Wnt5a Potentiates U46619-Induced platelet aggregation <Emphasis Type="Italic">via</Emphasis> the PI3K/Akt pathway

Platelet aggregation plays crucial roles in the formation of hemostatic plugs and thrombosis. Although it was recently shown that canonical Wnt signaling negatively regulates platelet aggregation, the role of non-canonical Wnt signaling remains unknown. Here, we observed that Wnt5a, one of the non-canonical Wnts, positively regulated platelet aggregation. Platelet aggregation was potentiated by the addition of Wnt5a to collagen-or U46619-induced rat platelet rich plasma (PRP). Treatment with Wnt5a to U46619-stimulated PRP resulted in an increase in the level of phosphorylated Akt, whereas phosphorylation of PKCδ and JNK1 was unaffected. In addition, inhibition of PI3K blocked the potentiating effect of Wnt5a. Taken together, these results suggest that Wnt5a potentiates U46619-induced platelet aggregation via the PI3K/Akt pathway.     

Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8

Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.     

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.     

Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation

Wnts are secreted signaling molecules that can transduce their signals through several different pathways. Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems. We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin. This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent. Furthermore, we provide evidence that Wnt-5a also acts in vivo to promote beta-catenin degradation in regulating mammalian limb development and possibly in suppressing tumor formation.     

Mitochondrial dynamics regulate melanogenesis through proteasomal degradation of MITF via ROS‐ERK activation

Mitochondrial dynamics control mitochondrial functions as well as their morphology. However, the role of mitochondrial dynamics in melanogenesis is largely unknown. Here, we show that mitochondrial dynamics regulate melanogenesis by modulating the ROS‐ERK signaling pathway. Genetic and chemical inhibition of Drp1, a mitochondrial fission protein, increased melanin production and mitochondrial elongation in melanocytes and melanoma cells. In contrast, down‐regulation of OPA1, a mitochondria fusion regulator, suppressed melanogensis but induced massive mitochondrial fragmentation in hyperpigmented cells. Consistently, treatment with CCCP, a mitochondrial fission chemical inducer, also efficiently repressed melanogenesis. Furthermore, we found that ROS production and ERK phosphorylation were increased in cells with fragmented mitochondria. And inhibition of ROS or ERK suppressed the antimelanogenic effect of mitochondrial fission in α‐MSH‐treated cells. In addition, the activation of ROS‐ERK pathway by mitochondrial fission induced phosphorylation of serine73 on MITF accelerating its proteasomal degradation. In conclusion, mitochondrial dynamics may regulate melanogenesis by modulating ROS‐ERK signaling pathway.     

c-Jun N-terminal kinase (JNK) cooperates with Gsk3β to regulate Dishevelled-mediated microtubule stability

Background  

Wnt factors are a large family of signaling molecules that play important roles in the regulation of cell fate specification, tissue polarity and cell movement. In the nervous system, Wnts also regulates the formation of neuronal connection acting as retrograde signals that regulate the remodeling of the axons prior to the assembly of the presynaptic apparatus. The scaffold protein Dishevelled (Dvl) mimics the effect of Wnt on the neuronal cytoskeleton by increasing the number of stable microtubule along the axon shaft and inducing the formation of looped microtubules (MT) at enlarged growth cones. A divergent Wnt-Dvl canonical pathway which bifurcates downstream of Gsk3β regulates MT dynamics.     

Mesd is a general inhibitor of different Wnt ligands in Wnt/LRP signaling and inhibits PC-3 tumor growth in vivo

Mesd is a specialized chaperone for Wnt co-receptor low-density lipoprotein receptor-related protein-5 (LRP5) and LRP6, which contain four β-propeller/epidermal growth factor modules, named E1 to E4 from N- to C-terminal, in their extracellular domains. Herein, we demonstrated that recombinant Mesd protein is a general Wnt inhibitor that blocks Wnt/β-catenin signaling induced not only by LRP6 E1-E2-binding Wnts but also by LRP6 E3-E4-binding Wnts. We also found that Mesd suppressed Wnt/β-catenin signaling induced by Wnt1 in prostate cancer PC-3 cells, and inhibited tumor growth in PC-3 xenograft model. Our results indicate that Mesd is a universal inhibitor of Wnt/LRP signaling on the cell surface.     

FHL2 anchors mitochondria to actin and adapts mitochondrial dynamics to glucose supply

Mitochondrial movement and distribution are fundamental to their function. Here we report a mechanism that regulates mitochondrial movement by anchoring mitochondria to the F-actin cytoskeleton. This mechanism is activated by an increase in glucose influx and the consequent O-GlcNAcylation of TRAK (Milton), a component of the mitochondrial motor-adaptor complex. The protein four and a half LIM domains protein 2 (FHL2) serves as the anchor. FHL2 associates with O-GlcNAcylated TRAK and is both necessary and sufficient to drive the accumulation of F-actin around mitochondria and to arrest mitochondrial movement by anchoring to F-actin. Disruption of F-actin restores mitochondrial movement that had been arrested by either TRAK O-GlcNAcylation or forced direction of FHL2 to mitochondria. This pathway for mitochondrial immobilization is present in both neurons and non-neuronal cells and can thereby adapt mitochondrial dynamics to changes in glucose availability.     

The PINK1/Parkin pathway: a mitochondrial quality control system?

Significant insight into the mechanisms that contribute to dopaminergic neurodegeneration in Parkinson disease has been gained from the analysis of genes linked to rare heritable forms of parkinsonism such as PINK1 and parkin, loss-of-function mutations of which cause autosomal recessive parkinsonism. PINK1 encodes a mitochondrially targeted Ser/Thr kinase and parkin encodes a ubiquitin-protein ligase. Functional studies of PINK1 and Parkin in animal and cellular model systems have shown that both proteins play important roles in maintaining mitochondrial integrity. Genetic studies of PINK1 and Parkin orthologs in flies have shown that PINK1 acts upstream from Parkin in a common pathway that appears to regulate mitochondrial morphology. Mitochondrial morphology is regulated by mitochondrial fission and fusion-promoting proteins, and is important in a variety of contexts, including mitochondrial trafficking and mitochondrial quality control. In particular, mitochondrial fission appears to promote the segregation of terminally dysfunctional mitochondria for degradation in the lysosome through a process termed mitophagy. Recent work has shown that Parkin promotes the degradation of dysfunctional mitochondria in vertebrate cell culture. Here we postulate a model whereby the PINK1/Parkin pathway regulates mitochondrial dynamics in an effort to promote the turnover of damaged mitochondria.     

Wnt Trafficking: New Insights into Wnt Maturation,Secretion and Spreading

Proteins of the Wnt family are secreted signaling molecules that regulate multiple processes in animal development and control tissue homeostasis in the adult. Wnts spread over considerable distances to regulate gene expression in cells located at distant sites. Paradoxically, Wnts are poorly mobile because of their posttranslational modification with lipids. Recent evidence suggests that several pathways exist that are capable of transforming hydrophobic, insoluble Wnts into long‐range signaling molecules. Furthermore, the discovery of Wntless as a protein specifically required for the secretion of Wnt suggests that Wnt trafficking through the secretory pathway is already under special scrutiny. Here, we review recent data on the molecular machinery that controls Wnt secretion and discuss how Wnts can be mobilized for long‐range signaling.     

Anti-apoptotic action of Wnt5a in dermal fibroblasts is mediated by the PKA signaling pathways

Wnts are secreted glycoproteins that control diverse biological processes, such as proliferation, differentiation, and apoptosis. We here found that Wnt5a inhibited apoptosis induced by serum deprivation in primary-cultured human dermal fibroblasts. Anti-apoptotic activity of Wnt5a was not inhibited by a dickkopf-1 (DKK), which blocks the canonical Wnt pathway. On the other hand, loss of function of protein kinase A (PKA), induced by treatment with PKA inhibitors, siRNA-mediated knocking down of endogenous PKA catalytic subunits, or enforced expression of dominant-negative PKA inhibited the Wnt5a anti-apoptotic activity, indicating the involvement of PKA in the Wnt5a anti-apoptotic activity. In agreement, phosphorylation levels of a cAMP response element binding protein (CREB), a representative downstream effector of PKA, the activation of which is known to lead to the pro-survival effects, was elevated by Wnt5a. In addition, Wnt5a increased the nuclear beta-catenin level and treatment with imatinib or ionomycin, either of which blocks the beta-catenin pathway, reduced the anti-apoptotic activity of Wnt5a, together suggesting the simultaneous involvement of the beta-catenin-mediated pathway in the Wnt5a anti-apoptotic activity. Based on another finding indicating that Wnt5a upregulated PKA-mediated phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) at serine 9 that caused inactivation of GSK-3beta and subsequently resulted in activation of the beta-catenin pathway, we have speculated that the Wnt5a anti-apoptotic activity may be partially mediated by PKA-mediated phosphorylation of GSK-3beta and subsequent activation of the beta-catenin pathway.     

Wnt signaling and forebrain development

Components of the Wnt signaling pathway are expressed in a tightly regulated and spatially specific manner during development of the forebrain, and Wnts are key regulators of regional forebrain identity. Wnt signaling from the cortical hem regulates the expansion and cell-type specification of the adjacent neuroepithelium and, in conjunction with Bmp, Fgf, and Shh signaling, controls dorsal-ventral forebrain patterning. Subsequently, Wnt signaling dynamically regulates the behavior of cortical progenitor cells, initially promoting the expansion of radial glia progenitor cells and later inducing neurogenesis by promoting terminal differentiation of intermediate progenitor cells. A role for Wnt signaling in cell-type specification has also been proposed.     

Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain

One of the earliest manifestations of anteroposterior pattering in the developing brain is the restricted expression of Six3 and Irx3 in the anterior and posterior forebrain, respectively. Consistent with the role of Wnts as posteriorizing agents in neural tissue, we found that Wnt signaling was sufficient to induce Irx3 and repress Six3 expression in forebrain explants. The position of the zona limitans intrathalamica (zli), a boundary-cell population that develops between the ventral (vT) and dorsal thalamus (dT), is predicted by the apposition of Six3 and Irx3 expression domains. The expression patterns of several inductive molecules are limited by the zli, including Wnt3, which is expressed posterior to the zli in the dT. Wnt3 and Wnt3a were sufficient to induce the dT marker Gbx2 exclusively in explants isolated posterior to the presumptive zli. Blocking the Wnt response allowed the induction of the vT-specific marker Dlx2 in prospective dT tissue. Misexpression of Six3 in the dT induced Dlx2 expression and inhibited the expression of both Gbx2 and Wnt3. These results demonstrate a dual role for Wnt signaling in forebrain development. First, Wnts directed the initial expression of Irx3 and repression of Six3 in the forebrain, delineating posterior and anterior forebrain domains. Later, continued Wnt signaling resulted in the induction of dT specific markers, but only in tissues that expressed Irx3.     

Mitochondrial quality control by the ubiquitin-proteasome system

Mitochondria perform multiple functions critical to the maintenance of cellular homoeostasis and their dysfunction leads to disease. Several lines of evidence suggest the presence of a MAD (mitochondria-associated degradation) pathway that regulates mitochondrial protein quality control. Internal mitochondrial proteins may be retrotranslocated to the OMM (outer mitochondrial membrane), multiple E3 ubiquitin ligases reside at the OMM and inhibition of the proteasome causes accumulation of ubiquitinated proteins at the OMM. Reminiscent of ERAD [ER (endoplasmic reticulum)-associated degradation], Cdc48 (cell division cycle 42)/p97 is recruited to stressed mitochondria, extracts ubiquitinated proteins from the OMM and presents ubiquitinated proteins to the proteasome for degradation. Recent research has provided mechanistic insights into the interaction of the UPS (ubiquitin-proteasome system) with the OMM. In yeast, Vms1 [VCP (valosin-containing protein) (p97)/Cdc48-associated mitochondrial-stress-responsive 1] protein recruits Cdc48/p97 to the OMM. In mammalian systems, the E3 ubiquitin ligase parkin regulates the recruitment of Cdc48/p97 to mitochondria, subsequent mitochondrial protein degradation and mitochondrial autophagy. Disruption of the Vms1 or parkin systems results in the hyper-accumulation of ubiquitinated proteins at mitochondria and subsequent mitochondrial dysfunction. The emerging MAD pathway is important for the maintenance of cellular and therefore organismal viability.     

The long and the short of Wnt signaling in C. elegans

The simplicity of C. elegans makes it an outstanding system to study the role of Wnt signaling in development. Many asymmetric cell divisions in C. elegans require the Wnt/beta-catenin asymmetry pathway. Recent studies confirm that SYS-1 is a structurally and functionally divergent beta-catenin, and implicate lipids and retrograde trafficking in maintenance of WRM-1/beta-catenin asymmetry. Wnts also regulate short-range events such as spindle rotation and gastrulation, and a PCP-like pathway regulates asymmetric divisions. Long-range, cell non-autonomous Wnt signals regulate vulval induction. Both short-range and long-range Wnt signal s are regulated by recycling of MIG-14/Wntless via the retromer complex. These studies indicate that C. elegans continues to be useful for identifying new, conserved mechanisms underlying Wnt signaling in metazoans.