Identification of a novel crosstalk between casein kinase 2α and NPM-ALK in ALK-positive anaplastic large cell lymphoma

It was previously reported that β-catenin contributes to the tumorigenesis of ALK-positive anaplastic large cell lymphoma (ALK⁺ALCL), and the oncogenic effects of β-catenin in these tumors are promoted by NPM-ALK, an abnormal fusion protein characteristic of ALK⁺ALCL. In this study, we hypothesized that NPM-ALK promotes the oncogenic activity of β-catenin via its functional interactions with the Wnt canonical pathway (WCP). To test this hypothesis, we examined if NPM-ALK modulates the gene expression of various members in the WCP. Using a Wnt pathway-specific oligonucleotide array and Western blots, we found that the expression of casein kinase 2α (CK2α) was substantially downregulated in ALK⁺ALCL cells in response to siRNA knockdown of NPM-ALK. CK2α is biologically important in ALK⁺ALCL, as its inhibition using 4,5,6,7-tetrabromobenzotriazole or siRNA resulted in a significant decrease in cell growth and a substantial decrease in the β-catenin protein level. Furthermore, CK2α co-immunoprecipitated with NPM-ALK and regulated its level of serine phosphorylation, a feature previously shown to correlate with the oncogenic potential of this fusion protein. To conclude, this study has revealed a novel crosstalk between NPM-ALK and CK2α, and our data supports the model that these two molecules work synergistically to promote the tumorigenicity of these lymphomas.     

The NADPH oxidase NOX5 protects against apoptosis in ALK-positive anaplastic large-cell lymphoma cell lines

Reactive oxygen species (ROS) are key modulators of apoptosis and carcinogenesis. One of the important sources of ROS is NADPH oxidases (NOXs). The isoform NOX5 is highly expressed in lymphoid tissues, but it has not been detected in any common Hodgkin or non-Hodgkin lymphoma cell lines. In diverse, nonlymphoid malignant cells NOX5 exerts an antiapoptotic effect. Apoptosis suppression is the hallmark feature of a rare type of lymphoma, termed anaplastic lymphoma kinase-positive (ALK⁺) anaplastic large-cell lymphoma (ALCL), and a major factor in the therapy resistance and relapse of ALK⁺ ALCL tumors. We applied RT-PCR and Western blot analysis to detect NOX5 expression in three ALK⁺ ALCL cell lines (Karpas-299, SR-786, SUP-M2). We investigated the role of NOX5 in apoptosis by small-interfering RNA (siRNA)-mediated gene silencing and chemical inhibition of NOX5 using FACS analysis and examining caspase 3 cleavage in Karpas-299 cells. We used immunohistochemistry to detect NOX5 in ALK⁺ ALCL pediatric tumors. NOX5 mRNA was uniquely detected in ALK⁺ ALCL cells, whereas cell lines of other lymphoma classes were devoid of NOX5. Transfection of NOX5-specific siRNA and chemical inhibition of NOX5 abrogated calcium-induced superoxide production and increased caspase 3-mediated apoptosis in Karpas-299 cells. Immunohistochemistry revealed focal NOX5 reactivity in pediatric ALK⁺ ALCL tumor cells. These results indicate that NOX5-derived ROS contribute to apoptosis blockage in ALK⁺ ALCL cell lines and suggest NOX5 as a potential pharmaceutical target to enhance apoptosis and thus to suppress tumor progression and prevent relapse in pediatric ALK⁺ ALCL patients that resist classical therapeutic approaches.     

Involvement of Grb2 Adaptor Protein in Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK)-mediated Signaling and Anaplastic Large Cell Lymphoma Growth

Most anaplastic large cell lymphomas (ALCL) express oncogenic fusion proteins derived from chromosomal translocations or inversions of the anaplastic lymphoma kinase (ALK) gene. Frequently ALCL carry the t(2;5) translocation, which fuses the ALK gene to the nucleophosmin (NPM1) gene. The transforming activity mediated by NPM-ALK fusion induces different pathways that control proliferation and survival of lymphoma cells. Grb2 is an adaptor protein thought to play an important role in ALK-mediated transformation, but its interaction with NPM-ALK, as well as its function in regulating ALCL signaling pathways and cell growth, has never been elucidated. Here we show that active NPM-ALK, but not a kinase-dead mutant, bound and induced Grb2 phosphorylation in tyrosine 160. An intact SH3 domain at the C terminus of Grb2 was required for Tyr¹⁶⁰ phosphorylation. Furthermore, Grb2 did not bind to a single region but rather to different regions of NPM-ALK, mainly Tyr^152–156, Tyr⁵⁶⁷, and a proline-rich region, Pro^415–417. Finally, shRNA knockdown experiments showed that Grb2 regulates primarily the NPM-ALK-mediated phosphorylation of SHP2 and plays a key role in ALCL cell growth.     

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.     

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     

Studies of Phosphoproteomic Changes Induced by Nucleophosmin-Anaplastic Lymphoma Kinase (ALK) Highlight Deregulation of Tumor Necrosis Factor (TNF)/Fas/TNF-related Apoptosis-induced Ligand Signaling Pathway in ALK-positive Anaplastic Large Cell Lymphoma

The oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), found exclusively in a subset of ALK-positive anaplastic large cell lymphoma, promotes tumorigenesis by exerting its constitutively active tyrosine kinase activity. Thus, characterization of the NPM-ALK-induced changes in the phosphoproteome will likely provide insights into the biology of this oncoprotein. To achieve this goal, we used a strategy of combining sequential affinity purification of phosphopeptides and LC/MS. GP293 cells transfected with either NPM-ALK or an NPM-ALK mutant with decreased tyrosine kinase activity (negative control) were used. We identified 506 phosphoproteins detectable in NPM-ALK-expressing cells but not in the negative control. Bioinformatics analysis revealed that these phosphoproteins carry a wide diversity of biological functions, some of which have not been described in association with NPM-ALK, such as the tumor necrosis factor (TNF)/Fas/tumor necrosis factor-related apoptosis-induced ligand (TRAIL) signaling pathway and the ubiquitin proteasome degradation pathway. In particular, modulations of the TNF/Fas/TRAIL pathway by NPM-ALK were supported by our antibody microarray data. Further validation of the TNF/Fas/TRAIL pathway was performed in ALK⁺ anaplastic large cell lymphoma (ALCL) cell lines with knockdown of NPM-ALK using short interference RNA, resulting in the loss of the tyrosine phosphorylation of tumor necrosis factor receptor-associated protein 1 (TRAP1) and receptor-interacting protein 1, two crucial TNF signaling molecules. Functional analyses revealed that knockdown of TRAP1 facilitated cell death induced by TRAIL or doxorubicin in ALK⁺ ALCL cells. This suggests that down-regulation of TRAP1 in combination with TRAIL or doxorubicin might be a potential novel therapeutic strategy for ALK⁺ ALCL. These findings demonstrated that our strategy allowed the identification of novel proteins downstream of NPM-ALK that contribute to the maintenance of neoplastic phenotype and holds great potential for future studies of cellular tyrosine kinases in normal states and diseases.Anaplastic lymphoma kinase (ALK)¹-positive anaplastic large cell lymphoma (ALK⁺ ALCL) is a subtype of T/null cell non-Hodgkin lymphoma that is characterized by its consistent expression of CD30 and anaplastic cytology (1). ALK⁺ ALCL is relatively uncommon among adults, accounting for ∼3% of adult non-Hodgkin lymphoma, whereas it is frequent in children, representing 10–20% of all lymphoma (2). Approximately 80% of ALK⁺ ALCL tumors carry the t(2;5)(p23;q35) chromosomal translocation that fuses the tyrosine kinase domain of ALK with the oligomerization domain of the nucleophosmin (NPM) protein. This chromosomal translocation results in the formation of the chimeric protein NPM-ALK (3–7). NPM-ALK has been shown to have propensity to dimerize, and this process triggers autophosphorylation of NPM-ALK and results in constitutive activation of the ALK tyrosine kinase. Once activated, NPM-ALK binds, phosphorylates, and constitutively activates a host of proteins involved in various cellular signaling pathways, including those of PI3K/RAC-α serine/threonine-protein kinase (AKT) (8, 9), JAK/STAT (10), mTOR (11–13), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)/ERK (14), and Ras/MAPK (15).As a tyrosine kinase, major attention was drawn to NPM-ALK in identifying the tyrosine phosphorylation, whereas other aspects of this oncogenic protein were not fully explored. Two LC/MS studies were performed previously (16, 17). Using quantitative LC/MS, Boccalatte et al. (16) reported 47 phosphoproteins in a panel of ALK⁺ ALCL cell lines that showed significant changes in their phosphorylation status upon ALK inhibition by short hairpin RNA or a tyrosine kinase inhibitor. Using LC/MS, Rush et al. (17) recently found ∼70 phosphoproteins from each of the two ALK⁺ ALCL cell lines, although whether the phosphorylation of these proteins is related to NPM-ALK was not determined. In both studies, only phosphotyrosine-containing proteins were captured by using a phosphotyrosine antibody immobilized to a solid phase prior to LC/MS analysis.However, mounting evidence has shown that NPM-ALK-induced signaling can culminate in the activation of oncogenic pathways via regulating serine/threonine phosphorylation (e.g. mTOR, c-Jun, nuclear interacting partner of ALK (NIPA), and AKT) (13, 18, 19). It is highly possible that NPM-ALK induces serine/threonine phosphorylation of downstream targets through recruitment and activation of various serine/threonine kinases like previously reported JNK and ERK (12, 18). In this regard, we have reported that NPM-ALK physically interacts with multiple serine/threonine kinases, including ribosomal protein S6 kinase α-3, serine/threonine/tyrosine kinase 1 (STYK1), serine/threonine-protein kinase PRP4, and serine/threonine-protein kinase PCTAIRE-3 (20). Therefore, comprehensive characterization of phosphoproteomic changes (i.e. serine, threonine, and tyrosine phosphorylation) induced by NPM-ALK will likely further our understanding of the pathobiology of NPM-ALK.Although comprehensive characterization of phosphoproteomic changes induced by oncogenic tyrosine kinases (such as NPM-ALK) is highly useful, this approach is met with multiple technical challenges. It is intrinsically difficult to detect phosphoproteins because the phosphopeptides produced from the phosphoproteins are generally present at a much lower amount compared with their native counterparts. In an attempt to overcome this difficulty, several enrichment approaches that are designed to isolate phosphorylated amino acid residues have been described, and they include IMAC through metal complexation with the phosphate group (21–23), acid-based interaction with titanium oxide (24–28), and solution charge-based enrichment by strong cation exchange chromatography (23, 29). Specific capture of phosphopeptides is possible by elimination of the phosphate group and subsequent introduction of an affinity tag (30) or conversion to methyl ester derivatives (21, 31, 32). In addition, several groups have combined various ion exchange approaches with IMAC to further enrich phosphopeptides prior to LC/MS/MS analysis (33–35). These works and others reported were mainly focused on improving the enrichment of phosphopeptides to facilitate the detection of as many phosphopeptides as possible. However, because tyrosine residues are present at much lower concentrations than serine or threonine (i.e. serine/threonine/tyrosine ratio is 1800:200:1), important proteins carrying phosphotyrosine residues often escape detection and are under-represented in the final results. To address this issue, several research groups recently reported the utilization of anti-phosphotyrosine antibodies to specifically enrich the phosphotyrosine-containing peptides within the digest (16, 17, 36) or intact protein prior to digestion (32). The use of anti-phosphotyrosine antibody alone, as used in the two previous studies of NPM-ALK (16, 17), is prone to nonspecific proteins binding to the anti-phosphotyrosine antibodies, resulting in a high background that may prevent the detection of the relatively weak signals of phosphotyrosine-containing peptides. Specific capture of phosphotyrosine-containing peptides has been enhanced by phosphotyrosine protein enrichment followed by IMAC (37, 38). Recently, Heck and co-workers (39) have reported an optimized enrichment method for phosphotyrosine peptides based on immunoaffinity purification. This method allowed a high level of enrichment; they were able to identify 1112 unique phosphotyrosine peptides derived from 4 mg of starting materials. However, biologically important proteins carrying phosphoserine and phosphothreonine residues were excluded with this approach. To identify both phosphotyrosine-containing proteins and phosphoserine-/phosphothreonine-containing proteins, parallel experiments involving in IMAC enrichment mainly for phosphoserine/phosphothreonine protein identification and anti-phosphotyrosine enrichment mainly for phosphotyrosine protein identification have been reported (23). However, this work required a doubled amount of the starting materials.To characterize the phosphoproteomic changes induced by NPM-ALK in a comprehensive and efficient manner, we developed a strategy and applied it to enrich phosphopeptides prior to the LC/MS analysis. The design of this enrichment strategy is based on three theoretical advantages. 1) It enables comprehensive identification of all three types of phosphorylated peptides. 2) It facilitates the detection of the phosphotyrosine-containing peptides despite their relatively low abundance compared with phosphoserine- and phosphothreonine-containing peptides. 3) It does not require parallel experiments; i.e. the same starting materials were used for profiling both phosphoserine-/phosphothreonine-containing proteins and phosphotyrosine-containing proteins. In the first step, all the phosphopeptides were enriched by using IMAC. In the second step, using an anti-phosphotyrosine specific antibody, we separated the phosphotyrosine pool from that of phosphoserine and phosphothreonine. Because of the prior enrichment by IMAC, the nonspecific protein binding to the phosphotyrosine antibody will be minimized. The phosphoserine/phosphothreonine pool was fractionated by strong cation exchange chromatography to facilitate their detection by LC/MS. Our results suggest that this strategy is highly useful in the comprehensive characterization of the phosphoproteome, particularly in enhancing our ability to detect phosphotyrosine peptides.One of the key objectives of this study was to comprehensively assess the phosphoproteomic changes induced by oncogenic kinase NPM-ALK that can serve as a proof of principle for future related biological studies. To achieve this goal, our experimental approach was designed to compare the qualitative phosphoproteomic changes between unaltered NPM-ALK- and NPM-ALK^FFF mutant-transfected cells, focusing on similar absolute changes in phosphorylation statuses, by using our strategy. We did not use a quantitative proteomics approach to analyze the phosphoproteomic changes induced by NPM-ALK because of the special property of this oncoprotein. Most of the previous reports (40–42) and our previous publication (43) have shown that phosphorylation of proteins downstream of NPM-ALK were only present in the presence of NPM-ALK; in the absence of NPM-ALK, the phosphorylation statuses of proteins downstream of NPM-ALK were lost. Using quantitative proteomics approaches, such as stable isotope labeling with amino acid in cell culture or isobaric tags for relative and absolute quantitation, will sacrifice the sensitivity of identification. Therefore, in this study, we used a qualitative phosphoproteomics approach instead of using a quantitative phosphoproteomics approach.In this study, we applied this protocol to compare the phosphorylation profile changes between the unaltered NPM-ALK- and NPM-ALK^FFF mutant-transfected cells and identify a set of phosphorylated proteins likely associated with NPM-ALK activity. It was found that the phosphoproteins identified regulate a diversity of key cellular pathways, some of which have never been reported to be associated with NPM-ALK. To document the biological relevance of these identified phosphoproteins, validation experiments were performed using NPM-ALK- and NPM-ALK^FFF mutant-transfected cells as well as ALK⁺ ALCL cells. The TNF/Fas/tumor necrosis factor-related apoptosis-induced ligand (TRAIL) signaling pathway was validated to be modulated by NPM-ALK in ALK⁺ ALCL. Functional analysis showed that knockdown of tumor necrosis factor receptor-associated protein 1 (TRAP1), one of the crucial TNF/Fas/TRAIL signaling molecules, enhanced TRAIL- or chemotherapeutic drug doxorubicin-induced cell death. These findings provide a potential therapeutic strategy for ALK⁺ ALCL and novel insight into the NPM-ALK-mediated lymphomagenesis.     

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.     

Phasic modulation of Wnt signaling enhances cardiac differentiation in human pluripotent stem cells by recapitulating developmental ontogeny

Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) offer immense value in studying cardiovascular regenerative medicine. However, intrinsic biases and differential responsiveness of hPSCs towards cardiac differentiation pose significant technical and logistic hurdles that hamper human cardiomyocyte studies. Tandem modulation of canonical and non-canonical Wnt signaling pathways may play a crucial role in cardiac development that can efficiently generate cardiomyocytes from pluripotent stem cells. Our Wnt signaling expression profiles revealed that phasic modulation of canonical/non-canonical axis enabled orderly recapitulation of cardiac developmental ontogeny. Moreover, evaluation of 8 hPSC lines showed marked commitment towards cardiac-mesoderm during the early phase of differentiation, with elevated levels of canonical Wnts (Wnt3 and 3a) and Mesp1. Whereas continued activation of canonical Wnts was counterproductive, its discrete inhibition during the later phase of cardiac differentiation was accompanied by significant up-regulation of non-canonical Wnt expression (Wnt5a and 11) and enhanced Nkx2.5⁺ (up to 98%) populations. These Nkx2.5⁺ populations transited to contracting cardiac troponin T-positive CMs with up to 80% efficiency. Our results suggest that timely modulation of Wnt pathways would transcend intrinsic differentiation biases of hPSCs to consistently generate functional CMs that could facilitate their scalable production for meaningful clinical translation towards personalized regenerative medicine.     

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.     

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.     

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.     

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.     

Protein complexes associated with β-catenin differentially influence the differentiation profile of neonatal and adult CD8+ T cells

The canonical Wnt signaling pathway is a master cell regulator involved in CD8⁺ T cell proliferation and differentiation. In human CD8⁺ T cells, this pathway induces differentiation into memory cells or a “stem cell memory like” population, which is preferentially present in cord blood. To better understand the role of canonical Wnt signals in neonatal or adult blood, we compared the proteins associated with β-catenin, in nonstimulated and Wnt3a-stimulated human neonatal and adult naive CD8⁺ T cells. Differentially recruited proteins established different complexes in adult and neonatal cells. In the former, β-catenin-associated proteins were linked to cell signaling and immunological functions, whereas those of neonates were linked to proliferation and metabolism. Wnt3a stimulation led to the recruitment and overexpression of Wnt11 in adult cells and Wnt5a in neonatal cells, suggesting a differential connexion with planar polarity and Wnt/Ca²⁺ noncanonical pathways, respectively. The chromatin immunoprecipitation polymerase chain reaction β-catenin was recruited to a higher level on the promoters of cell renewal genes in neonatal cells and of differentiation genes in those of adults. We found a preferential association of β-catenin with CBP in neonatal cells and with p300 in the adult samples, which could be involved in a higher self-renewal capacity of the neonatal cells and memory commitment in those of adults. Altogether, our results show that different proteins associated with β-catenin during Wnt3a activation mediate a differential response of neonatal and adult human CD8⁺ T cells.     

WNT-mediated relocalization of dishevelled proteins

Summary TheWnt family of proto-oncogenes encodes secreted signaling proteins that are required for mouse development. TheDrosophila Wnt homolog, thewingless (Wg) segment polarity gene, mediates a signal transduction pathway in which the downstream elements appear to be conserved through evolution. One such element, thedishevelled gene product, becomes hyperphosphorylated and translocates to the plasma membrane in response to Wg (Yanagawa et al., 1995). We report here that the mouseDishevelle-1 (Dvl-1) andDishevelled-2 genes encode proteins that are differentially localized inWnt-overexpressing PC12 cell lines (PC12/Wnt). WhereasDvl-1 andDvl-2 proteins are limited to the soluble fraction of parental PC12 cells, PC12/Wnt cells display a subset ofDvl-1 protein associated with the membrane andDvl-2 protein with the cytoskeletal fraction. These results suggest a conserved role forDvl inWnt/wg signal transduction.     

Activation of non-canonical Wnt/JNK pathway by Wnt3a is associated with differentiation fate determination of human bone marrow stromal (mesenchymal) stem cells

The canonical Wnt signaling pathway can determine human bone marrow stromal (mesenchymal) stem cell (hMSC) differentiation fate into osteoblast or adipocyte lineages. However, its downstream targets in MSC are not well characterized. Thus, using DNA microarrays, we compared global gene expression patterns induced by Wnt3a treatment in two hMSC lines: hMSC-LRP5^T253 and hMSC-LRP5^T244 cells carrying known mutations of Wnt co-receptor LRP5 (T253I or T244M) that either enhances or represses canonical Wnt signaling, respectively. Wnt3a treatment of hMSC activated not only canonical Wnt signaling, but also the non-canonical Wnt/JNK pathway through upregulation of several non-canonical Wnt components e.g. naked cuticle 1 homolog (NKD1) and WNT11. Activation of the non-canonical Wnt/JNK pathway by anisomycin enhanced osteoblast differentiation whereas its inhibition by SP600125 enhanced adipocyte differentiation of hMSC. In conclusion, canonical and non-canonical Wnt signaling cooperate in determining MSC differentiation fate.