Wnt/β-Catenin Signaling Regulates the Proliferation and Differentiation of Mesenchymal Progenitor Cells through the p53 Pathway

Objective

Mesenchymal progenitor cells (MPCs) are found in articular cartilage from normal controls and patients with osteoarthritis (OA). Nevertheless, the molecular mechanisms of the proliferation and differentiation of these cells remain unclear. In this study, we aimed to determine the involvement of Wnt/β-catenin signaling in regulating the proliferation and differentiation of MPCs.

Methods

MPCs were isolated from the articular cartilage of normal and OA patients. Cells were sorted by immunomagnetic cell separation. Cell proliferation capacity was evaluated using the MTT assay. Toluidine blue staining and immunostaining with anti-collagen II or anti-aggrecan antibodies were used to determine the chondrogenic differentiation capabilities of MPCs. The mRNA and protein expression of target genes were examined by quantitative real-time polymerase chain reaction and Western blotting, respectively. Knock-down of p53 expression was achieved with RNA interference.

Results

Most cells isolated from the normal and OA patients were CD105⁺ and CD166⁺ positive (Normal subjects: CD105⁺/CD166⁺, 94.6%±1.1%; OA: CD105⁺/CD166⁺, 93.5%±1.1%). MPCs derived from OA subjects exhibited decreased differentiation capabilities and enhanced Wnt/β-catenin activity. Inhibition of Wnt/β-catenin signaling promoted proliferation and differentiation, whereas activation of this pathway by treatment with rWnt3a protein decreased the proliferation and differentiation of normal MPCs. Additionally, Wnt/β-catenin signaling positively regulated p53 expression, and silencing of p53 increased proliferation and differentiation of MPCs.

Conclusions

Wnt/β-catenin regulated the proliferation and differentiation of MPCs through the p53 pathway.     

Interruption of Wnt Signaling in Müller Cells Ameliorates Ischemia-Induced Retinal Neovascularization

Retinal Müller cells are major producers of inflammatory and angiogenic cytokines which contribute to diabetic retinopathy (DR). Over-activation of the Wnt/β-catenin pathway has been shown to play an important pathogenic role in DR. However, the roles of Müller cell-derived Wnt/β-catenin signaling in retinal neovascularization (NV) and DR remain undefined. In the present study, mice with conditional β-catenin knockout (KO) in Müller cells were generated and subjected to oxygen-induced retinopathy (OIR) and streptozotocin (STZ)-induced diabetes. Wnt signaling was evaluated by measuring levels of β-catenin and expression of its target genes using immunoblotting. Retinal vascular permeability was measured using Evans blue as a tracer. Retinal NV was visualized by angiography and quantified by counting pre-retinal nuclei. Retinal pericyte loss was evaluated using retinal trypsin digestion. Electroretinography was performed to examine visual function. No abnormalities were detected in the β-catenin KO mice under normal conditions. In OIR, retinal levels of β-catenin and VEGF were significantly lower in the β-catenin KO mice than in littermate controls. The KO mice also had decreased retinal NV and vascular leakage in the OIR model. In the STZ-induced diabetic model, disruption of β-catenin in Müller cells attenuated over-expression of inflammatory cytokines and ameliorated pericyte dropout in the retina. These findings suggest that Wnt signaling activation in Müller cells contributes to retinal NV, vascular leakage and inflammation and represents a potential therapeutic target for DR.     

WIKI4, a Novel Inhibitor of Tankyrase and Wnt/?-Catenin Signaling

The Wnt/ß-catenin signaling pathway controls important cellular events during development and often contributes to disease when dysregulated. Using high throughput screening we have identified a new small molecule inhibitor of Wnt/ß-catenin signaling, WIKI4. WIKI4 inhibits expression of ß-catenin target genes and cellular responses to Wnt/ß-catenin signaling in cancer cell lines as well as in human embryonic stem cells. Furthermore, we demonstrate that WIKI4 mediates its effects on Wnt/ß-catenin signaling by inhibiting the enzymatic activity of TNKS2, a regulator of AXIN ubiquitylation and degradation. While TNKS has previously been shown to be the target of small molecule inhibitors of Wnt/ß-catenin signaling, WIKI4 is structurally distinct from previously identified TNKS inhibitors.     

In Vitro Effects of a Small-Molecule Antagonist of the Tcf/?-Catenin Complex on Endometrial and Endometriotic Cells of Patients with Endometriosis

Background

Our previous studies suggested that aberrant activation of Wnt/ß-catenin signaling might be involved in the pathophysiology of endometriosis. We hypothesized that inhibition of Wnt/ß-catenin signaling might result in inhibition of cell proliferation, migration, and/or invasion of endometrial and endometriotic epithelial and stromal cells of patients with endometriosis.

Objectives

The aim of the present study was to evaluate the effects of a small-molecule antagonist of the Tcf/ß-catenin complex (PKF 115–584) on cell proliferation, migration, and invasion of endometrial and endometriotic epithelial and stromal cells.

Methods

One hundred twenty-six patients (78 with and 48 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of PKF 115–584 on cell proliferation, migration, and invasion and on the Tcf/ß-catenin target genes were evaluated in endometrial epithelial and stromal cells of patients with and without endometriosis, and in endometrial and endometriotic epithelial and stromal cells of the same patients.

Results

The inhibitory effects of PKF 115–584 on cell migration and invasion in endometrial epithelial and stromal cells of patients with endometriosis prepared from the menstrual phase were significantly higher than those of patients without endometriosis. Levels of total and active forms of MMP-9 were significantly higher in epithelial and stromal cells prepared from menstrual endometrium in patients with endometriosis compared to patients without endometriosis. Treatment with PKF 115–584 inhibited MMP-9 activity to undetectable levels in both menstrual endometrial epithelial and stromal cells of patients with endometriosis. The number of invasive cells was significantly higher in epithelial and stromal cells of endometriotic tissue compared with matched eutopic endometrium of the same patients. Treatment with PKF 115–584 decreased the number of invasive endometriotic epithelial cells by 73% and stromal cells by 75%.

Conclusions

The present findings demonstrated that cellular mechanisms known to be involved in endometriotic lesion development are inhibited by targeting the Wnt/β-catenin pathway.     

Expression Characteristics of β-Catenin in Scallop Chlamys farreri Gonads and Its Role as a Potential Upstream Gene of Dax1 through Canonical Wnt Signalling Pathway Regulating the Spermatogenesis

β-catenin is a key signaling molecule in the canonical Wnt pathway, which is involved in animal development. However, little information has been reported for β-catenin in bivalves. In the present study, we cloned a homolog of β-catenin from the scallop Chlamys farreri and determined its expression characteristics. The full-length cDNA of β-catenin was 3,353 bp, including a 2,511 bp open reading frame that encoded a predicted 836 amino acid protein. Level of the β-catenin mRNA increased significantly (P<0.05) with C. farreri gonadal development and presented a sexually dimorphic expression pattern in the gonads, which was significantly high in ovaries detected by quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical analysis revealed that the β-catenin was mainly located in germ cells of the gonads, with obvious positive immune signals in the oogonia and oocytes of ovaries as well as in the spermatogonia and spermatocytes of testes, implying β-catenin might be involved in the gametogenesis of C. farreri. Furthermore, when 0.1 µg/mL and 0.2 µg/mL DKK-1 (an inhibitor of the canonical Wnt pathway) were added in vitro to culture medium containing testis cells of C. farreri, the expression of β-catenin decreased significantly detected by qRT-PCR (P<0.05), suggesting the canonical Wnt signal pathway exists in the scallop testis. Similarly, when 50 µM and 100 µM quercetin (an inhibitor of β-catenin) were added in vitro to the culture system, Dax1 expression was significantly down-regulated compared with controls (P<0.05), implying the β-catenin is an upstream gene of Dax1 and is involved in the regulation of C. farreri spermatogenesis.     

Ubiquitin-specific protease 53 promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

The ubiquitin protease pathway plays important role in human bone marrow-derived mesenchymal stem cell (hBMSC) differentiation, including osteogenesis. However, the function of deubiquitinating enzymes in osteogenic differentiation of hBMSCs remains poorly understood. In this study, we aimed to investigate the role of ubiquitin-specific protease 53 (USP53) in the osteogenic differentiation of hBMSCs. Based on re-analysis of the Gene Expression Omnibus database, USP53 was selected as a positive regulator of osteogenic differentiation in hBMSCs. Overexpression of USP53 by lentivirus enhanced osteogenesis in hBMSCs, whereas knockdown of USP53 by lentivirus inhibited osteogenesis in hBMSCs. In addition, USP53 overexpression increased the level of active β-catenin and enhanced the osteogenic differentiation of hBMSCs. This effect was reversed by the Wnt/β-catenin inhibitor DKK1. Mass spectrometry showed that USP53 interacted with F-box only protein 31 (FBXO31) to promote proteasomal degradation of β-catenin. Inhibition of the osteogenic differentiation of hBMSCs by FBXO31 was partially rescued by USP53 overexpression. Animal studies showed that hBMSCs with USP53 overexpression significantly promoted bone regeneration in mice with calvarial defects. These results suggested that USP53 may be a target for gene therapy for bone regeneration.Subject terms: Cell signalling, Mesenchymal stem cells     

Canonical Wnt/β-Catenin Signalling Is Essential for Optic Cup Formation

A multitude of signalling pathways are involved in the process of forming an eye. Here we demonstrate that β-catenin is essential for eye development as inactivation of β-catenin prior to cellular specification in the optic vesicle caused anophthalmia in mice. By achieving this early and tissue-specific β-catenin inactivation we find that retinal pigment epithelium (RPE) commitment was blocked and eye development was arrested prior to optic cup formation due to a loss of canonical Wnt signalling in the dorsal optic vesicle. Thus, these results show that Wnt/β-catenin signalling is required earlier and play a more central role in eye development than previous studies have indicated. In our genetic model system a few RPE cells could escape β-catenin inactivation leading to the formation of a small optic rudiment. The optic rudiment contained several neural retinal cell classes surrounded by an RPE. Unlike the RPE cells, the neural retinal cells could be β-catenin-negative revealing that differentiation of the neural retinal cell classes is β-catenin-independent. Moreover, although dorsoventral patterning is initiated in the mutant optic vesicle, the neural retinal cells in the optic rudiment displayed almost exclusively ventral identity. Thus, β-catenin is required for optic cup formation, commitment to RPE cells and maintenance of dorsal identity of the retina.     

Dok5 regulates proliferation and differentiation of osteoblast via canonical Wnt/β-catenin signaling

Objective:In bone tissue engineering, the use of osteoblastic seed cells has been widely adopted to mediate the osteogenic differentiation so as to prompt bone regeneration and repair. It is hypothesized that Dok5 can regulate the proliferation and differentiation of osteoblasts. In this study, the role of Dok5 in osteoblast proliferation and differentiation was investigated.Methods:A lentiviral vector to silence Dok5 was transferred to C3H10, 293T and C2C12 cells. CCK-8 assay was used to detect the cell proliferation. Cells were stained by ALP and AR-S staining. Western blot and RT-PCR were used to detect the expression levels of related factors.Results:Dok5 expression level was gradually up-regulated during the osteoblast differentiation. Dok5 silencing down-regulated the expression levels of osteogenic biosignatures OPN, OCN, and Runx2 and suppressed the osteogenesis. Additionally, the osteoblast proliferation and canonical Wnt/β-catenin signaling were suppressed upon Dok5 knockdown, β-catenin expression level was significantly down-regulated in the knockdown group, while the expression levels of GSK3-β and Axin, negative regulators in the Wnt signaling pathway, were up-regulated. Furthermore, overexpression of Dok5 promoted the proliferation and osteogenesis and activated the canonical Wnt/β-catenin signaling pathway.Conclusion:Dok5 may regulate the osteogenic proliferation and differentiation via the canonical Wnt/β-catenin signaling pathway.     

Tax1 binding protein 3 regulates osteogenic and adipogenic differentiation through inactivating Wnt/β-catenin signalling

Tax1 binding protein 3 (Tax1bp3) is a PDZ domain-containing protein that is overexpressed in cancer. Previous studies recognized Tax1bp3 as an inhibitor of β-catenin. Till now it is not known whether Tax1bp3 regulates osteogenic and adipogenic differentiation of mesenchymal progenitor cells. In the current study, the data showed that Tax1bp3 was expressed in bone and was increased in the progenitor cells when induced toward osteoblast and adipocyte differentiation. The overexpression of Tax1bp3 in the progenitor cells inhibited osteogenic differentiation and conversely stimulated adipogenic differentiation, and the knockdown of Tax1bp3 affected the differentiation of the progenitor cells oppositely. Ex vivo experiments using the primary calvarial osteoblasts from osteoblast-specific Tax1bp3 knock-in mice also demonstrated the anti-osteogenic and pro-adipogenic function of Tax1bp3. Mechanistic investigations revealed that Tax1bp3 inhibited the activation of canonical Wnt/β-catenin and bone morphogenetic proteins (BMPs)/Smads signalling pathways. Taken together, the current study has provided evidences demonstrating that Tax1bp3 inactivates Wnt/β-catenin and BMPs/Smads signalling pathways and reciprocally regulates osteogenic and adipogenic differentiation from mesenchymal progenitor cells. The inactivation of Wnt/β-catenin signalling may be involved in the reciprocal role of Tax1bp3.     

Stimulation of wnt/ß-catenin pathway in human CD8(+) T lymphocytes from blood and lung tumors leads to a shared young/memory phenotype

Cancer can be treated by adoptive cell transfer (ACT) of T lymphocytes. However, how to optimally raise human T cells to a differentiation state allowing the best persistence in ACT is a challenge. It is possible to differentiate mouse CD8⁺ T cells towards stem cell-like memory (T_SCM) phenotype upon TCR stimulation with Wnt/ß-catenin pathway activation. Here, we evaluated if T_SCM can be obtained from human mature CD8⁺ T cells following TCR and Wnt/ß-catenin activation through treatment with the chemical agent 4,6-disubstituted pyrrolopyrimidine (TWS119), which inhibits the glycogen synthase kinase-3β (GSK-3β), key inhibitor of the Wnt pathway. Human CD8⁺ T cells isolated from peripheral blood or tumor-infiltrating lymphocytes (TIL), and treated with TWS119 gave rise to CD62L⁺CD45RA⁺ cells, indicative of early differentiated stage, also expressing CD127 which is normally found on memory cells, and CD133, an hematopoietic stem cell marker. T_SCM cells raised from either TIL or blood secreted numerous inflammatory mediators, but in lower amounts than those measured without TWS119. Finally, generated T_SCM CD8⁺ T cells expressed elevated Bcl-2 and no detectable caspase-3 activity, suggesting increased persistence. Our data support a role for Wnt/ß-catenin pathway in promoting the T_SCM subset in human CD8⁺ T cells from TIL and the periphery, which are relevant for ACT.     

The SULFs,Extracellular Sulfatases for Heparan Sulfate,Promote the Migration of Corneal Epithelial Cells during Wound Repair

Corneal epithelial wound repair involves the migration of epithelial cells to cover the defect followed by the proliferation of the cells to restore thickness. Heparan sulfate proteoglycans (HSPGs) are ubiquitous extracellular molecules that bind to a plethora of growth factors, cytokines, and morphogens and thereby regulate their signaling functions. Ligand binding by HS chains depends on the pattern of four sulfation modifications, one of which is 6-O-sulfation of glucosamine (6OS). SULF1 and SULF2 are highly homologous, extracellular endosulfatases, which post-synthetically edit the sulfation status of HS by removing 6OS from intact chains. The SULFs thereby modulate multiple signaling pathways including the augmentation of Wnt/ß-catenin signaling. We found that wounding of mouse corneal epithelium stimulated SULF1 expression in superficial epithelial cells proximal to the wound edge. Sulf1^−/−, but not Sulf2^−/−, mice, exhibited a marked delay in healing. Furthermore, corneal epithelial cells derived from Sulf1^−/− mice exhibited a reduced rate of migration in repair of a scratched monolayer compared to wild-type cells. In contrast, human primary corneal epithelial cells expressed SULF2, as did a human corneal epithelial cell line (THCE). Knockdown of SULF2 in THCE cells also slowed migration, which was restored by overexpression of either mouse SULF2 or human SULF1. The interchangeability of the two SULFs establishes their capacity for functional redundancy. Knockdown of SULF2 decreased Wnt/ß-catenin signaling in THCE cells. Extracellular antagonists of Wnt signaling reduced migration of THCE cells. However in SULF2- knockdown cells, these antagonists exerted no further effects on migration, consistent with the SULF functioning as an upstream regulator of Wnt signaling. Further understanding of the mechanistic action of the SULFs in promoting corneal repair may lead to new therapeutic approaches for the treatment of corneal injuries.     

Wnt7a Regulates Multiple Steps of Neurogenesis

Although Wnt7a has been implicated in axon guidance and synapse formation, investigations of its role in the early steps of neurogenesis have just begun. We show here that Wnt7a is essential for neural stem cell self-renewal and neural progenitor cell cycle progression in adult mouse brains. Loss of Wnt7a expression dramatically reduced the neural stem cell population and increased the rate of cell cycle exit in neural progenitors in the hippocampal dentate gyrus of adult mice. Furthermore, Wnt7a is important for neuronal differentiation and maturation. Loss of Wnt7a expression led to a substantial decrease in the number of newborn neurons in the hippocampal dentate gyrus. Wnt7a^−/− dentate granule neurons exhibited dramatically impaired dendritic development. Moreover, Wnt7a activated β-catenin and its downstream target genes to regulate neural stem cell proliferation and differentiation. Wnt7a stimulated neural stem cell proliferation by activating the β-catenin–cyclin D1 pathway and promoted neuronal differentiation and maturation by inducing the β-catenin–neurogenin 2 pathway. Thus, Wnt7a exercised critical control over multiple steps of neurogenesis by regulating genes involved in both cell cycle control and neuronal differentiation.