Inhibition of GSK3ß Stimulates BMP Signaling and Decreases SOST Expression Which Results in Enhanced Osteoblast Differentiation.

Both bone morphogenetic protein (BMP) and Wnt signaling have significant roles in osteoblast differentiation and the interaction between BMP and Wnt signaling is well known. Sclerostin is an important inhibitor of bone formation, inhibiting Wnt signaling and downstream effects of BMP such as alkaline phosphatase activity and matrix mineralization in vitro. However, little is known about the effect of BMP and Wnt signaling interaction on the regulation of SOST, the gene encoding sclerostin. Possibly, uncoupling of osteoblast differentiation regulators and SOST expression could increase osteoblast differentiation. Therefore, we investigated the effect of BMP and Wnt signaling interaction on the expression of SOST and the subsequent effect on osteoblast differentiation. Human osteosarcoma cells (SaOS-2) and murine pre-osteoblast cells (KS483) were treated with different concentrations of Wnt3a, a specific GSK3ß inhibitor (GIN) and BMP4. Both Wnt3a and GIN increased BMP4-induced BMP signaling and BMP4 increased Wnt3a and GIN-induced Wnt signaling. However, the effect of GIN was much stronger. Quantitative RT-PCR analysis showed that SOST expression dose-dependently decreased with increasing Wnt signaling, while BMP4 induced SOST expression. GIN significantly decreased the BMP4-induced SOST expression. This resulted in an increased osteoblast differentiation as measured by ALP activity in the medium and matrix mineralization. We conclude that GSK3ß inhibition by GIN caused an uncoupling of BMP signaling and SOST expression, resulting in an increased BMP4-induced osteoblast differentiation. This effect can possibly be used in clinical practice to induce local bone formation, for example, fracture healing or osseointegration of implants.

Wnt3a induces exosome secretion from primary cultured rat microglia.

BACKGROUND: Microglia, the immune effector cells of the CNS and the signaling molecule Wnt, both play critical roles in neurodevelopment and neurological disease. Here we describe the inducible release of exosomes from primary cultured rat microglia following treatment with recombinant carrier-free Wnt3a. RESULTS: Wnt3a was internalised into microglia, being detectable in early endosomes, and secreted in exosomes through a GSK3-independent mechanism. Electron microscopy demonstrated that exosomes were elliptical, electron-dense (100 nm) vesicles that coalesced with time in vitro. In contrast to microglia, primary cortical neurons released exosomes constitutively and the quantity of exosomes released was not altered by Wnt3a treatment. The proteomic profile of the microglial-derived exosomes was characterised using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and the vesicles were found to be associated with proteins involved in cellular architecture, metabolism, protein synthesis and protein degradation including ß-actin, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunits and ubiquitin (45 proteins in total). Unlike lipopolysaccharide, Wnt3a did not induce a neurotoxic, pro-inflammatory phenotype in primary microglia. CONCLUSION: These findings reveal a novel mechanism through which Wnt3a signals in microglia resulting in the release of exosomes loaded with proteinaceous cargo.

Delivery of exogenous proteins by mesenchymal stem cells attenuates early memory deficits in a murine model of Alzheimer's disease.

A promising intervention for Alzheimer's disease (AD) would ideally target key pathological factors that are involved in AD pathogenesis. Soluble factors produced by engrafted mesenchymal stem cells (MSCs) mediate potential therapeutic effects in AD. However, these therapeutic benefits are largely hampered by the limited paracrine capacity of MSCs. In this study, we used adenovirus-mediated gene transduction of bone marrow MSCs to deliver exogenous proteins into the brain of APPswe/PSEN1dE9 (APP/PS1) mice in the early stage of impairment. We observed that engrafted MSCs carrying exogenous (C-X3-C motif) ligand 1 (CX3CL1) alone reduced the production of the inflammatory cytokine TNF-ɑ and improved synapse-related protein expression but not cognitive function. Transplantation of MSCs carrying CX3CL1 and Wnt3a (CX3CL1-Wnt3a-MSC) significantly attenuated the learning and memory impairment when compared with a control group. The improvement of neurobehavioral functions in APP/PS1 mice treated with CX3CL1-Wnt3a-MSC was related to the inhibition of microglial neurotoxicity and promotion of hippocampal neurogenesis. Transplantation of CX3CL1-Wnt3a-MSC also regulated phosphoinositide 3-kinase/activated protein kinase B (PI3K/AKT) signaling to inhibit the activity of glycogen synthase kinase 3 beta (GSK3ß). Taken together, these results indicate that the delivery of exogenous proteins via MSCs can modulate microglial function and enhance neurogenesis, thereby providing new insights into AD intervention.

Local Wnt3a treatment restores bone regeneration in large osseous defects after surgical debridement of osteomyelitis.

Impaired bone homeostasis caused by osteomyelitis provokes serious variations in the bone remodeling process, thereby involving multiple inflammatory cytokines to activate bone healing. We have previously established a mouse model for post-traumatic osteomyelitis and studied bone regeneration after sufficient debridement. Moreover, we could further characterize the postinfectious inflammatory state of bony defects after debridement with elevated osteoclasts and decreased bone formation despite the absence of bacteria. In this study, we investigated the positive effects of Wnt-pathway modulation on bone regeneration in our previous established mouse model. This was achieved by local application of Wnt3a, a recombinant activator of the canonical Wnt-pathway. Application of Wnt3a could enhance new bone formation, which was verified by histological and µ-CT analysis. Moreover, histology and western blots revealed enhanced osteoblastogenesis and downregulated osteoclasts in a RANKL-dependent manner. Further analysis of Wnt-pathway showed downregulation after bone infections were reconstituted by application of Wnt3a. Interestingly, Wnt-inhibitory proteins Dickkopf 1 (DKK1), sclerostin, and secreted frizzled protein 1 (sFRP1) were upregulated simultaneously to Wnt-pathway activation, indicating a negative feedback for active form of Beta-catenin. In this study, we could demonstrate enhanced bone formation in defects caused by post-traumatic osteomyelitis after Wnt3a application. KEY MESSAGES: Osteomyelitis decreases bone regeneration Wnt3a restores bone healing after infection Canonical Wnt-pathway activation with negative feedback.

ß-Catenin-Dependent Wnt Signaling: A Pathway in Acute Cutaneous Wounding.

BACKGROUND: Acute wound healing is a dynamic process that results in the formation of scar tissue. The mechanisms of this process are not well understood; numerous signaling pathways are thought to play a major role. Here, the authors have identified ß-catenin-dependent Wnt signaling as an early acute-phase reactant in acute wound healing and scar formation. METHODS: The authors created 6-mm full-thickness excisional cutaneous wounds on adult ß-catenin-dependent Wnt signal (BAT-gal) reporter mice. The expression of canonical Wnt after wounding was analyzed using X-gal staining and quantitative real-time polymerase chain reaction. Next, recombinant mouse Wnt3a (rmWnt3a) was injected subcutaneously to the wound edge, daily. The mice were killed at stratified time points, up to 15 days after injury. Histologic analysis, quantitative real-time polymerase chain reaction, and Western blot were performed. RESULTS: Numerous individual Wnt ligands increased in expression after wounding, including Wnt3a, Wnt4, Wnt10a, and Wnt11. A specific pattern of Wnt activity was observed, localized to the hair follicle and epidermis. Mice injected with rmWnt3a exhibited faster wound closure, increased scar size, and greater expression of fibroblast growth factor receptor-2 and type I collagen. CONCLUSIONS: The authors' data suggest that ß-catenin-dependent Wnt signaling expression increases shortly after cutaneous wounding, and exogenous rmWnt3a accelerates reepithelialization, wound matrix maturation, and scar formation. Future experiments will focus on the intersection of Wnt signaling and other known profibrotic cytokines.

Neuroprotective and regenerative roles of intranasal Wnt-3a administration after focal ischemic stroke in mice.

Wnt signaling is a conserved pathway involved in expansion of neural progenitors and lineage specification during development. However, the role of Wnt signaling in the post-stroke brain has not been well-elucidated. We hypothesized that Wnt-3a would play an important role for neurogenesis and brain repair. Adult male mice were subjected to a focal ischemic stroke targeting the sensorimotor cortex. Mice that received Wnt-3a (2 µg/kg/day, 1 h after stroke and once a day for the next 2 days, intranasal delivery) had reduced infarct volume compared to stroke controls. Wnt-3a intranasal treatment of seven days upregulated the expression of brain-derived growth factor (BDNF), increased the proliferation and migration of neuroblasts from the subventricular zone (SVZ), resulting in increased numbers of newly formed neurons and endothelial cells in the peri-infarct zone. Both the molecular and cellular effects of Wnt-3a were blocked by the Wnt specific inhibitors XAV-939 or Dkk-1. In functional assays, Wnt-3a treatment enhanced the local cerebral blood flow (LCBF) in the peri-infarct, as well as improved sensorimotor functions in a battery of behavioral tests. Together, our data demonstrates that the Wnt-3a signaling can act as a dual neuroprotective and regenerative factor for the treatment of ischemic stroke.

Gelatin device for the delivery of growth factors involved in endochondral ossification.

Controlled release drug delivery systems are well established as oral and implantable dosage forms. However, the controlled release paradigm can also be used to present complex soluble signals responsible for cellular organization during development. Endochondral ossification (EO), the developmental process of bone formation from a cartilage matrix is controlled by several soluble signals with distinct functions that vary in structure, molecular weight and stability. This makes delivering them from a single vehicle rather challenging. Herein, a gelatin-based delivery system suitable for the delivery of small molecules as well as recombinant human (rh) proteins (rhWNT3A, rhFGF2, rhVEGF, rhBMP4) is reported. The release behavior and biological activity of the released molecules was validated using analytical and biological assays, including cell reporter systems. The simplicity of fabrication of the gelatin device should foster its adaptation by the diverse scientific community interested in interrogating developmental processes, in vivo.

Dysregulation of Fra1 expression by Wnt/ß-catenin signalling promotes glioma aggressiveness through epithelial-mesenchymal transition.

Aberrant expression of Fos-related antigen-1 (Fra1) is commonly elevated in various malignant cancers and is strongly implicated in invasion and metastasis. However, the molecular mechanisms underlying its dysregulation in human glioma remain poorly understood. In the present study, we demonstrate that up-regulation of Fra1 plays a crucial role in the glioma aggressiveness and epithelial-mesenchymal transition (EMT) activated by Wnt/ß-catenin signal pathway. In glioma cells, activation of Wnt/ß-catenin signalling by Wnt3a administration obviously induced EMT and directly activated the transcription of Fra1. Phenotype experiments revealed that up-regulation of Fra1 induced by Wnt/ß-catenin signalling drove the EMT of glioma cells. Furthermore, it was found that the cisplatin resistance acquired by Wnt/ß-catenin signalling activation depended on increased expression of Fra1. Analysis of clinical specimens verified a positive correlation between Fra1 and ß-catenin as well as a poor prognosis in glioma patients with double-high expressions of them. These findings indicate that an aberrant Wnt/ß-catenin signalling leads to the EMT and drug resistance of glioma via Fra1 induction, which suggests novel therapeutic strategies for the malignant disease.

Wnt signaling promotes axonal regeneration following optic nerve injury in the mouse.

Adult mammalian CNS axons generally do not regenerate, creating an obstacle to effective repair and recovery after neuronal injury. The canonical Wnt/ß-catenin signaling pathway is an essential signal transduction cascade that regulates axon growth and neurite extension in the developing mammalian embryo. In this study, we investigated whether a Wnt/ß-catenin signaling activator could be repurposed to induce regeneration in the adult CNS after axonal injury. We used a retinal ganglion cell (RGC) axon crush injury model in a transgenic Wnt reporter mouse, and intravitreal injections were used to deliver Wnt3a or saline to the RGC cell bodies within the retina. Our findings demonstrated that Wnt3a induced Wnt signaling in RGCs and resulted in significant axonal regrowth past the lesion site when measured at two and four weeks post-injury. Furthermore, Wnt3a-injected eyes showed increased survival of RGCs and significantly higher pattern electroretinography (PERG) amplitudes compared to the control. Additionally, Wnt3a-induced axonal regeneration and RGC survival were associated with elevated activation of the transcription factor Stat3, and reducing expression of Stat3 using a conditional Stat3 knock-out mouse line led to diminished Wnt3a-dependent axonal regeneration and RGC survival. Therefore, these findings reveal a novel role for retinal Wnt signaling in axonal regrowth and RGC survival following axonal injury, which may lead to the development of novel therapies for axonal regeneration.

Dishevelled stability is positively regulated by PKCζ-mediated phosphorylation induced by Wnt agonists.

Dishevelled (Dvl) proteins are central mediators of both canonical and non-canonical Wnt signaling. It is well known that, upon Wnt stimulation, Dvl becomes phosphorylated. However, how Wnt-induced phosphorylation of Dvl is regulated and its consequences are poorly understood. Here we found that Dvl proteins are overexpressed in colon cancer cells. In addition, we found that Wnt3a treatment rapidly induces hyperphosphorylation and stabilization of Dvl2 and Dvl3. The latter can be blocked by inhibition of Protein Kinase C (PKC)α, PKCδ, and PKCζ isoforms. We also found that Wnt3a-induced phosphorylation of Dvl3 by PKCζ is required to avoid Dvl3 degradation via proteasome. This demonstrated, to our knowledge for the first time, that hyperphosphorylation of Dvl by PKCζ results in Dvl stabilization. This is clear contrast with the consequences reported to date of CK1δ/ε-mediated Dvl phosphorylation upon Wnt treatment. Mapping the interaction domain between PKCζ and Dvl3 indicated that, although the Dvl-DIX domain is required to stabilize PKCζ-phosphorylated Dvl, it is not the region phosphorylated by this kinase. Our data show that the Dvl-DEP domain, required for specific interaction with PKCζ, is the site phosphorylated by this kinase, and also probably the Dvl-C terminus. Our findings suggest a model of positive regulation of PKCζ-mediated Dvl signaling activity, to produce a strong and sustained response to Wnt3a treatment by stabilizing Dvl protein levels.

[Effects of Wnt3a on osteogenic differentiation of dental pulp stem cells].

Objective: To investigate the effect of Wnt3a on osteogenic differentiation of human dental pulp stem cells (DPSC). Methods: DPSCs were subjected to different concentrations of Wnt3a (0, 5, 20, 50 and 100 µg/L) and at seven days after culture the alkaline phosphatase (ALP) activity was tested. Mineralized nodule formation was examined by alizarin red staining. Osteogenic-related gene expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type Ⅰ (COL-Ⅰ), Runt-related transcription factor-2 (RUNX2) was examined by quantitative real-time PCR (qPCR). Results: After seven days of induction by DPSC, Wnt3a protein could inhibit the ALP activity (concentration 0: 1.076±0.203, 5 µg/L: 0.828±0.118, 20 µg/L: 0.505±0.044, 50 µg/L: 0.499±0.038, 100 µg/L: 0.483±0.060). The expression of OCN in 5 µg/L Wnt3a group (0.092±0.005) was lower than that in culture medium (0.858±0.190)(P<0.05). Alizarin red staining showed that 5 µg/L Wnt3a had no mineralization induction effect on DPSC. Conclusions: Wnt3a could inhibit osteogenic differentiation of dental pulp stem cells.

Generation of cortical neurons from human induced-pluripotent stem cells by biodegradable polymeric microspheres loaded with priming factors.

Ischemic stroke is often associated with loss of cortical neurons leading to various neurological deficits. A cell replacement based on stem cell transplantation to repair the damaged brain requires the generation of specific neuronal subtypes. Recently, induced pluripotent stem cells have been used to generate various subtypes of neurons in vitro for transplantation in stroke-damaged brains. However, whether these cells can be primed as neuronal precursors to become cortical projection neurons by means of biomaterials releasing differentiation factors is not known. Here, we report that microspheres of biodegradable poly(ester-amide) composed of adipic acid, L-phenyl-alanine and 1,4-butanediol, loaded with differentiation factors, can be used to fate human induced pluripotent stem cell-derived long-term expandable neuroepithelial-like stem cells to cortical projection neurons. The three factors, Wnt3A, BMP4 and cyclopamine, were released from loaded microspheres over at least one month following biphasic dynamic time course, promoting cortical differentiation of the cells in vitro. Microspheres did not evoke significant inflammatory response after transplantation into intact rodent brain. Our study shows the potential of biodegradable polymer microspheres to promote neuronal differentiation by continuous release of factors, thereby creating the appropriate microenvironment. This new strategy may improve the efficacy of stem cell-based therapeutic approaches.

Integrated analysis of the Wnt responsive proteome in human cells reveals diverse and cell-type specific networks.

Wnt signalling is a fundamentally important signalling pathway that regulates many aspects of metazoan development and is frequently dysregulated in cancer. Although many of the core components of the Wnt signalling pathway, such as ß-catenin, have been extensively studied, the broad systems level responses of the mammalian cell to Wnt signalling are less well understood. In addition, the cell- or tissue-specific protein networks that modulate Wnt signalling in the diverse tissues or developmental stages in which it functions remain to be defined. To address these questions, we undertook a broad survey of the Wnt response in different human cell lines using both interaction and expression proteomics approaches. Our data reveal both similar and divergent responses of pathways and processes in the three cell-lines analyzed as well as a marked attenuation of the response to exogenous Wnt treatment in cells harbouring a stabilizing (activating) mutation of ß-catenin. We also identify cell-type specific components of the Wnt signalling network and find that by integrating expression and interaction proteomics data a more complete description of the Wnt interaction network can be achieved. Finally, our results attest to the power of LC-MS/MS to reveal novel cellular responses in even relatively well studied biological pathways such as Wnt signalling.