Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis

Chondrocytes and osteoblasts are two primary cell types in the skeletal system that are differentiated from common mesenchymal progenitors. It is believed that osteoblast differentiation is controlled by distinct mechanisms in intramembranous and endochondral ossification. We have found that ectopic canonical Wnt signaling leads to enhanced ossification and suppression of chondrocyte formation. Conversely, genetic inactivation of beta-catenin, an essential component transducing the canonical Wnt signaling, causes ectopic formation of chondrocytes at the expense of osteoblast differentiation during both intramembranous and endochondral ossification. Moreover, inactivation of beta-catenin in mesenchymal progenitor cells in vitro causes chondrocyte differentiation under conditions allowing only osteoblasts to form. Our results demonstrate that beta-catenin is essential in determining whether mesenchymal progenitors will become osteoblasts or chondrocytes regardless of regional locations or ossification mechanisms. Controlling Wnt/beta-catenin signaling is a common molecular mechanism underlying chondrocyte and osteoblast differentiation and specification of intramembranous and endochondral ossification.     

A Wnt1-regulated genetic network controls the identity and fate of midbrain-dopaminergic progenitors in vivo

Midbrain neurons synthesizing the neurotransmitter dopamine play a central role in the modulation of different brain functions and are associated with major neurological and psychiatric disorders. Despite the importance of these cells, the molecular mechanisms controlling their development are still poorly understood. The secreted glycoprotein Wnt1 is expressed in close vicinity to developing midbrain dopaminergic neurons. Here, we show that Wnt1 regulates the genetic network, including Otx2 and Nkx2-2, that is required for the establishment of the midbrain dopaminergic progenitor domain during embryonic development. In addition, Wnt1 is required for the terminal differentiation of midbrain dopaminergic neurons at later stages of embryogenesis. These results identify Wnt1 as a key molecule in the development of midbrain dopaminergic neurons in vivo. They also suggest the Wnt1-controlled signaling pathway as a promising target for new therapeutic strategies in the treatment of Parkinson's disease.     

Wnt signalling requires MTM-6 and MTM-9 myotubularin lipid-phosphatase function in Wnt-producing cells

Wnt proteins are lipid-modified glycoproteins that have important roles in development, adult tissue homeostasis and disease. Secretion of Wnt proteins from producing cells is mediated by the Wnt-binding protein MIG-14/Wls, which binds Wnt in the Golgi network and transports it to the cell surface for release. It has recently been shown that recycling of MIG-14/Wls from the plasma membrane to the trans-Golgi network is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is still poorly understood. In this study, we report the identification of MTM-6 and MTM-9 as novel regulators of MIG-14/Wls trafficking in Caenorhabditis elegans. MTM-6 and MTM-9 are myotubularin lipid phosphatases that function as a complex to dephosphorylate phosphatidylinositol-3-phosphate, a central regulator of endosomal trafficking. We show that mutation of mtm-6 or mtm-9 leads to defects in several Wnt-dependent processes and demonstrate that MTM-6 is required in Wnt-producing cells as part of the MIG-14/Wls-recycling pathway. This function is evolutionarily conserved, as the MTM-6 orthologue DMtm6 is required for Wls stability and Wg secretion in Drosophila. We conclude that regulation of endosomal trafficking by the MTM-6/MTM-9 myotubularin complex is required for the retromer-dependent recycling of MIG-14/Wls and Wnt secretion.     

Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes

Canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity, but the molecular mechanism by which this is achieved remains unclear. Here, we report that Wnt signaling triggers the sequestration of GSK3 from the cytosol into multivesicular bodies (MVBs), so that this enzyme becomes separated from its many cytosolic substrates. Endocytosed Wnt colocalized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition, endogenous GSK3 activity decreased in the cytosol, and GSK3 became protected from protease treatment inside membrane-bounded organelles. Cryoimmunoelectron microscopy showed that these corresponded to MVBs. Two proteins essential for MVB formation, HRS/Vps27 and Vps4, were required for Wnt signaling. The sequestration of GSK3 extended the half-life of many other proteins in addition to β-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3 phosphorylation sites. We conclude that multivesicular endosomes are essential components of the Wnt signal-transduction pathway.     

The p110delta isoform of PI 3-kinase negatively controls RhoA and PTEN

Inactivation of PI 3-kinase (PI3K) signalling is critical for tumour suppression by PTEN. This is thought to be a unidirectional relationship in which PTEN degrades the lipids produced by PI3K, thus controlling cell proliferation, survival and migration. We now show that this relationship is in fact bidirectional, whereby PI3K reciprocally controls PTEN. We report that the p110delta PI3K negatively regulates PTEN, through a pathway involving inhibition of RhoA. Inactivation of p110delta in macrophages led to reduced Akt and Rac1 activation, but paradoxically to increased RhoA and PTEN activity. Partial inactivation of p190RhoGAP and a reduced binding of cytoplasmic RhoA to the cyclin-dependent kinase inhibitor p27 both contributed to the increased RhoA-GTP levels upon p110delta inactivation. Pharmacological inhibition of ROCK, a downstream effector kinase of RhoA, restored all signalling and functional defects of p110delta inactivation, including Akt phosphorylation, chemotaxis and proliferation. This work identifies the RhoA/ROCK pathway as a major target of p110delta-mediated PI3K signalling, and establishes for the first time that PI3K controls itself, via a feedback loop involving PTEN.     

Shifted, the Drosophila ortholog of Wnt inhibitory factor-1, controls the distribution and movement of Hedgehog

We here identify and characterize an extracellular modulator of Hedgehog signaling in Drosophila, Shifted. Shifted is required for high levels of long-range signaling in the developing wing imaginal disc. Surprisingly, shifted encodes the only Drosophila ortholog of the secreted vertebrate protein Wnt Inhibitory Factor-1 (WIF-1), whose known role is to bind to extracellular Wnts and inhibit their activity. However, Shifted does not regulate Hedgehog signaling by affecting Wingless or Wnt signaling. We show instead that Shifted is a secreted protein that acts over a long distance and is required for the normal accumulation of Hh protein and its movement in the wing. Our data further indicate that Shf interacts with Hh and the heparan sulfate proteoglycans. Therefore, we propose that Shf stabilizes the interaction between Hh and the proteoglycans, an unexpected role for a member of the WIF-1 family.     

Epithelial sheet movement requires the cooperation of c-Jun and MAP3K1

Epithelial sheet movement is an essential morphogenetic process during mouse embryonic eyelid closure in which Mitogen-Activated Protein 3 Kinase 1 (MAP3K1) and c-Jun play a critical role. Here we show that MAP3K1 associates with the cytoskeleton, activates Jun N-terminal kinase (JNK) and actin polymerization, and promotes the eyelid inferior epithelial cell elongation and epithelium protrusion. Following epithelium protrusion, c-Jun begins to express and acts to promote ERK phosphorylation and migration of the protruding epithelial cells. Homozygous deletion of either gene causes defective eyelid closure, but non-allelic non-complementation does not occur between Map3k1 and c-Jun and the double heterozygotes have normal eyelid closure. Results from this study suggest that MAP3K1 and c-Jun signal through distinct temporal-spatial pathways and that productive epithelium movement for eyelid closure requires the consecutive action of MAP3K1-dependent cytoskeleton reorganization followed by c-Jun-mediated migration.     

Contrasting responses of lymphoid progenitors to canonical and noncanonical Wnt signals

The Wnt family of secreted glycoproteins has been implicated in many aspects of development, but its contribution to blood cell formation is controversial. We overexpressed Wnt3a, Wnt5a, and Dickkopf 1 in stromal cells from osteopetrotic mice and used them in coculture experiments with highly enriched stem and progenitor cells. The objective was to learn whether and how particular stages of B lymphopoiesis are responsive to these Wnt family ligands. We found that canonical Wnt signaling, through Wnt3a, inhibited B and plasmacytoid dendritic cell, but not conventional dendritic cell development. Wnt5a, which can oppose canonical signaling or act through a different pathway, increased B lymphopoiesis. Responsiveness to both Wnt ligands diminished with time in culture and stage of development. That is, only hematopoietic stem cells and very primitive progenitors were affected. Although Wnt3a promoted retention of hematopoietic stem cell markers, cell yields and dye dilution experiments indicated it was not a growth stimulus. Other results suggest that lineage instability results from canonical Wnt signaling. Lymphoid progenitors rapidly down-regulated RAG-1, and some acquired stem cell-staining characteristics as well as myeloid and erythroid potential when exposed to Wnt3a-producing stromal cells. We conclude that at least two Wnt ligands can differentially regulate early events in B lymphopoiesis, affecting entry and progression in distinct differentiation lineages.     

SNX3 controls Wingless/Wnt secretion through regulating retromer-dependent recycling of Wntless

Drosophila Wingless (Wg) acts as a morphogen during development. Wg secretion is controlled by a seven-pass transmembrane cargo Wntless (Wls). We have recently identified retromer as a key regulator involved in Wls trafficking. As sorting nexin (SNX) molecules are essential components of the retromer complex, we hypothesized that specific SNX(s) is required for retromer-mediated Wnt secretion. Here, we generated Drosophila mutants for all of the eight snx members, and identified Drosophila SNX3 (DSNX3) as an essential molecule required for Wg secretion. We show that Wg secretion and its signaling activity are defective in Dsnx3 mutant clones in wing discs. Wg levels in the culture medium of Dsnx3-depleted S2 cells are also markedly reduced. Importantly, Wls levels are strikingly reduced in Dsnx3 mutant cells, and overexpression of Wls can rescue the Wg secretion defect observed in Dsnx3 mutant cells. Moreover, DSNX3 can interact with the retromer component Vps35, and co-localize with Vps35 in early endosomes. These data indicate that DSNX3 regulates Wg secretion via retromer-dependent Wls recycling. In contrast, we found that Wg secretion is not defective in cells mutant for Drosophila snx1 and snx6, two components of the classical retromer complex. Ectopic expression of DSNX1 or DSNX6 fails to rescue the Wg secretion defect in Dsnx3 mutant wing discs and in Dsnx3 dsRNA-treated S2 cells. These data demonstrate the specificity of the DSNX3-retromer complex in Wls recycling. Together, our findings suggest that DSNX3 acts as a cargo-specific component of retromer, which is required for endocytic recycling of Wls and Wg/Wnt secretion.     

Impaired cardiac function and IGF-I response in myocytes from calmodulin-diabetic mice: role of Akt and RhoA

This study characterized the cardiac contractile function and IGF-I response in a transgenic diabetic mouse model. Mechanical properties were evaluated in cardiac myocytes from OVE26 diabetic and FVB wild-type mice, including peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR(90)) and maximal velocity of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) was evaluated as Ca(2+)-induced Ca(2+) release [difference in fura 2 fluorescent intensity (Delta FFI)] and fluorescence decay rate (tau). Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a, phospholamban (PLB), Na(+)-Ca(2+) exchanger (NCX), GLUT4, and the serine-threonine kinase Akt were assessed by Western blot. RhoA and IGF-I/IGF-I receptor mRNA levels were determined by RT-PCR and Northern blot. OVE26 myocytes displayed decreased PS, +/-dL/dt, and Delta FFI associated with prolonged TPS, TR(90), and tau. SERCA2a, NCX, and Akt activation were reduced, whereas PLB and RhoA were enhanced in OVE26 hearts. GLUT4 was unchanged. IGF-I enhanced PS and Delta FFI in FVB but not OVE26 myocytes. IGF-I mRNA was increased, but IGF-I receptor mRNA was reduced in OVE26 hearts and livers. These results validate diabetic cardiomyopathy in OVE26 mice due to reduced SERCA2, NCX, IGF-I response, and Akt activation associated with enhanced RhoA level, suggesting a therapeutic potential for Akt and RhoA.     

Quantitative phosphoproteome profiling of Wnt3a-mediated signaling network: indicating the involvement of ribonucleoside-diphosphate reductase M2 subunit phosphorylation at residue serine 20 in canonical Wnt signal transduction

The complexity of canonical Wnt signaling comes not only from the numerous components but also from multiple post-translational modifications. Protein phosphorylation is one of the most common modifications that propagates signals from extracellular stimuli to downstream effectors. To investigate the global phosphorylation regulation and uncover novel phosphoproteins at the early stages of canonical Wnt signaling, HEK293 cells were metabolically labeled with two stable isotopic forms of lysine and were stimulated for 0, 1, or 30 min with purified Wnt3a. After phosphoprotein enrichment and LC-MS/MS analysis, 1057 proteins were identified in all three time points. In total 287 proteins showed a 1.5-fold or greater change in at least one time point. In addition to many known Wnt signaling transducers, other phosphoproteins were identified and quantitated, implicating their involvement in canonical Wnt signaling. k-Means clustering analysis showed dynamic patterns for the differential phosphoproteins. Profile pattern and interaction network analysis of the differential phosphoproteins implicated the possible roles for those unreported components in Wnt signaling. Moreover 100 unique phosphorylation sites were identified, and 54 of them were quantitated in the three time points. Site-specific phosphopeptide quantitation revealed that Ser-20 phosphorylation on RRM2 increased upon 30-min Wnt3a stimulation. Further studies with mutagenesis, the Wnt reporter gene assay, and RNA interference indicated that RRM2 functioned downstream of beta-catenin as an inhibitor of Wnt signaling and that Ser-20 phosphorylation of RRM2 counteracted its inhibition effect. Our systematic profiling of dynamic phosphorylation changes responding to Wnt3a stimulation not only presented a comprehensive phosphorylation network regulated by canonical Wnt signaling but also found novel molecules and phosphorylation involved in Wnt signaling.     

Interplay between Wnt2 and Wnt2bb controls multiple steps of early foregut-derived organ development

The vertebrate liver, pancreas and lung arise in close proximity from the multipotent foregut endoderm. Tissue-explant experiments uncovered instructive signals emanating from the neighbouring lateral plate mesoderm, directing the endoderm towards specific organ fates. This suggested that an intricate network of signals is required to control the specification and differentiation of each organ. Here, we show that sequential functions of Wnt2bb and Wnt2 control liver specification and proliferation in zebrafish. Their combined specific activities are essential for liver specification, as their loss of function causes liver agenesis. Conversely, excess wnt2bb or wnt2 induces ectopic liver tissue at the expense of pancreatic and anterior intestinal tissues, revealing the competence of intestinal endoderm to respond to hepatogenic signals. Epistasis experiments revealed that the receptor frizzled homolog 5 (fzd5) mediates part of the broader hepatic competence of the alimentary canal. fzd5 is required for early liver formation and interacts genetically with wnt2 as well as wnt2bb. In addition, lack of both ligands causes agenesis of the swim bladder, the structural homolog of the mammalian lung. Thus, tightly regulated spatiotemporal expression of wnt2bb, wnt2 and fzd5 is central to coordinating early liver, pancreas and swim bladder development from a multipotent foregut endoderm.     

Formation of AP-3 transport intermediates requires Vps41 function

Transport of a subset of membrane proteins to the yeast vacuole requires the function of the AP-3 adaptor protein complex. To define the molecular requirements of vesicular transport in this pathway, we used a biochemical approach to analyse the formation and content of the AP-3 transport intermediate. A vam3tsf (vacuolar t-SNARE) mutant blocks vesicle docking and fusion with the vacuole and causes the accumulation of 50-130-nanometre membrane vesicles, which we isolated and showed by biochemical analysis and immunocytochemistry to contain both AP-3 adaptors and alkaline phosphatase (ALP) pathway cargoes. Inactivation of AP-3 or the protein Vps41 blocks formation of this vesicular intermediate. Vps41 binds to the AP-3 delta-adaptin subunit, suggesting that they function together in the formation of ALP pathway transport intermediates at the late Golgi.     

Destruction complex function in the Wnt signaling pathway of Drosophila requires multiple interactions between Adenomatous polyposis coli 2 and Armadillo

The tumor suppressor Adenomatous polyposis coli (APC) negatively regulates Wnt signaling through its activity in the destruction complex. APC binds directly to the main effector of the pathway, β-catenin (βcat, Drosophila Armadillo), and helps to target it for degradation. In vitro studies demonstrated that a nonphosphorylated 20-amino-acid repeat (20R) of APC binds to βcat through the N-terminal extended region of a 20R. When phosphorylated, the phospho-region of an APC 20R also binds βcat and the affinity is significantly increased. These distinct APC-βcat interactions suggest different models for the sequential steps of destruction complex activity. However, the in vivo role of 20R phosphorylation and extended region interactions has not been rigorously tested. Here we investigated the functional role of these molecular interactions by making targeted mutations in Drosophila melanogaster APC2 that disrupt phosphorylation and extended region interactions and deletion mutants missing the Armadillo binding repeats. We tested the ability of these mutants to regulate Wnt signaling in APC2 null and in APC2 APC1 double-null embryos. Overall, our in vivo data support the role of phosphorylation and extended region interactions in APC2's destruction complex function, but suggest that the extended region plays a more significant functional role. Furthermore, we show that the Drosophila 20Rs with homology to the vertebrate APC repeats that have the highest affinity for βcat are functionally dispensable, contrary to biochemical predictions. Finally, for some mutants, destruction complex function was dependent on APC1, suggesting that APC2 and APC1 may act cooperatively in the destruction complex.     

Convergence of Wnt and Notch signaling controls ovarian cancer cell survival

In the last 40 years ovarian cancer mortality rates have slightly declined and, consequently, it continues to be the fifth cause of cancer death in women. In the present study, we showed that β-catenin signaling is involved in the functions of ovarian cancer cells and interacts with the Notch system. Wnt and Notch systems showed to be prosurvival for ovarian cancer cells and their inhibition impaired cell proliferation and migration. We also demonstrated that the inhibition of β-catenin by means of two molecules, XAV939 and ICG-001, decreased the proliferation of the IGROV1 and SKOV3 ovarian cancer cell lines and that ICG-001 increased the percentage of IGROV1 cells undergoing apoptosis. The simultaneous inhibition of β-catenin and Notch signaling, by using the DAPT inhibitor, decreased ovarian cancer cell proliferation to the same extent as targeting only the Wnt/β-catenin pathway. A similar effect was observed in IGROV1 cell migration with ICG-001 and DAPT. ICG-001 increased the Notch target genes Hes-1 and Hey-1 and increased Jagged1 expression. However, no changes were observed in Dll4 or Notch 1 and 4 expressions. Our results suggest that Notch and β-catenin signaling co-operate in ovarian cancer to ensure the proliferation and migration of cells and that this could be achieved, at least partly, by the upregulation of Notch Jagged1 ligand in the absence of Wnt signaling. We showed that the Wnt pathway crosstalks with Notch in ovarian cancer cell functions, which may have implications in ovarian cancer therapeutics.     

Contribution of marrow-derived progenitors to vascular and cardiac regeneration

Adult bone marrow is a rich reservoir of tissue-specific pluripotent stem and progenitor cells. Accumulating evidence suggest that these cells have the potential of contributing to tissue revascularization and cardiac regeneration. Physiological stress results in the release of specific chemokines and cytokines that promote mobilization of stem cells to the peripheral circulation. Incorporation of these mobilized cells contributes to formation of functional vasculature and sets up stage for tissue regeneration. Vascular Endothelial Growth Factor (VEGF) through interaction with its receptors VEGFR2 and VEGFR1 expressed on endothelial and hematopoietic stem cells promote recruitment of these cells into the sites of tissue injury accelerating vascular healing. Similarly, subset of CD34 + marrow derived cells are mobilized and recruited to the ischemic myocardium, differentiating into cardiac and vascular cells, restoring cardiac function. Identification of cellular mediators and tissue specific chemocytokines that facilitate selective recruitment of marrow-derived stem and progenitor cells to specific organs, will open up new avenues to accelerate cardiovascular regeneration and tissue revascularization.     

SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding

The salt tolerance gene SOS3 (for salt overly sensitive3) of Arabidopsis is predicted to encode a calcium binding protein with an N-myristoylation signature sequence. Here, we examine the myristoylation and calcium binding properties of SOS3 and their functional significance in plant tolerance to salt. Treatment of young Arabidopsis seedlings with the myristoylation inhibitor 2-hydroxymyristic acid caused the swelling of root tips, mimicking the phenotype of the salt-hypersensitive mutant sos3-1. In vitro translation assays with reticulocyte showed that the SOS3 protein was myristoylated. Targeted mutagenesis of the N-terminal glycine-2 to alanine prevented the myristoylation of SOS3. The functional significance of SOS3 myristoylation was examined by expressing the wild-type myristoylated SOS3 and the mutated nonmyristoylated SOS3 in the sos3-1 mutant. Expression of the myristoylated but not the nonmyristoylated SOS3 complemented the salt-hypersensitive phenotype of sos3-1 plants. No significant difference in membrane association was observed between the myristoylated and nonmyristoylated SOS3. Gel mobility shift and (45)Ca(2)+ overlay assays demonstrated that SOS3 is a unique calcium binding protein and that the sos3-1 mutation substantially reduced the capacity of SOS3 to bind calcium. The resulting mutant SOS3 protein was not able to interact with the SOS2 protein kinase and was less capable of activating it. Together, these results strongly suggest that both N-myristoylation and calcium binding are required for SOS3 function in plant salt tolerance.