular guidance cues. Attractive guidance cues are proposed to stimulate cytoplasmic signaling pathways that promote actin polymerization (Huber et al. 2003). Thus the direction of axon outgrowth is directly linked to the extracellular gradient of the guidance cue; i.e., there is greater extension on the side of the neuron that is closest to the source of the cue. Netrins are bifunctional guidance cues that are attractive to some axons but repulsive to others. Studies have shown that the axon response to netrin is determined by the composition of netrin receptors on the cell surface and the internal state of the growth cone (Round and Stein 2007). The UNC-6 (netrin) guidance cue in Caenorhabditis elegans interacts with the UNC-40 (DCC) receptor to mediate attraction (Hedgecock et al. 1990; Ishii et al. 1992; Chan et al. 1996). The AVM and HSN neurons are useful for studying UNC-40-mediated responses to UNC-6. The cell bodies of these neurons are situated on the lateral body wall and send a single axon ventrally during larval development.In AVM and HSN, a signaling module comprising UNC-6, UNC-40, phosphoinositide 3-kinase (PI3K), Rac, and MIG-10 (lamellipodin) is thought to transmit the directional information provided by the graded distribution of extracellular guidance cues to the internal cellular machinery that promotes directed outgrowth (Adler et al. 2006; Chang et al. 2006; Quinn et al. 2006, 2008). MIG-10 appears to provide an important link because this family of proteins can interact with proteins that promote actin polymerization, and it is associated with asymmetric concentrations of f-actin and microtubules in turning growth cones (Krause et al. 2004; Quinn et al. 2008). MIG-10 is observed as asymmetrically localized to the ventral site of axon outgrowth in developing HSN neurons. This MIG-10 localization is sensitive to the source of UNC-6. Normally, the source of UNC-6 is ventral; in the absence of UNC-6, there is an equal distribution of MIG-10 along the cell surface, whereas ectopic UNC-6 expression from dorsal muscles causes dorsal MIG-10 localization (Adler et al. 2006). The UNC-40 receptor is also asymmetrically localized in HSN, and this localization is also dependent on UNC-6 (Adler et al. 2006). UNC-40 signaling activates Rac GTPase, and MIG-10 interacts specifically with the activated Rac (Quinn et al. 2008). Therefore, the asymmetric activation of Rac through UNC-40 recruits asymmetric MIG-10 localization.By activating or directing components to the surface nearest the UNC-6 source, the asymmetric distribution of UNC-6 could polarize the neuron. However, an alternative idea is suggested from studies of chemotaxing cells. This model predicts that chemoattractant signaling involves two different elements: one that activates the intrinsic ability of cells to generate asymmetry and another that biases the orientation of the asymmetry (Wedlich-Soldner and Li 2003). The polarization signal does not depend on the spatial information provided by the chemoattractant gradient, whereas the orientation signal does. The asymmetric localization of the UNC-40 and MIG-10 signaling complex is suggestive of the segregation of signaling components into separate “front” and “rear” regions during chemotactic cell migration (Weiner 2002; Mortimer et al. 2008). It is hypothesized that this segregation is accomplished through short-range positive feedback mechanisms that promote the local production or recruitment of signaling molecules. In addition, a long-range inhibition mechanism globally increases the degradation of these molecules. Together such mechanisms could strongly amplify the asymmetric distribution of molecules needed for directed movement. This model has been put forth to explain why chemotactic cells polarize and move in a random direction when encountering a uniform chemoattractant concentration. Although the chemoattractant receptors may be uniformly stimulated across the surface of the cells, randomly oriented asymmetry can be established through these mechanisms.If the AVM and HSN neurons behave similarly to chemotactic cells, then uniformly stimulating UNC-40 receptors might similarly cause nonspecific asymmetric UNC-40 localization and axon migrations in varying directions. However, this is difficult to test in vivo. Unlike exposing chemotactic cells to a uniform concentration of a chemotractant in vitro, there is no reliable way to ensure that a neuron in vivo is exposed to a uniform concentration of UNC-6. The pseudocoelomic cavity of C. elegans is fluid filled, and UNC-6 expression patterns are spatially and temporally complex (Wadsworth et al. 1996). How the distribution of UNC-6 is affected by interactions with the extracellular matrix and cell surfaces is unknown.Using a genetic approach, we have found an UNC-40 mutation that triggers randomly oriented neuronal asymmetry. On the basis of the models proposed for chemotactic cells, we suggest that there is an UNC-6/UNC-40-mediated signal that specifically induces the neuron''s intrinsic ability to polarize. The UNC-40 mutation activates this signal; however, a second signal, which normally would concurrently orient asymmetry relative to the UNC-6 gradient, is not activated. Single amino acid changes within the UNC-6 ligand can enhance or suppress the randomly oriented asymmetry phenotype caused by the UNC-40 mutation. This suggests that specific UNC-40 conformations uncouple the activation of the different signals.     

Casein Kinase 2 Dependent Phosphorylation of Neprilysin Regulates Receptor Tyrosine Kinase Signaling to Akt

Neprilysin (NEP) is a type II membrane metalloproteinase that cleaves physiologically active peptides at the cell surface thus regulating the local concentration of these peptides available for receptor binding and signal transduction. In addition, the cytoplasmic N-terminal domain of NEP interacts with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) thereby regulating intracellular signaling via Akt. Thus, NEP serves dual functions in extracellular and intracellular signal transduction. Here, we show that NEP undergoes phosphorylation at serine residue 6 within the N-terminal cytoplasmic domain. In vitro and cell culture experiments demonstrate that Ser 6 is efficiently phosphorylated by protein kinase CK2. The phosphorylation of the cytoplasmic domain of NEP inhibits its interaction with PTEN. Interestingly, expression of a pseudophosphorylated NEP variant (Ser6Asp) abrogates the inhibitory effect of NEP on insulin/insulin-like growth factor-1 (IGF-1) stimulated activation of Akt. Thus, our data demonstrate a regulatory role of CK2 in the interaction of NEP with PTEN and insulin/IGF-1 signaling.