Overexpression Of Wnt5a Research Articles

Osteogenic potency of stem cell-based genetic engineering targeting Wnt3a and Wnt9a

Abstract Bone engineering is a promising therapeutic approach to correct skeletal defects, and genetically-modified stem cells have been implicated in engineering new bone. However, the use of genetically-modified human mesenchymal stem cells targeting an osteogenic growth factor Wnt is not yet investigated. In the present study, a proliferation assay and the alkaline phosphatase (ALP) activity and expression of runt-related transcription factor 2 (Runx2) and osteocalcin (OC) transcripts were investigated to examine the effect of Wnt2 overexpression, Wnt3a overexpression, and Wnt9a knockdown on cell proliferation and osteoblast differentiation of bone marrowderived mesenchymal stem cells (BMSCs). The results showed that the expression of Wnt2 and Wnt3a was up-regulated throughout the osteoblast differentiation period of BMSCs, whereas that of Wnt9a was down-regulated. Overexpression of Wnt3a stimulated cell proliferation while knockdown of Wnt9a increased the ALP activity and the expression of Runx2 and OC. Double transfection producing Wnt3a overexpression and Wnt9a knockdown simultaneously resulted in up-regulation of osteoblast differentiation markers, i.e., the ALP activity and the Runx2 expression. In conclusion, simultaneous genetic modification of Wnt3a overexpression and Wnt9a knockdown enhances osteoblast differentiation of BMSCs, suggesting its osteogenic potency to regenerate new bone in vivo.

Secreted Frizzled-Related Protein-3 (sFRP3) Is Produced by Myeloma Cells and Augments Wnt3a-Induced Differentiation of Mesenchymal Stem Cells and OPG Production in Osteoblasts

Abstract 808 Background:Bone disease is one of the most debilitating complications in patients with multiple myeloma (MM). The molecular mechanisms by which MM triggers bone disease are not fully understood. We have previously demonstrated that Dkk1 is highly expressed in primary MM plasma cells, and associated with bone disease in MM patients by inhibiting Wnt signaling-promoted mesenchymal stem cell differentiation and osteoprotegerin production in osteoblast cells. We have also reported that increase in Wnt signaling in the bone marrow microenvironment by overexpression of Wnt3a in myeloma cells or administration of rWnt3a, or indirectly increasing Wnt signaling by administration of anti-Dkk1 neutralizing antibody also decreased in osteoclast numbers. However, Dkk1 is less frequently expressed in MM cell lines that are derived mostly from late stage of MM; and injection of these MM cell lines into human fetal bone also is able to induce bone lesion in MM animal model. These results indicate that additional factors may be involved in induction of the bone disease at the stage of the disease. The members of the sFRPs family of secreted proteins (including sFRP-1, -2, -3 and -4) directly bind to Wnts, thereby preventing Wnts from binding to the cellular Wnt receptor complex. It has also been reported that sFRP-1 and -2 augment canonical Wnt3a activated signaling in fibroblast. MM cells from pateints with advanced bone lesions express sFRP2 mRNA. Like sFRP2, sFRP3 mRNA is highly expressed in MM plasma cells, but it's function in MM bone disease remains unknown. We sought to investigate the role of sFRP3 in MM-triggered bone lesions using the osteoblast (OB) cell lines CH3T1/2 and C2C12, and serum from MM pateints those MM cells expressed high level of sFRP3. Methods/Results:RT-PCR analysis showed that sFRP3 is expressed in primary MM plasma cells and certain MM cell lines. Recombinant sFRP3 protein did not inhibit, but synergized with Wnt3a to increase beta-catenin protein, while Dkk1 significantly inhibited this process. Similarly, sFRP3 treatment of OB cells increase Wnt-3a-induced TCF transcript activity in OB cells transfected with TOPflash luciferase report constructs. sFRP3 also increased MSC differentiation, as evidenced by increase in alkaline phosphatase activity (ALP) and increased in mineralization by Alizarin red staining. sFRP3 treatment also increases OPG mRNA and protein production in these cells. Similar to sFRP3, sFRP1 and sFRP2 synergistically acted with Wnt3a to induce MSC differentiation and OPG expression in osteoblasts, while Dkk1 significantly inhibited these processes. To confirm the synergistic effects of sFRPs with canonical Wnt signaling on MSC differentiation, we employed R-podin1, a well-known agonist of canonical Wnt signaling. Treatments of MSC cells with R-podin1 led to increase in beta-catenin protein and TCF transcriptional activity and in ALP activity, and increase in OPG mRNA and protein. Pretreatment of the cells with sFRP2 and sFPP3 proteins further enhanced the function of R-podin1. In contrast, Dkk1 protein showed negative effect on R-Spodin1 functions, indicating that sFRP2 and sFRP3 synergized with R-Spodin1 to induce activation of canonical Wnt signaling and subsequent MSC differentiation and OPG production. Conclusion:Taken together, these data suggest that sFRP2 and sFRP3 augment canonical Wnt signaling to induce MSC differentiation and indirectly inhibit osteoclastogenesis by regulating OPG in MSC cells. These results also indicate that Dkk1 may be most important in MM-induced bone disease. Disclosures:Barlogie:Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

Canonical Wnt signaling is involved in switching from cell proliferation to myogenic differentiation of mouse myoblast cells

BackgroundWnt/β-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and β-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of β-catenin signaling during myogenic differentiation remain unknown.ResultsUsing C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of β-catenin/Tcf complex formation, reduced basal β-catenin in the cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased both cytosolic and membrane-bound β-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation.ConclusionsThese results suggest that various Wnt ligands control subcellular β-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via β-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation.

Wnt Signaling Regulates Neuronal Differentiation of Cortical Intermediate Progenitors

Cortical intermediate progenitors (IPs) comprise a secondary neuronal progenitor pool that arises from radial glia (RG). IPs are essential for generating the correct number of cortical neurons, but the factors that regulate the expansion and differentiation of IPs in the embryonic cortex are essentially unknown. In this study, we show that the Wnt-β-catenin pathway (canonical Wnt pathway) regulates IP differentiation into neurons. Upregulation of Wnt-β-catenin signaling by overexpression of Wnt3a in the neocortex induced early differentiation of IPs into neurons and the accumulation of these newly born neurons at the subventricular zone/intermediate zone border. Long-term overexpression of Wnt3a led to cortical dysplasia associated with the formation of large neuronal heterotopias. Conversely, downregulation of Wnt-β-catenin signaling with Dkk1 during mid and late stages of neurogenesis inhibited neuronal production. Consistent with previous reports, we show that Wnt-β-catenin signaling also promotes RG self-renewal. Thus, our findings show differential effects of the Wnt-β-catenin pathway on distinct groups of cortical neuronal progenitors: RG self-renewal and IP differentiation. Moreover, our findings suggest that dysregulation of Wnt signaling can lead to developmental defects similar to human cortical malformation disorders.

Differentiation of human neural progenitor cells regulated by Wnt-3a

Wnt ligands play pivotal roles in the control of cell growth and differentiation during central nervous system development via the Wnt signaling pathway. In this study, we investigated the effects of Wnt-3a and β-catenin on the differentiation of ReNcell VM human neural progenitor cells. After overexpression of Wnt-3a or mutant-stabilized β-catenin in ReNcell VM cells, their effects on TCF-mediated transcription, Wnt target gene expression and differentiation into neuronal and glial cells were investigated. Our results show that activation of Wnt/β-catenin signaling increases TCF-mediated transcription and the expression of the Wnt target genes Axin2, LEF1 and CyclinD1 in ReNcell VM cells. In contrast to mutant-stabilized β-catenin, Wnt-3a increases neurogenesis during the differentiation of ReNcell VM cells. Thus, our data suggest that neurogenesis induced by Wnt-3a is independent of the transcriptional activity of Wnt/β-catenin pathway in ReNcell VM cells.

Segregation of myoblast fusion and muscle-specific gene expression by distinct ligand-dependent inactivation of GSK-3β

Myogenic differentiation involves myoblast fusion and induction of muscle-specific gene expression, which are both stimulated by pharmacological (LiCl), genetic, or IGF-I-mediated GSK-3β inactivation. To assess whether stimulation of myogenic differentiation is common to ligand-mediated GSK-3β inactivation, myoblast fusion and muscle-specific gene expression were investigated in response to Wnt-3a. Moreover, crosstalk between IGF-I/GSK-3β/NFATc3 and Wnt/GSK-3β/β-catenin signaling was assessed. While both Wnt-3a and LiCl promoted myoblast fusion, muscle-specific gene expression was increased by LiCl, but not by Wnt-3a or β-catenin over-expression. Furthermore, LiCl and IGF-I, but not Wnt-3a, increased NFATc3 transcriptional activity. In contrast, β-catenin-dependent transcriptional activity was increased by Wnt-3a and LiCl, but not IGF-I. These results for the first time reveal a segregated regulation of myoblast fusion and muscle-specific gene expression following stimulation of myogenic differentiation in response to distinct ligand-specific signaling routes of GSK-3β inactivation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-010-0467-7) contains supplementary material, which is available to authorized users.

Wnt5a promotes differentiation of human dental papilla cells

To investigate the role of Wnt5a in the process of differentiation of human dental papilla cells (HDPCs). Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of Wnt5a on the differentiation of HDPCs. The effect of Wnt5a on HDPCs differentiation was determined by ALP activity assay, ALP staining and mineral induction assay. Mineralization-related gene expressions were assessed by RT-PCR. Immunostaining revealed Wnt5a expression in the odontoblast layer and dental papilla tissue. Over-expression of Wnt5a by transfecting HDPCs with an Wnt5a-carrying construct increased ALPase activity and the formation of mineralized nodules of HDPCs. RT-PCR analysis showed that the expressions of mineralization-related genes, such as bone sialoprotein, collagen type I, osteonectin, osteopontin (OCN), dentine matrix protein-1 were up-regulated by Wnt5a. Wnt5a promoted differentiation of HDPCs.

Wnt5a signaling is involved in the aggressiveness of prostate cancer and expression of metalloproteinase

Wnt5a is a representative ligand that activates the beta-catenin-independent pathway in Wnt signaling. Although it has been reported that abnormal activation of the Wnt/beta-catenin-dependent pathway is often observed in human prostate cancer, the involvement of the beta-catenin-independent pathway in this cancer is unclear. Abnormal expression of Wnt5a and beta-catenin was observed in 27 (28%) and 49 (50%) of 98 prostate cancer cases, respectively, by immunohistochemical analyses. Simultaneous expression of Wnt5a and beta-catenin was observed in only five cases, suggesting their exclusive expression. The positive detection of Wnt5a was correlated with high Gleason scores and biochemical relapse of prostate cancer, but that of beta-catenin was not. Knockdown and overexpression of Wnt5a in human prostate cancer cell lines reduced and stimulated, respectively, their invasion activities, and the invasion activity required Frizzled2 and Ror2 as Wnt receptors. Wnt5a activated Jun-N-terminal kinase through protein kinase D (PKD) and the inhibition of PKD suppressed Wnt5a-dependent cell migration and invasion. In addition, Wnt5a induced the expression of metalloproteinase-1 through the recruitment of JunD to its promoter region. These results suggest that Wnt5a promotes the aggressiveness of prostate cancer and that its expression is involved in relapse after prostatectomy.

WNT5A Expression in Ameloblastoma and Its Roles in Regulating Enamel Epithelium Tumorigenic Behaviors

Odontogenic tumors originate from the remains of migrating enamel epithelium after the completion of normal tooth genesis. These enamel epithelium remnants exhibit the ability to recapitulate the events that occur during tooth formation. Several lines of evidence suggest that aberrance in the signaling pathways similar to the ones that are used during tooth development, including the WNT pathway, might be the cause of odontogenic tumorigenesis and maintenance. In this study we demonstrated that WNT5A expression was intense in both the epithelial component of ameloblastomas, the most common epithelial odontogenic tumor, and in this tumor's likely precursor cell, the enamel epithelium located at the cervical loop of normal developing human tooth buds. Additionally, when WNT5A was overexpressed in enamel epithelium cells (LS-8), the clones expressing high levels of WNT5A (S) exhibited characteristics of tumorigenic cells, including growth factor independence, loss of anchorage dependence, loss of contact inhibition, and tumor formation in immunocompromised mice. Moreover, overexpression of WNT5A drastically increased LS-8 cell migration and actin reorganization when compared with controls. Suppression of endogenous WNT5A in LS-8 cells (AS) greatly impaired their migration and AS cells failed to form significant actin reorganization and membrane protrusion was rarely seen. Taken together, our data indicate that WNT5A signaling is important in modulating tumorigenic behaviors of enamel epithelium cells in ameloblastomas.

Up-regulation of Wnt5a gene expression in the nitrofen-induced hypoplastic lung

Purpose The pathogenesis of pulmonary hypoplasia in nitrofen-induced congenital diaphragmatic hernia (CDH) still remains unclear. Wnt signaling pathways play a critical role in lung development. Whereas canonical Wnt signaling regulates branching morphogenesis during early lung development, the noncanonical Wnt5a controls late lung morphogenesis, including patterning of distal airway and vascular tubulogenesis (alveolarization). Overexpression of Wnt5a in transgenic mice and in the chick has been reported to result in severe pulmonary hypoplasia. We designed this study to test the hypothesis that the pulmonary Wnt5a gene expression is up-regulated in late stages of lung morphogenesis in CDH. Methods Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15, D18, and D21 and divided into 3 groups: control; nitrofen without CDH, CDH(−); and nitrofen with CDH, CDH(+) (n = 8 at each time-point, respectively). Wnt5a pulmonary gene expression was analyzed by real-time reverse transcription polymerase chain reaction. Immunohistochemistry was performed to evaluate Wnt5a protein expression at each time-point. Results Pulmonary relative mRNA expression levels of Wnt5a were significantly increased in CDH(−) and CDH(+) at D18 (1.61 ± 0.92 and 1.81 ± 1.20, respectively) and D21 (2.40 ± 0.74* and 2.65 ± 0.35*, respectively) compared to controls at D18 and D21 (0.90 ± 0.17* and 1.69 ± 0.53**, respectively) (* P < .05, ** P < .001 vs control ). Strong Wnt5a immunoreactivity was seen in the distal epithelium at D18 and D21 in nitrofen-induced hypoplastic lung compared to controls. Conclusion Up-regulation of pulmonary Wnt5a gene expression in the late lung morphogenesis may interfere with patterning of alveolarization, causing pulmonary hypoplasia in the nitrofen-induced CDH.

Wnt/β-catenin signalling pathway following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

The Wnt/β-catenin signaling pathway plays an important role in development, tissue homeostasis, and regeneration. Inappropriate activation of the Wnt pathway is linked to a wide range of human cancers. The purpose of this study was to characterize the Wnt/β-catenin signaling pathway as depicted by the expression of Wnt1, Frizzled-1, Wnt5a, Frizzled-5 and β-catenin during 4NQO-induced rat tongue carcinogenesis by immunohistochemistry. Male Wistar rats were distributed into three groups of 10 animals each and treated with 4NQO solution at 50 ppm through their drinking water for 4, 12, and 20 weeks. Ten animals were used as control group. No histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure; however, an overexpression of Wnt5a was noticed when compared to control group ( p < 0.05). The Wnt1 showed significant differences ( p < 0.05) in pre-neoplastic lesions at 12 weeks following carcinogen exposure. In well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO, Wnt1 was expressed in the majority of the dysplasic cells and tumor cells. This was statistically significant ( p < 0.05). No significant differences ( p > 0.05) were found in expression of Frizzled-1, Frizzled-5 or β-catenin following oral carcinogenesis. Taken together, our results support the belief that expression of Wnt1 and Wnt5a is related to malignant transformation and conversion of oral mucosa.

WNT5A inhibits human dental papilla cell proliferation and migration

WNT proteins are a large family of cysteine-rich secreted molecules that are linked to both canonical and non-canonical signal pathways, and have been implicated in oncogenesis and tissue development. Canonical WNT proteins have been proven to play critical roles in tooth development, while little is known about the role of non-canonical WNT proteins such as WNT5A. In this study, WNT5A was localized to human dental papilla tissue and human dental papilla cells (HDPCs) cultured in vitro, using immunochemistry and RT-PCR. Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of WNT5A on HDPCs. The BrdU incorporation assay, the MTT assay and flow cytometric analysis showed that over-expression of Wnt5a strongly inhibited the proliferation of HDPCs in vitro. Wound healing and transwell migration assays indicated that over-expression of WNT5A reduced migration of HDPCs. In conclusion, our results showed that WNT5A negatively regulates both proliferation and migration of HDPCs, suggesting its important role in odontogenesis via controlling the HDPCs.

Variation in WNT genes expression in different subtypes of chronic lymphocytic leukemia

The Wnt molecules are a family of secretory glycoproteins implicated in proliferation and differentiation of both normal and malignant cells. Despite extensive investigation of the WNT genes expression profile in various tumors, little is known about their expression in chronic lymphocytic leukemia (CLL). In this study, the expression profile of 14 WNT genes was investigated in a large number of Iranian patients with CLL. Semi-quantitative RT-PCR was performed on peripheral blood leukemic cells obtained from 62 patients with CLL. Peripheral blood mononuclear cells isolated from 11 age matched normal subjects served as control to determine baseline expression level of these genes. Our results have demonstrated significant up-regulation of WNT-3, WNT-4, WNT-5B, WNT-7B, WNT-9A, WNT-10A, and WNT-16B in patients with CLL compared to normal subjects (p < 0.05 to p < 0.0001). WNT gene expression analysis in different CLL subtypes showed a similar pattern of expression in progressive and indolent clinical subtypes. Over-expression of WNT-5A and WNT-9A genes was observed in patients with no mutation in their immunoglobulin (Ig) variable region heavy chain (Ig VH) genes compared to those with mutated Ig VH genes. Comparison between patients expressing VH1 (n = 9), VH3 (n = 40) and VH4 (n = 12) gene families, revealed down-regulation of WNT-3 and WNT-9A in VH3 positive patients. Our results indicate up-regulation of many members of the WNT gene family in CLL suggesting involvement of the Wnt canonical and/or noncanonical signaling pathways in CLL tumorigenesis.

Wnt7a Activates the Planar Cell Polarity Pathway to Drive the Symmetric Expansion of Satellite Stem Cells

Satellite cells in skeletal muscle are a heterogeneous population of stem cells and committed progenitors. We found that quiescent satellite stem cells expressed the Wnt receptor Fzd7 and that its candidate ligand Wnt7a was upregulated during regeneration. Wnt7a markedly stimulated the symmetric expansion of satellite stem cells but did not affect the growth or differentiation of myoblasts. Silencing of Fzd7 abrogated Wnt7a binding and stimulation of stem cell expansion. Wnt7a signaling induced the polarized distribution of the planar cell polarity effector Vangl2. Silencing of Vangl2 inhibited Wnt7a action on satellite stem cell expansion. Wnt7a overexpression enhanced muscle regeneration and increased both satellite cell numbers and the proportion of satellite stem cells. Muscle lacking Wnt7a exhibited a marked decrease in satellite cell number following regeneration. Therefore, Wnt7a signaling through the planar cell polarity pathway controls the homeostatic level of satellite stem cells and hence regulates the regenerative potential of muscle.

Activation of Wnt signalling in stroma from pancreatic cancer identified by gene expression profiling

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant desmoplastic stroma. Interactions between cancer and stromal cells play a critical role in tumour invasion, metastasis and chemoresistance. Therefore, we hypothesized that gene expression profile of the stromal components of pancreatic carcinoma is different from chronic pancreatitis and reflects the interaction with the tumour. We investigated the gene expression of eleven stromal tissues from PDAC, nine from chronic pancreatitis and cell lines of stromal origin using the Affymetrix U133 GeneChip set. The tissue samples were microdissected, the RNA was extracted, amplified and labelled using a repetitive in vitro transcription protocol. Differentially expressed genes were identified and validated using quantitative RT-PCR and immuno-histochemistry. We found 255 genes to be overexpressed and 61 genes to be underexpressed within the stroma of pancreatic carcinoma compared to the stroma of chronic pancreatitis. Analysis of the involved signal transduction pathways revealed a number of genes associated with the Wnt pathway of which the differential expression of SFRP1 and WNT5a was confirmed using immunohistochemistry. Moreover, we could demonstrate that WNT5a expression was induced in fibroblasts during cocultivation with a pancreatic carcinoma cell line. The identified differences in the expression profile of stroma cells derived from tumour compared to cells of inflammatory origin suggest a specific response of the tissue surrounding malignant cells. The overexpression of WNT5a, a gene involved in the non canonical Wnt signalling and chondrocyte development might contribute to the strong desmoplastic reaction seen in pancreatic cancer.

Proteasome Inhibitor, Bortezomib Induces Mesenchymal Stem Cells toward Osteoblast Differentiation through Wnt-Independent Activation of Beta-catenin/TCF Signaling

In multiple myeloma (MM), the proteasome inhibitor bortezomib induces mesenchymal stem cells (MSC) toward osteoblast differentiation. However, it is full unclear about the mechanism(s) underlying bortezomib in this process. Wnt/beta-catenin pathway plays a pivotal role in osteoblast differenciation and bone development. We have demonstrated that inhibition of Wnt/beta-catenin signaling by MM-derived Dkk1 suppresses osteoblast progenitor cell differentiation into osteoblasts (Qiang et al, Bone 2008) and deregulate RANKL and OPG expression in osteoblast cells (Qiang et al Blood 2008a). Increase Wnt signaling by overexpression of Wnt3a in myeloma cells diminished MM-trigged bone lesion in mouse model (Qiang et al Blood 2008b). In the present study we revealed that bortezomib promotes MSC differentiation into osteoblast cells via Wnt-independent activation of beta-catenin/TCF signaling. E-cadherin pull-down assay and subsequently immunoblotting analysis demonstrated that bortezomib induced increases in both free and active forms of beta-catenin protein in cytoplasm and nuclear in bell-shaped dose- and time-dependent manner in mouse and human osteoblast progenitor cell lines including C2C12, C3H10T1/2, Saos-2 and MG63. Similar results were illustrated in primary human 2 cases of normal MSC and MSC from 8 cases of MM pateints. Bortezomib induced increase in ubiquitinated beta-catenin was evidenced by obvious seen slow migration bands of beta-catenin protein in SDS-PAGE gel analysis indicating that bortezomib increased beta-catenin protein by modification of proteasome-mediated degradation of beta-catenin. Increase in cytoplasm and nuclear beta-catenin protein response to bortezomib treatment in the osteoblast cell lines and 4 cases MM derived MSC was further confirmed by immunofluorescent analysis. RT-PCT analysis of TCF family revealed that abundant TCF1 and TCF4 mRNA were expressed in all tested cell lines and in a primary normal MSC, and MM-derived MSC. Bortezomib treatment also resulted in TCF transcriptional activity in bell-shaped, dose-dependent pattern as determined by luciferase activity in these cells transfected with TOPflash plasmid DNAs. Maximal responses to bortezomib were seen at 12.5 nM for both C2C12 (p<0.001) and MG63 (P<0.01), 25 nM for C3H10T1/2 (p<0.001) (p<0.00001) compared with non-stimulation control. These results suggest that transcriptional activation was a downstream effect of bortezomib in osteoblast progenitor cell lines and MM derived MSC. Bortezomib induced increases in beta-catenin protein and TCF transcriptional activity were independent of modification expression of extracellular 19 members of Wnt family ligands, 10 members of Frizzled receptor family, LRP5/6 co-receptors, and antagonists of 4 members of Dkk and sFRP family, respectively, as determined by RT-PCR analysis. Bortezomib did not increase intracellular Dvl-3 proteins, a downstream target of Wnt pathway. Lithium chloride, an inhibitor of GSK3beta did not synergized bortezomib induced increases in beta-catenin protein or TCF transcriptional activity indicating that bortezomib active beta-catenin/TCF signaling independent of activity of GSK3beta. Blocking the catenin/TCF signaling by expressing dominant–activation of beta- negative TFC attenuated bortezomib-induced matrix mineralization indicating that bortezomib induced MSC differentiation into osteoblast through activation of beta-catenin/TCF signaling. Data from experiments in comparison with biological effect of bortezomib with Wnt3a demonstrated that bortezomib did not have effect on OPG and RANKL in these cells, while Wnt3a induces OPG mRNA and protein, but inhibited RANKL expression indicating that bortezomib may have not effect on osteoclastogensis. These results provide insights into a clinically relevant mechanism of action of bortezomib and as such a rationale for its use in the treatment of diseases related to suppression of Wnt/beta-catenin/TCF signaling.

Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration

Satellite cells represent the stem cell population of adult skeletal muscle. The molecular mechanisms that control the proliferation of satellite cells are not well understood. In this study, we show that in response to injury, myofibres activate Wnt ligand transcription and activate a reporter cell line that is sensitive to the canonical Wnt-signalling pathway. Activated satellite cells on isolated cultured myofibres show robust expression of activated-beta-catenin (Act-beta-Cat), a key downstream transcriptional coactivator of canonical Wnt signalling. We provide evidence that the Wnt family of secreted glycoproteins act on satellite cells in a ligand-specific manner. Overexpression of Wnt1, Wnt3a or Wnt5a protein causes a dramatic increase in satellite-cell proliferation. By contrast, exposure of satellite cells to Wnt4 or Wnt6 diminishes this process. Moreover, we show that the prolonged satellite-cell quiescence induced by inhibitory Wnt is reversible and exposing inhibited satellite cells to stimulatory Wnt signalling restores their proliferation rate. Stimulatory Wnt proteins induce premature satellite cell BrdU incorporation as well as nuclear translocation of Act-beta-Cat. Finally, we provide evidence that the Act-beta-Cat translocation observed in single fibres during in vitro culture also occurs in cases of acute and chronic skeletal muscle regeneration in rodents and humans. We propose that Wnt proteins may be key factors that regulate the rate of satellite-cell proliferation on adult muscle fibres during the wound-healing response.

Wnt/β-Catenin Signaling Suppresses Rapsyn Expression and Inhibits Acetylcholine Receptor Clustering at the Neuromuscular Junction

The dynamic interaction between positive and negative signals is necessary for remodeling of postsynaptic structures at the neuromuscular junction. Here we report that Wnt3a negatively regulates acetylcholine receptor (AChR) clustering by repressing the expression of Rapsyn, an AChR-associated protein essential for AChR clustering. In cultured myotubes, treatment with Wnt3a or overexpression of beta-catenin, the condition mimicking the activation of the Wnt canonical pathway, inhibited Agrin-induced formation of AChR clusters. Moreover, Wnt3a treatment promoted dispersion of AChR clusters, and this effect was prevented by DKK1, an antagonist of the Wnt canonical pathway. Next, we investigated possible mechanisms underlying Wnt3a regulation of AChR clustering in cultured muscle cells. Interestingly, we found that Wnt3a treatment caused a decrease in the protein level of Rapsyn. In addition, Rapsyn promoter activity in cultured muscle cells was inhibited by the treatment with Wnt3a or beta-catenin overexpression. Forced expression of Rapsyn driven by a promoter that is not responsive to Wnt3a prevented the dispersing effect of Wnt3a on AChR clusters, suggesting that Wnt3a indeed acts to disperse AChR clusters by down-regulating the expression of Rapsyn. The role of Wnt/beta-catenin signaling in dispersing AChR clusters was also investigated in vivo by electroporation of Wnt3a or beta-catenin into mouse limb muscles, where ectopic Wnt3a or beta-catenin caused disassembly of postsynaptic apparatus. Together, these results suggest that Wnt/beta-catenin signaling plays a negative role for postsynaptic differentiation at the neuromuscular junction, probably by regulating the expression of synaptic proteins, such as Rapsyn.

Increasing Wnt3a Signaling in Myeloma Cells Inhibits the Osteolytic Phenotype and Tumor Growth In Vivo through Regulation of the RANKL/OPG Axis in Osteoblasts and Indirectly Regulates Osteoclastogenesis.

We have demonstrated that canonical and non-canonical Wnt signaling occurs in myeloma cells (Qiang et al., 2005) and overexpression of Wnt3a in myeloma cells inhibits the osteolytic phenotype and also tumor growth in vivo (Qiang et al Blood, Abstract #3420, 2006). To further investigate the mechanisms that contribute to this process we have expanded our in vivo data by showing that while H929 cells stably expressing Wnt3a (H929/W3a) leads to reduced tumor growth in the in-vivo SCID-hu bone graft model compared with H929 vector alone transfected control cells (H929/EV), there was no significant difference in the subcutaneous growth of the two cell lines in SCID mice. Taken together these data suggests that alteration of the human bone marrow microenvironment is central to Wnt-mediated reduction in tumor growth in bone. We next employed an in-vitro co-culture model in which the mouse osteoprogenitor cell line, C2C12, and human osteoblast cell line, Saos-2 were co-cultured with either H929/Wnt3 or H929/EV cells. QPCR analysis demonstrated that osteoprotegerin (OPG) mRNA expression (relative OPG mRNA to GAPDH) in C2C12 cells co-cultured with H929/W3a was significantly elevated compared with H929/EV (mean±SD: 14.34±0.97 vs 8.43±0.16; P<0.001). ELISA analysis showed that OPG protein levels in the cell culture supernatant were also significantly higher (71.02 ± 6.178 vs 0 pg/ml; P<0.001). Similar results in OPG mRNA and protein levels were observed in Saos-2 cells co-cultured with H929/W3a relative to H929/EV. Furthermore, treatment of C2C12 cells with recombinant Wnt3a protein induced both OPG mRNA (48.1 ±1.2 vs 1.0±0.5; P<0.001) and protein levels (1767.03 ± 44.8 vs 1.11 ± 0.03 p< 0.0001) compared with vehicle alone. These results suggest that forced expression of a canonical Wnt ligand by MM cells might promote OPG transcription in osteoblast progenitors in-vivo. To further confirm the role of Wnt signaling in regulation of OPG and RANKL transcription, we produced C2C12 cells that stably express Dkk1. These clones showed a significant inhibition of Wnt3a induced OPG mRNA (22.2± 2.3 vs 1.7±0.35; p<0.001) and protein (73.3 ± 18.0 vs. 0 pg/ml; p<0.01) compared with vector control. In contrast, RANKL mRNA (5.1±0.9 vs 1.0± 0.5, p<0.01) and protein (9.3±3.8 vs. 0 pg/ml; p<0.01) were increased in Dkk1 expressing clones compared with control. Moreover, supernatant from C2C12 clones stably expressing a DN-beta-catenin (DNBC/C2C12) contained a significantly higher level of RANKL (17.3± 3.5 pg/ml vs. 0±0; P<0.001) and a dramatically lower level of OPG protein (0±0 vs. 431.186 pg/ml; P<0.001) compared with control. Finally, the numbers of multinuclear TRAP-positive osteoclasts were significantly more abundant in culture containing supernatant from DNBC/C2C12 than that from vector control, while Wnt3a exposure had no effect on osteoclast formation in-vitro. Taken together, these data suggest that Wnt ligand-mediated inhibition of myeloma cell growth, and inhibition of osteolytic lesions, in-vivo may result from upregulation of OPG and loss of RANKL in osteoblast progenitors, which subsequently diminishes osteoclast formation. Results of these studies provide new insights into mechanism by which Wnts may serve as an important indirect regulator of myeloma growth and osteoclast formation, and as such, targeting Wnt signaling may be an new therapeutic strategy for controlling myeloma growth and associated bone disease.

Role of Wnt5a in the proliferation of human glioblastoma cells

Wnt5a operates as either a tumor suppressor or a tumor stimulator, according to tumor type. The functions of Wnt5a in human glioblastoma (GBM) have yet to be determined. We initially evaluated the expression of Wnt5a in human glioma. The results of immunohistochemical analyses have revealed that Wnt5a expression was higher in human GBM than in normal brain tissue and low-grade astrocytoma. In order to assess the role of Wnt5a on proliferation in human glioblastoma cells, we employed U87MG and GBM-05, a newly established GBM cell line. GBM-05 was established from a patient diagnosed with GBM. GBM-05 cells were shown to express Nestin, but did not express GFAP and Map2ab. GBM-05 cells formed infiltrating brain tumors after being intracerebrally transplanted into nude mice, and xenotransplanted GBM-05 cells were observed to differentiate into neuronal and astrocyte lineages. Wnt5a expression in the xenotransplanted tumors was higher than that detected in the surrounding brain tissues. The overexpression of Wnt5a increased the proliferation of GBM-05 and U87MG in vitro. By way of contrast, the downregulation of Wnt5a expression as the result of RNA interference reduced proliferation from GBM-05 and U87MG cells in vitro, and reduced tumorigenicity in vivo. Our data indicate that Wnt5a signaling is an important regulator in the proliferation of human glioma cells.