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Objective: We aimed to evaluate the prognostic performance of circulating Klotho in coronary atherosclerotic disease (CAD), and to further explore the effect of Klotho on stress-mediated endothelial senescence and underlying mechanism., Methods: A cohort of 295 patients had a 12-month follow-up for major adverse cardiovascular events (MACE). Serum Klotho was detected by enzyme linked immunosorbent assay. Cell viability, SA-β-Gal staining, the expression of P53 and P16 were analyzed for endothelial senescence. Oxidative stress was evaluated by measurement of reactive oxygen species, superoxide dismutase and malondialdehyde. LC3, P62, Wnt3a, GSK-3β and mTOR were analyzed by western blotting. Autophagosome formation was detected by adenovirus transfection., Results: In epidemiological analysis, low Klotho (≤295.9 pg/ml) was significantly associated with MACE risk (HR=2.266, 95 %CI 1.229-4.176). In experimental analysis, Klotho alleviated endothelial senescence and oxidative stress caused by Ang-II exposure; Klotho restored impaired autophagic flux to ameliorate Ang-II induced endothelial senescence; Ang-II activated Wnt3a/GSK-3β/mTOR signaling to inhibit autophagy, whereas Klotho restored autophagy through blockade of Wnt3a/GSK-3β/mTOR signaling; Klotho ameliorated endothelial senescence by suppressing Wnt3a/GSK-3β/mTOR pathway under Ang-II exposure., Conclusions: Prognostic significance of Klotho in CAD is potentially ascribed to its anti-endothelial senescence effect via autophagic flux restoration by inhibiting Wnt3a/ GSK-3β/mTOR signaling., Competing Interests: Conflicts of interest The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Stroke is a life-threatening disease with limited therapeutic options. Damage to the blood-brain barrier (BBB) is the key pathological feature of ischemic stroke. This study explored the role of the bradykinin (BK)/bradykinin 1 receptor (B1R) and its mechanism of action in the BBB. Human brain microvascular endothelial cells (BMECs) were used to test for cellular responses to BK by using the Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine staining, enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence, cellular permeability assays, and western blotting to evaluate cell viability, cytokine production, and reactive oxygen species (ROS) levels in vitro. A BBB induced by middle cerebral artery occlusion was used to evaluate BBB injuries, and the role played by BK/B1R in ischemic/reperfusion (I/R) was explored in a rat model. Results showed that BK reduced the viability of BMECs and increased the levels of proinflammatory cytokines (interleukin 6 [IL-6], IL-18, and monocyte chemoattractant protein-1) and ROS. Additionally, cellular permeability was increased by BK treatment, and the expression of tight junction proteins (claudin-5 and occludin) was decreased. Interestingly, Wnt3a expression was inhibited by BK and exogenous Wnt3a restored the effects of BK on BMECs. In an in vivo I/R rat model, knockdown of B1R significantly decreased infarct volume and inflammation in I/R rats. Our results suggest that BK might be a key inducer of BBB injury and B1R knockdown might provide a beneficial effect by upregulating Wnt3a., (© 2022 The Authors. Journal of Biochemical and Molecular Toxicology published by Wiley Periodicals LLC.)

We investigated whether BMP4, FGF8, and/or WNT3a on neural crest-like cells (NCLC) derived from mouse induced pluripotent stem (miPS) cells will promote differentiation of odontoblasts-like cells. After the miPS cells matured into embryonic body (EB) cells, they were cultured in a neural induction medium to produce NCLC. As the differentiation of NCLC were confirmed by RT-qPCR, they were then disassociated and cultured with a medium containing, BMP4, FGF8, and/or WNT3a for 7 and 14 days. The effect of these stimuli on NCLC were assessed by RT-qPCR, ALP staining, and immunocytochemistry. The cultured EB cells presented a significant increase of Snai1, Slug, and Sox 10 substantiating the differentiation of NCLC. NCLC stimulated with more than two stimuli significantly increased the odontoblast markers Dmp-1, Dspp, Nestin, Alp, and Runx2 expression compared to control with no stimulus. The expression of Dmp-1 and Dspp upregulated more when FGF8 was combined with WNT3a. ALP staining was positive in groups containing BMP4 and fluorescence was observed in immunocytochemistry of the common significant groups between Dmp-1 and Dspp. After stimulation, the cell morphology demonstrated a spindle-shaped cells with long projections resembling odontoblasts. Simultaneous BMP4, FGF8, and WNT3a stimuli significantly differentiated NCLC into odontoblast-like cells., (© 2022. The Author(s) under exclusive licence to The Japanese Society for Clinical Molecular Morphology.)

Osteogenic differentiation, the process by which bone marrow mesenchymal stem/stromal (a.k.a. skeletal stem) cells and osteoprogenitors form osteoblasts, is a critical event for bone formation during development, fracture repair, and tissue maintenance. Extra cellular and intracellular signaling pathways triggering osteogenic differentiation are relatively well known; however, the ensuing change in cell energy metabolism is less clearly defined. We and others have previously reported activation of mitochondria during osteogenic differentiation. To further elucidate the involved bioenergetic mechanisms and triggers, we tested the effect of osteogenic media containing ascorbate and β-glycerol phosphate, or various osteogenic hormones and growth factors on energy metabolism in long bone (ST2)- and calvarial bone (MC3T3-E1)-derived osteoprogenitors. We show that osteogenic media and differentiation factors, Wnt3a and BMP2, stimulate mitochondrial oxidative phosphorylation (OxPhos) with little effect on glycolysis. The activation of OxPhos occurs acutely, suggesting a metabolic signaling change rather than protein expression change. To this end, we found that the observed mitochondrial activation is Akt dependent. Akt is activated by osteogenic media, Wnt3a, and BMP2, leading to increased phosphorylation of various mitochondrial Akt targets, a phenomenon known to stimulate OxPhos. In sum, our data provide comprehensive analysis of cellular bioenergetics during osteoinduction in cells of two different origins (mesenchyme vs neural crest) and identify Wnt3a and BMP2 as physiological stimulators of mitochondrial respiration through Akt activation.

Little is known about the effect of wnt-3a on motor nerve function and its specific molecular mechanisms after spinal cord injury (SCI). This study demonstrates that the downregulated expression levels of caspases-3, caspases-9 and chondroitin sulfate proteoglycan (CSPG) proteins and number of proportion of transferase UTP nick end labeling (TUNEL)-positive neurons by wnt-3a treatment. Then, Nissl and hematoxylin-eosin (HE) staining showed that wnt-3a significantly reduced the loss of spinal anterior horn motor neurons and promoted repair of injured spinal cord tissues after SCI. The above factors constructed a favorable microenvironment for the recovery of motor nerve function after SCI. To elucidate the molecular mechanism of neuroprotection of wnt-3a on SCI, the study showed that the expression levels of Beclin-1 and light chain (LC)3-II/I in spinal cord neurons were significantly improved by wnt-3a after SCI in vitro and vivo experiments, while the effect of wnt-3a was inhibited after mechanistic target of rapamycin (mTOR) signaling pathway being activated by MHY-1485. Besides, the level of p70S6K phosphorylation was inhibited by wnt-3a treatment, on the contrary, the level of p70S6K protein was elevated by wnt-3a, indicating that wnt-3a significantly activated neuronal autophagy by inhibiting mTOR signaling pathway after SCI. To further verify the correlation between neuroprotection of wnt-3a and autophagy, we found that after the rats and spinal cord neurons were combined treatment with wnt-3a and MHY-1485, the neuroprotection of wnt-3a on SCI was significantly inhibited. This study is the first to report that wnt-3a improves functional recovery through autophagy activation via inhibiting the mTOR signaling pathway after SCI., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: Nanog is expressed in adult endothelial cells (ECs) at a low-level, however, its functional significance is not known. The goal of our study was to elucidate the role of Nanog in adult ECs using a genetically engineered mouse model system. Approach and Results: Biochemical analyses showed that Nanog is expressed in both adult human and mouse tissues. Primary ECs isolated from adult mice showed detectable levels of Nanog, Tert (telomerase reverse transcriptase), and eNos (endothelial nitric oxide synthase). Wnt3a (Wnt family member 3A) increased the expression of Nanog and hTERT (human telomerase reverse transcriptase) in ECs and increased telomerase activity in these cells. In a chromatin immunoprecipitation experiment, Nanog directly bound to the hTERT and eNOS promoter/enhancer DNA elements, thereby regulating their transcription. Administration of low-dose tamoxifen to ROSA mT/mG ::Nanog fl/+ ::Cdh5 CreERT2 mice induced deletion of a single Nanog allele, simultaneously labeling ECs with green fluorescent protein and resulting in decreased Tert and eNos levels. Histological and morphometric analyses of heart tissue sections prepared from these mice revealed cell death, microvascular rarefaction, and increased fibrosis in cardiac vessels. Accordingly, EC-specific Nanog -haploinsufficiency resulted in impaired EC homeostasis and angiogenesis. Conversely, re-expression of cDNA encoding the hTERT in Nanog-depleted ECs, in part, restored the effect of loss of Nanog., Conclusions: We showed that low-level Nanog expression is required for normal EC homeostasis and angiogenesis in adulthood.

Wnt3a-coated beads can induce asymmetric divisions of mouse embryonic stem cells (mESCs), resulting in one self-renewed mESC and one differentiating epiblast stem cell. This provides an opportunity for studying histone inheritance pattern at a single-cell resolution in cell culture. Here, we report that mESCs with Wnt3a-bead induction display nonoverlapping preexisting (old) versus newly synthesized (new) histone H3 patterns, but mESCs without Wnt3a beads have largely overlapping patterns. Furthermore, H4K20me2/3, an old histone-enriched modification, displays a higher instance of asymmetric distribution on chromatin fibers from Wnt3a-induced mESCs than those from non-induced mESCs. These locally distinct distributions between old and new histones have both cellular specificity in Wnt3a-induced mESCs and molecular specificity for histones H3 and H4. Given that post-translational modifications at H3 and H4 carry the major histone modifications, our findings provide a mammalian cell culture system to study histone inheritance for maintaining stem cell fate and for resetting it during differentiation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intestinal epithelial barrier dysfunction is a risk factor in the pathogenesis of Crohn's disease (CD); however, no corrective FDA-approved therapies exist. We used an enteroid (EnO)-based system in two murine models of experimental CD, SAMP1/YitFc (SAMP) and TNF ΔARE/+ (TNF). While severely inflamed SAMP mice do not generate EnOs, "inflammation-free" SAMP mice form EnO structures with impaired morphology and reduced intestinal stem cell (ISC) and Paneth cell viability. We validated these findings in TNF mice concluding that inflammation in intestinal tissues impedes EnO generation and suppressing inflammation by steroid administration partially rescues impaired formation in SAMP mice. We generated the first high-resolution transcriptional profile of the SAMP ISC niche demonstrating that alterations in multiple key pathways contribute to niche defect and targeting them may partially rescue the phenotype. Furthermore, we correlated the defects in formation and the rescue of EnO formation to reduced viability of ISCs and Paneth cells., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: This study aimed to investigate the effects of combined activin A and Wnt3a treatment on definitive endoderm (DE) differentiation from human parthenogenetic embryonic stem cells (hPESCs)., Methods: hPESCs on human foreskin fibroblast feeder layers were induced to differentiate into DE using a combination of 50 ng/ml activin A and 25 ng/ml Wnt3a. Expression of the DE markers CXCR4, E-cadherin (ECD), Sox17, and Goosecoid (Gsc) were examined using flow cytometry and real-time quantitative PCR., Results: The combination of activin A and Wnt3a significantly enhanced the percentages of CXCR4 + , ECD + , Sox17 + , and Gsc + cells, culminating on day 2 of induction. This combined use promoted DE differentiation from hPESCs in vitro ., Conclusions: Through the combination treatment using activin A and Wnt3a, DE differentiation from hPESCs culminated at 48 h, which can be regarded as the optimal time-point to induce differentiation of endodermal cells such as pancreatic, liver, and intestinal cells., (© 2020 by the Association of Clinical Scientists, Inc.)

Precision-cut intestinal slices (PCIS) is an ex vivo culture technique that found its applications in toxicology, drug transport and drug metabolism testing, as well as in fibrosis research. The main limiting factor of PCIS as experimental model is the relatively short viability of tissue slices. Here, we describe a strategy for extending the life-span of PCIS during culture using medium that is routinely used for growing intestinal organoids. Mouse and rat PCIS cultured in standard medium progressively showed low ATP/protein content and severe tissue degradation, indicating loss of tissue viability. In turn, organoid medium, containing epithelial growth factor (EGF), Noggin and R-spondin, maintained significantly higher ATP/protein levels and better preserved intestinal architecture of mouse PCIS at 96 h. In contrast, organoid medium that additionally contained Wnt, had a clear positive effect on the ATP content of rat PCIS during 24 h of culture, but not on slice histomorphology. Our proof-of-concept study provides early evidence that employing organoid medium for PCIS culture improved tissue viability during extended incubation. Enabling lasting PCIS cultures will greatly widen their range of applications in predicting long-term intestinal toxicity of xenobiotics and elucidating their mechanism of action, among others., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Our goal was to evaluate alveolar bone healing in OVX mice, and to assess the functional utility of a WNT-based treatment to accelerate healing in mice with an osteoporotic-like bony phenotype., Introduction: Is osteoporosis a risk factor for dental procedures? This relatively simple question is exceedingly difficult to answer in a clinical setting, for two reasons. First, as an age-related disease, osteoporosis is frequently accompanied by age-related co-morbidities that can contribute to slower tissue repair. Second, the intervals at which alveolar bone repair are assessed in a clinical study are often measured in months to years. This study aimed to evaluate alveolar bone repair in ovariectomized (OVX) mice and provide preclinical evidence to support a WNT-based treatment to accelerate alveolar bone formation., Methods: OVX was performed in young mice to produce an osteoporotic-like bone phenotype. Thereafter, the rate of extraction socket healing and osteotomy repair was assessed. A liposomal WNT3A treatment was tested for its ability to promote alveolar bone formation in this OVX-induced model of bone loss., Results: Bone loss was observed throughout the murine skeleton, including the maxilla, and mirrored the pattern of bone loss observed in aged mice. Injuries to the alveolar bone, including tooth extraction and osteotomy site preparation, both healed significantly slower than the same injuries produced in young controls. Given sufficient time, however, all injuries eventually healed. In OVX mice, osteotomies healed significantly faster if they were treated with L-WNT3A., Conclusions: Alveolar bone injuries heal slower in OVX mice that exhibit an osteoporotic-like phenotype. The rate of alveolar bone repair in OVX mice can be significantly promoted with local delivery of L-WNT3A.

The network of Wingless/Int-1 (WNT)-induced signaling pathways includes β-catenin-dependent and -independent pathways. β-Catenin regulates T cell factor/lymphoid enhancer-binding factor (TCF/LEF)-mediated gene transcription, and in response to WNTs, β-catenin signaling is initiated through engagement of a Frizzled (FZD)/LDL receptor-related protein 5/6 (LRP5/6) receptor complex. FZDs are G protein-coupled receptors, but the question of whether heterotrimeric G proteins are involved in WNT/β-catenin signaling remains unanswered. Here, we investigate whether acute activation of WNT/β-catenin signaling by purified WNT-3A requires functional signaling through heterotrimeric G proteins. Using genome editing, we ablated expression of G s /G olf /G q /G 11 /G 12 /G 13 /G z in HEK293 (ΔG7) cells, leaving the expression of pertussis toxin (PTX)-sensitive G i/o proteins unchanged, to assess whether WNT-3A activates WNT/β-catenin signaling in WT and ΔG7 cells devoid of functional G protein signaling. We monitored WNT-3A-induced activation by detection of phosphorylation of LDL receptor-related protein 6 (LRP6), electrophoretic mobility shift of the phosphoprotein Dishevelled (DVL), β-catenin stabilization and dephosphorylation, and TCF-dependent transcription. We found that purified, recombinant WNT-3A efficiently induces WNT/β-catenin signaling in ΔG7 cells in both the absence and presence of G i/o -blocking PTX. Furthermore, cells completely devoid of G protein expression, so called Gα-depleted HEK293 cells, maintain responsiveness to WNT-3A with regard to the hallmarks of WNT/β-catenin signaling. These findings corroborate the concept that heterotrimeric G proteins are not required for this FZD- and DVL-mediated signaling branch. Our observations agree with previous results arguing for FZD conformation-dependent functional selectivity between DVL and heterotrimeric G proteins. In conclusion, WNT/β-catenin signaling through FZDs does not require the involvement of heterotrimeric G proteins., (© 2019 Bowin et al.)

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.

The small intestine plays an important role in the absorption and metabolism of oral drugs. In the current evaluation system, it is difficult to predict the precise absorption and metabolism of oral drugs. In this study, we generated small intestinal epithelial-like cells from human induced pluripotent stem cells (hiPS-SIECs), which could be applied to drug absorption and metabolism studies. The small intestinal epithelial-like cells were efficiently generated from human induced pluripotent stem cell by treatment with WNT3A, R-spondin 3, Noggin, EGF, IGF-1, SB202190, and dexamethasone. The gene expression levels of small intestinal epithelial cell (SIEC) markers were similar between the hiPS-SIECs and human adult small intestine. Importantly, the gene expression levels of colonic epithelial cell markers in the hiPS-SIECs were much lower than those in human adult colon. The hiPS-SIECs generated by our protocol exerted various SIEC functions. In conclusion, the hiPS-SIECs can be utilized for evaluation of drug absorption and metabolism., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The objective of our experiments was to identify new therapeutic strategies to stimulate dentin formation in an adult tooth. To address this objective, we evaluated dentin production in 2 acute trauma models: one involving a pulp exposure and the other involving a superficial dentin injury. Molecular, cellular, and histologic analyses revealed that in response to a severe injury, where the pulp is exposed to the oral cavity, cell death is rampant and the repair response initiates from surviving pulp cells and, to a lesser extent, surviving odontoblasts. When an injury is superficial, as in the case of a dentin injury model, then disturbances are largely confined to pulp tissue immediately underneath the damaged dentin tubules. We found that the pulp remained vital and innervated; primary odontoblasts upregulated HIF1α; and the rate of mineralization was significantly increased. A tamoxifen-inducible Axin2 CreERT2/+ ; R26R mTmG/+ reporter strain was then used to demonstrate that a population of long-lived Wnt-responsive odontoblasts, which secreted dentin throughout the life of the animal, were responsible for depositing new dentin in response to a superficial injury. Amplifying Wnt signaling in the pulp stimulates dentin secretion, and in the dentin injury model, we show that a liposomal formulation of human WNT3A protein passes through dentinal tubules and is capable of upregulating Wnt signaling in the pulp. These data provide strong proof of concept for a therapeutic pulp-capping material to stimulate Wnt signaling in odontoblasts and thus improve the pulp repair response.

After ischemic stroke, apoptosis of neurons is a primary factor in determining outcome. Wnt3a is a naturally occurring protein that has been shown to have protective effects in the brain for traumatic brain injury. Although wnt3a has been investigated in the phenomena of neurogenesis, anti-apoptosis, and anti-inflammation, it has never been investigated as a therapy for stroke. We hypothesized that the potential neuroprotective agent wnt3a would reduce infarction and improve behavior following ischemic stroke by attenuating neuronal apoptosis and promoting cell survival through the Frizzled-1/PIWI1a/FOXM1 pathway in middle cerebral artery occlusion (MCAO) rats. A total of 229 Sprague Dawley rats were assigned to male, female, and 9-month-old male MCAO or sham groups followed by reperfusion 2 h after MCAO. Animals assigned to MCAO were either given wnt3a or its control. To explore the downstream signaling of wnt3a, the following interventions were given: Frizzled-1 siRNA, PIWI1a siRNA, and PIWI1a-clustered regularly interspaced short palindromic repeats, along with the appropriate controls. Post-MCAO assessments included neurobehavioral tests, infarct volume, Western blot, and immunohistochemistry. Endogenous levels of wnt3a and Frizzled-1/PIWI1a/FOXM1 were lowered after MCAO. The administration of intranasal wnt3a, 1 h after MCAO, increased PIWIL1a and FOXM1 expression through Frizzled-1, reducing brain infarction and neurological deficits at 24 and 72 h. Frizzled-1 and PIWI1a siRNAs reversed the protective effects of wnt3a after MCAO. Restoration of PIWI1a after knockdown of Frizzled-1 increased FOXM1 survival protein and reduced cleaved caspase-3 levels. In summary, wnt3a decreases neuronal apoptosis and improves neurological deficits through Frizzled-1/PIWI1a/FOXM1 pathway after MCAO in rats. Therefore, wnt3a is a novel intranasal approach to decrease apoptosis after stroke. SIGNIFICANCE STATEMENT Only 5% of patients receive recombinant tissue plasminogen activator after stroke, and few qualify for mechanical thrombectomy. No neuroprotective agents have been successfully translated to promote neuronal survival in stroke. Thus, using a clinically relevant rat model of stroke, middle cerebral artery occlusion, we explored a novel intranasal administration of wnt3a. wnt3a naturally occurs in the body and crosses the blood-brain barrier, supporting the clinically translatable approach of intranasal administration. Significant neuronal apoptosis occurs during stroke, and wnt3a shows promise due to its antiapoptotic effects. We investigated whether wnt3a mediates its poststroke effects via Frizzled-1 and the impact on its downstream signaling molecules, PIWI1a and FOXM1, in apoptosis. Elucidating the mechanism of wnt3a will identify additional pharmacological targets and further understanding of stroke., (Copyright © 2018 the authors 0270-6474/18/386787-15$15.00/0.)

Background: Klotho is a well-known anti-aging hormone, which serves as a suppressor of aging through a variety of mechanisms. Aging of skeletal muscle is concomitant with a decrease in muscle stem cell function resulting in impaired regeneration., Methods: Here we investigate the functional role of the anti-aging hormone Klotho for muscle stem cell function after cardiotoxin-induced injury of skeletal muscle using a klotho hypomorphic mouse line, which is characterized by a premature aging phenotype. Furthermore, we perform floating single myofiber cultures with their adjacent muscle stem cells to investigate the interplay between canonical Wnt signaling and Klotho function., Results: We demonstrate that muscle stem cell numbers are significantly decreased in klotho hypomorphic mice. Furthermore, we show that muscle stem cell function is also severely impaired upon loss of klotho expression, in culture and during regeneration in vivo. Moreover, we demonstrate that addition of recombinant Klotho protein inhibits aberrant excessive Wnt signaling in aged muscle stem cells thereby restoring their functionality., Conclusions: The anti-aging hormone Klotho counteracts aberrant canonical Wnt signaling in muscle stem cells and might be one of the naturally occurring inhibitors of canonical Wnt signaling in skeletal muscle.

Stem cells residing in the periodontal ligament (PDL) support the homeostasis of the periodontium, but their in vivo identity, source(s), and function(s) remain poorly understood. Here, using a lineage-tracing mouse strain, we identified a quiescent Wnt-responsive population in the PDL that became activated in response to tooth extraction. The Wnt-responsive population expanded by proliferation, then migrated from the PDL remnants that remained attached to bundle bone, into the socket. Once there, the Wnt-responsive progeny upregulated osteogenic protein expression, differentiated into osteoblasts, and generated the new bone that healed the socket. Using a liposomal WNT3A protein therapeutic, we showed that a single application at the time of extraction was sufficient to accelerate extraction socket healing 2-fold. Collectively, these data identify a new stem cell population in the intact periodontium that is directly responsible for alveolar bone healing after tooth removal.

Cementum is a calcified, avascular connective tissue that laminates the root of a tooth and plays a pivotal role in the development, homeostasis, and regeneration of a periodontal tissue. As a potential treatment for periodontal tissue defects in the patient with chronic periodontitis, much attention has been paid to tissue engineering combined with mesenchymal stem cells for regenerating periodontal tissues including cementum. However, limited information is available for the molecular factors that have impacts on the differentiation of mesenchymal stem cells into cementoblasts. Here, we focus on the effect of Wnt3a as a potential inducer and tested the effect of this protein in vitro using human bone marrow-derived mesenchymal stem cells. It was found that, when cells were cultured in an osteogenic medium containing Wnt3a, cementoblast-specific genes, such as cementum protein 1 and cementum attachment protein, as well as bone-related genes were significantly upregulated. These results suggest that Wnt3a promotes differentiation of the cells into cementoblast-like cells. Further experiments were carried out using inhibitors to gain deeper insights into molecular mechanisms underlying the observed differentiation. As a result, we conclude that Wnt3a-triggered differentiation into cementoblast-like cells is the consequence of the activation of the canonical Wnt signaling pathway with possible involvement of the non-canonical pathway.

Tumor: tumor microenvironment (TME) interactions are critical for tumor progression and the composition and structure of the local extracellular matrix (ECM) are key determinants of tumor metastasis. We recently reported that activation of Wnt/beta-catenin signaling in Ewing sarcoma cells induces widespread transcriptional changes that are associated with acquisition of a metastatic tumor phenotype. Significantly, ECM protein-encoding genes were found to be enriched among Wnt/beta-catenin induced transcripts, leading us to hypothesize that activation of canonical Wnt signaling might induce changes in the Ewing sarcoma secretome. To address this hypothesis, conditioned media from Ewing sarcoma cell lines cultured in the presence or absence of Wnt3a was collected for proteomic analysis. Label-free mass spectrometry was used to identify and quantify differentially secreted proteins. We then used in silico databases to identify only proteins annotated as secreted. Comparison of the secretomes of two Ewing sarcoma cell lines revealed numerous shared proteins, as well as a degree of heterogeneity, in both basal and Wnt-stimulated conditions. Gene set enrichment analysis of secreted proteins revealed that Wnt stimulation reproducibly resulted in increased secretion of proteins involved in ECM organization, ECM receptor interactions, and collagen formation. In particular, Wnt-stimulated Ewing sarcoma cells up-regulated secretion of structural collagens, as well as matricellular proteins, such as the metastasis-associated protein, tenascin C (TNC). Interrogation of published databases confirmed reproducible correlations between Wnt/beta-catenin activation and TNC and COL1A1 expression in patient tumors. In summary, this first study of the Ewing sarcoma secretome reveals that Wnt/beta-catenin activated tumor cells upregulate secretion of ECM proteins. Such Wnt/beta-catenin mediated changes are likely to impact on tumor: TME interactions that contribute to metastatic progression., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Traumatic brain injury (TBI) is a prevalent disorder, but no effective therapies currently exist. An underlying pathophysiology of TBI includes the pathological elevation of autophagy. β-Catenin, a downstream mediator of the canonical Wnt pathway, is a repressor of autophagy. The Wnt/β-catenin pathway plays a crucial role in cell proliferation and neuronal plasticity/repair in the adult brain. We hypothesized that activation of this pathway could promote neuroprotection and neural regeneration following TBI. In the controlled cortical impact (CCI) model of TBI in C57BL/6 mice (total n = 160), we examined intranasal application of recombinant Wnt3a (2 μg/kg) in a short-term (1 dose/day for 2 days) and long-term (1 dose/day for 7 days) regimen. Immunohistochemistry was performed at 1 to 14 days post-TBI to assess cell death and neurovascular regeneration. Western blotting measured canonical Wnt3a activity, expression of growth factors, and cell death markers. Longitudinal behavior assays evaluated functional recovery. In short-term experiments, Wnt3a treatment with a 60-min delay post-TBI suppressed TBI-induced autophagic activity in neurons (44.3 ± 6.98 and 4.25 ± 2.53 LC3+/NeuN+ double positive cells in TBI+Saline and TBI+Wnt3a mice, respectively; p < 0.0001, n = 5/group), reduced autophagic markers light chain 3 (LC3)-II and Beclin-1, as well as injury markers caspase-3 and matrix metalloproteinase 9 (MMP-9). The Wnt3a treatment reduced cell death and contusion volume (0.72 ± 0.07 mm 2 and 0.26 ± 0.04 mm 2 in TBI+Saline and TBI+Wnt3a mice, respectively; p < 0.001, n = 5/group). The 7-day Wnt3a treatment increased levels of β-catenin and growth factors glial-derived growth factor (GDNF) and vascular endothelial growth factor (VEGF). This chronic Wnt3a therapy augmented neurogenesis (0.52 ± 0.09 and 1.25 ± 0.13 BrdU+/NeuN+ co-labeled cells in TBI+Saline mice and TBI+Wnt3a mice, respectively; p < 0.01, n = 6/group) and angiogenesis (0.26 ± 0.07 and 0.74 ± 0.13 BrdU+/GLUT1+ co-labeled cells in TBI+Saline and TBI+Wnt3a mice, respectively; p = 0.014, n = 6/group). The treatment improved performance in the rotarod test and adhesive removal test. Targeting the Wnt pathway implements a unique combination of protective and regenerative approaches after TBI.

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 β-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 β-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 β-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.

Purpose: Wnt/β-catenin signaling in the trabecular meshwork (TM) is required for maintaining normal intraocular pressure (IOP), although the mechanism(s) behind this are unknown. We hypothesize that Wnt/β-catenin signaling regulates IOP via β-catenin's effects on cadherin junctions., Methods: Nonglaucomatous primary human TM (NTM) cells were treated with or without 100 ng/ml Wnt3a, 1 μg/ml sFRP1, or both for 4 to 48 hours. Cells were immunostained for β-catenin, total cadherins, or cadherin isoforms. Membrane proteins or whole-cell lysates were isolated for Western immunoblotting and probed for cadherin isoforms. RNA was extracted for cDNA synthesis and qPCR analysis of cadherin expression. Some NTM cells were cultured on electric plates for cell impedance assays. Ad5.CMV recombinant adenoviruses encoding K-cadherin, and/or sFRP1 were injected into eyes of 4- to 6-month-old female BALB/cJ mice (n = 8-10). Conscious IOPs were assessed for 35 days., Results: Upon Wnt3a treatment, total cadherin expression increased and β-catenin accumulated at the TM cell membrane and on processes formed between TM cells. qPCR showed that Wnt3a significantly increased K-cadherin expression in NTM cells (P < 0.01, n = 3), and Western immunoblotting showed that Wnt3a increased K-cadherin in NTM cells, which was inhibited by the addition of sFRP1. Cell impedance assays showed that Wnt3a treatment increased transcellular resistance and anti-K-cadherin siRNA decreased transcellular resistance (P < 0.001, n = 4-6). Our in vivo study showed that K-cadherin significantly decreased sFRP1-induced ocular hypertension (P < 0.05, n = 6). Western immunoblotting also showed that K-cadherin alleviated sFRP1-induced β-catenin decrease in mouse anterior segments., Conclusions: Our results suggest that cadherins play important roles in the regulation of TM homeostasis and IOP via the Wnt/β-catenin pathway.

Dermal papilla (DP) regulates the growth and cycling of hair follicles. Cultured DP cells are useful for the study of their role in relation to hair growth and regeneration. However, cultivation of human DP cells is tedious and difficult. In addition, cultured DP cells possess a relatively short replicative life span, requiring immortalized human DP cell lines. We previously established an immortalized human DP cell line, SV40T-hTERT-DPC, by introducing human telomerase reverse transcriptase (hTERT) gene into the transformed cell line, SV40T-DPC. In this study, we co-transfected the simian virus 40 large T antigen (SV40T-Ag) and hTERT into DP cells from scalp hair follicles from a male with androgenetic alopecia and established five immortalized DP cell lines and named KNU-101, KNU-102, KNU-103, KNU-201 and KNU-202. We then evaluated tumorigenicity, expression of DP markers, responses to androgen, Wnt3a and BMP4, and expression of DP signature genes. These cell lines displayed early passage morphology and maintained responses to androgen, Wnt and BMP. Furthermore, these cell lines expressed DP markers and DP signature genes. KNU cell lines established in this study are potentially useful sources for hair research., (Copyright © 2018 Elsevier Inc. All rights reserved.)

mTORC1 signaling not only plays important physiological roles in the regulation of proliferation and osteogenic differentiation of BMSCs, but also mediates exogenous Wnt-induced protein anabolism and osteoblast differentiation. However, the downstream effectors of the mTORC1 signaling in the above processes are still poorly understood. In this study, we explored the specific role of S6K1, one of the major targets of the mTORC1 pathway, in BMSCs self-renewal and osteogenic differentiation. We first found that S6K1 was active in primary mouse bone marrow stromal cells, and further activated upon osteogenic induction. We then determined the effects of S6K1 inhibition by LY2584702 Tosylate, a selective inhibitor of S6K1 (hereafter S6KI), using both primary mouse bone marrow stromal cells and ST2 cells. Colony-Forming Unit-Fibroblast (CFU-F) assays showed that S6KI dramatically reduced the total number of colonies formed in primary BMSCs cultures. Under the basal osteogenic culture condition, S6KI significantly inhibited mRNA expression of osteoblast marker genes (Sp7, Bglap, Ibsp, and Col1a1), ALP activity and matrix mineralization. Upon Wnt3a treatments, S6KI inhibited Wnt3a-induced osteoblast differentiation and expression of protein anabolism genes in ST2 cells, but to a much lesser degree than rapamycin (a specific inhibitor of mTORC1 signaling). Collectively, our findings have demonstrated that pharmacological inhibition of S6K1 impaired self-renewal and osteogenic differentiation of BMSCs, but only partially suppressed exogenous Wnt3a-induced osteoblast differentiation and protein anabolism., (© 2017 Wiley Periodicals, Inc.)

The Wnt/ β -catenin pathway is crucial in normal development and throughout life, but aberrant activation of this pathway has been linked to kidney fibrosis, although the mechanisms involved remain incompletely determined. Here, we investigated the role of Wnt/ β -catenin in regulating macrophage activation and the contribution thereof to kidney fibrosis. Treatment of macrophages with Wnt3a exacerbated IL-4- or TGF β 1-induced macrophage alternative (M2) polarization and the phosphorylation and nuclear translocation of STAT3 in vitro Conversely, inhibition of Wnt/ β -catenin signaling prevented these IL-4- or TGF β 1-induced processes. In a mouse model, induced deletion of β -catenin in macrophages attenuated the fibrosis, macrophage accumulation, and M2 polarization observed in the kidneys of wild-type littermates after unilateral ureter obstruction. This study shows that activation of Wnt/ β -catenin signaling promotes kidney fibrosis by stimulating macrophage M2 polarization., (Copyright © 2018 by the American Society of Nephrology.)

Human perivascular stem/stromal cells (hPSC) are a multipotent mesenchymogenic stromal cell population defined by their perivascular locale. Recent studies have demonstrated the high potential for clinical translation of this fluorescence-activated cell sorting (FACS)-derived cell population for autologous bone tissue engineering. However, the mechanisms underlying the osteogenic differentiation of PSC are incompletely understood. The current study investigates the roles of canonical and noncanonical Wnt signaling in the osteogenic and adipogenic differentiation of PSC. Results showed that both canonical and noncanonical Wnt signaling activity transiently increased during PSC osteogenic differentiation in vitro. Sustained WNT3A treatment significantly decreased PSC osteogenic differentiation. Conversely, sustained treatment with Wnt family member 16 (WNT16), a mixed canonical and noncanonical ligand, increased osteogenic differentiation in a c-Jun N-terminal kinase (JNK) pathway-dependent manner. Conversely, WNT16 knockdown significantly diminished PSC osteogenic differentiation. Finally, WNT16 but not WNT3A increased the adipogenic differentiation of PSC. These results indicate the importance of regulation of canonical and noncanonical Wnt signaling for PSC fate and differentiation. Moreover, these data suggest that WNT16 plays a functional and necessary role in PSC osteogenesis.

Aberrant activation of the canonical Wingless type (Wnt) signaling pathway plays a key role in the development of hypertrophic scars and keloids, and this aberrant activation of Wnt pathway can be a potential target for the development of novel anti-fibrotic agents. In this study, we evaluated the anti-fibrotic potential of a soluble Wnt decoy receptor (sLRP6E1E2)-expressing non-replicating adenovirus (Ad; dE1-k35/sLRP6E1E2) on human dermal fibroblasts (HDFs), keloid fibroblasts (KFs), and keloid tissue explants. Higher Wnt3a and β-catenin expression was observed in the keloid region compared to the adjacent normal tissues. The activity of β-catenin and mRNA expression of type-I and -III collagen were significantly decreased following treatment with dE1-k35/sLRP6E1E2 in HDFs and KFs. The expression of LRP6, β-catenin, phosphorylated glycogen synthase kinase 3 beta, Smad 2/3 complex, and TGF-β1 were decreased in Wnt3a- or TGF-β1-activated HDFs, following administration of dE1-k35/sLRP6E1E2. Moreover, dE1-k35/sLRP6E1E2 markedly inhibited nuclear translocation of both β-catenin and Smad 2/3 complex. The expression levels of type-I and -III collagen, fibronectin, and elastin were also significantly reduced in keloid tissue explants after treatment with dE1-k35/sLRP6E1E2. These results indicate that Wnt decoy receptor-expressing Ad can degrade extracellular matrix in HDFs, KFs, and primary keloid tissue explants, and thus it may be beneficial for treatment of keloids.

Wnt proteins preferentially activate either β-catenin-dependent or β-catenin-independent signals, but the activity of a particular Wnt also depends on cellular context and receptor availability. We previously reported that Wnt-3a induces neural differentiation of human embryonic stem cell-derived neural stem cells (NSCs) in a β-catenin-independent manner by activating a signal involving JNK and the AP-1 family member ATF-2. Here, we report the results of a gene silencing approach to identify the Wnt receptors that mediate this response to Wnt-3a. Silencing of ROR2 increased neuronal differentiation, as measured by expression of the genes DCX, NEUROD1, and NGN1, suggesting ROR2 signals normally prevent differentiation. Silencing of the other Wnt receptors singly did not affect Wnt-3a-induced neuronal differentiation. However, pairwise silencing of ROR1 and FZD4 or FZD5 and of LRP6 and FZD4 or FZD5 inhibited neuronal differentiation, as detected by reductions in the expression of neuronal genes and immunocytochemical detection of DCX, NEUROD1 and DCX. Ectopic expression of these receptors in HEK 293 cells increased ATF2-dependent transcription. In addition, ROR1 coimmunoprecipitated with FZD4 and LRP6 in transfected HEK 293 cells and colocalized with FZD4 and with LRP6 at the cell surface of transfected L cells. Wnt-3a did not appear to affect these interactions but did alter the interactions between LRP6 and FZD4/5. Together, these observations highlight roles for ROR1, LRP6, FZD4, and FZD5 in neural stem cell differentiation and provide support for a model in which dynamic interactions among these receptors mediate Wnt-3a activation of ATF2 signaling.

Signalling downstream of Activin/Nodal (ActA) and Wnt can induce endoderm differentiation and also support self-renewal in pluripotent cells. Here we find that these apparently contradictory activities are fine-tuned by insulin. In the absence of insulin, the combination of these cytokines supports endoderm in a context-dependent manner. When applied to naive pluripotent cells that resemble peri-implantation embryos, ActA and Wnt induce extra-embryonic primitive endoderm (PrE), whereas when applied to primed pluripotent epiblast stem cells (EpiSC), these cytokines induce gastrulation-stage embryonic definitive endoderm. In naive embryonic stem cell culture, we find that insulin complements LIF signalling to support self-renewal; however, when it is removed, LIF, ActA and Wnt signalling not only induce PrE differentiation, but also support its expansion. Self-renewal of these PrE cultures is robust and, on the basis of gene expression, these cells resemble early blastocyst-stage PrE, a naive endoderm state able to make both visceral and parietal endoderm.

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.

Background & Aims: Neural stem and progenitor cells from the enteric nervous system (ENS) might serve as a source of cells for treatment of neurogastrointestinal disorders. Before we can use these cells, we must increase our understanding of the signaling mechanisms that regulate proliferation and differentiation. We systematically evaluated the effects of canonical Wnt signaling on proliferation and differentiation of cultured ENS progenitor cells from neonatal mice and humans., Methods: We isolated ENS progenitors from tunica muscularis of the small intestine of newborn (postnatal day 0) wild-type C57BL/6 mice as well as from Wnt1-Cre2 reporter mice. We also obtained intestinal tissue samples from infants (2 and 7 months old) undergoing surgery for imperforate anus or focal intestinal perforation and isolated ENS cells. ENS cells were cultured under proliferation conditions leading to formation of 3-dimensional spheres, which we activated with Wnt3a and SB216763 in order to activate the β-catenin-dependent canonical Wnt pathway. We used immunoblot and quantitative polymerase chain reaction to evaluate the molecular response to Wnt stimuli and immunohistochemistry, proliferation, and cell death assays to identify new neurons., Results: In proliferating enterospheres derived from ENS progenitor cells, we verified the expression of Wnt receptors frizzled 1-10 and the co-receptors low-density lipoprotein receptor-related proteins 5 and 6. Pharmacologic stimulation with Wnt agonists led to intracellular accumulation of Wnt-dependent β-catenin and up-regulated expression of known Wnt target genes axin2, lef1, and lgr5. Activation of the canonical Wnt pathway promoted growth of ENS cell spheres during cell expansion and increased the number of newborn neurons derived from mouse and human progenitor cells., Conclusions: In studies of human and mouse ENS progenitors, we found activation of the Wnt signaling pathway to promote neurogenesis of the ENS in vitro. The neurogenic effect of Wnt agonists on ENS progenitors supports their use in generation of cell pools for autologous cell replacement therapies., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) modulates many functions in endothelial cells. S-nitrosylation (SNO) of cysteine residues on β-catenin by eNOS-derived NO has been shown to influence intercellular contacts between endothelial cells. However, the implication of SNO in the regulation of β-catenin transcriptional activity is ill defined. Here, we report that NO inhibits the transcriptional activity of β-catenin and endothelial cell proliferation induced by activation of Wnt/β-catenin signaling. Interestingly, induction by Wnt3a of β-catenin target genes, such as the axin2 gene, is repressed in an eNOS-dependent manner by vascular endothelial growth factor (VEGF). We identified Cys466 of β-catenin as a target for SNO by eNOS-derived NO and as the critical residue for the repressive effects of NO on β-catenin transcriptional activity. Furthermore, we observed that Cys466 of β-catenin, located at the binding interface of the β-catenin-TCF4 transcriptional complex, is essential for disruption of this complex by NO. Importantly, Cys466 of β-catenin is necessary for the inhibitory effects of NO on Wnt3a-stimulated proliferation of endothelial cells. Thus, our data define the mechanism responsible for the repressive effects of NO on the transcriptional activity of β-catenin and link eNOS-derived NO to the modulation by VEGF of Wnt/β-catenin-induced endothelial cell proliferation., (Copyright © 2017 American Society for Microbiology.)

In canonical pathway, Wnt3A has been known to stabilize β-catenin through the dissociation between β-catenin and glycogen synthase kinase-3β (GSK-3β) that suppresses the phosphorylation and degradation of β-catenin. In non-canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross-talk between canonical and non-canonical pathways by Wnt signaling; however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK-3β and accumulation of β-catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh-RhoA and Tat-C3 abolished both the phosphorylation of GSK-3β and accumulation of β-catenin. Y27632, an inhibitor of Rho-associated coiled coil kinase (ROCK) and si-ROCK inhibited both GSK-3β phosphorylation and β-catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK-3β in vitro. In addition, Wnt3A-induced cell proliferation and migration, which were inhibited by Tat-C3 and Y27632. Taken together, we propose the cross-talk between canonical and non-canonical signaling pathways of Wnt3A, which induces GSK-3β phosphorylation and β-catenin accumulation through RhoA and ROCK activation. J. Cell. Physiol. 232: 1104-1113, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Aim: To enhance survival and generation of definitive endoderm cells from human embryonic stem cells in a simple and reproducible system., Main Methods: Definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs) was induced under a chemical-defined condition withdrawn insulin supplement and serum albumin. We dissected influence of "alternative growth factors", WNT3A, BMP4 and bFGF in activin A-driven differentiation by detection of DE-associated genes expression and cell viability. Expression of DE-associated SOX17 and FOXA2 genes was analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Quantitative evaluation of DE efficiency was performed by flow cytometry analysis of CXCR4-expressed cell population. Cell viability during DE differentiation was analyzed by an Annexin V/PI double staining test., Key Findings: Supplementation with WNT3A, BMP4 or bFGF promoted DE generation in a dose- and time-dependent manner. Cell apoptosis elicited by activin A was significantly ameliorated by a cocktail with WNT3A, BMP4 and bFGF. This allowed for sustained cell viability without insulin-containing supplements, thereby indirectly improving the efficiency of DE generation. Therefore, the cocktail containing is optimal for efficient DE generation in the presence of activin A and an insulin/albumin-free condition., Significance: This optimal condition facilitates the balance between the productivity and the viability maintenance, and could be valuable for mass production of DE with minimal variation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

Aim: To fabricate PEGylated liposomes which preserve the activity of hydrophobic Wnt3A protein, and to demonstrate their efficacy in promoting expansion of osteoprogenitors from human bone marrow., Methods: PEGylated liposomes composed of several synthetic lipids were tested for their ability to preserve Wnt3A activity in reporter and differentiation assays. Single-molecule microspectroscopy was used to test for direct association of protein with liposomes., Results: Labeled Wnt3A protein directly associated with all tested liposome preparations. However, Wnt3A activity was preserved or enhanced in PEGylated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes but not in PEGylated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. PEGylated Wnt3A liposomes associated with skeletal stem cell populations in human bone marrow and promoted osteogenesis., Conclusion: Active Wnt protein-containing PEGylated liposomes may have utility for systemic administration for bone repair.

The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5 + crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt. Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5 + CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5 + CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5 + CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5 + CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5 + CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.

Wnt signalling proteins are essential for culture of human organ stem cells in organoids, but most Wnt protein formulations are poorly active in serum-free media. Here we show that purified Wnt3a protein is ineffective because it rapidly loses activity in culture media due to its hydrophobic nature, and its solubilization requires a detergent, CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), that interferes with stem cell self-renewal. By stabilizing the Wnt3a protein using phospholipids and cholesterol as carriers, we address both problems: Wnt activity remains stable in serum-free media, while non-toxic carriers allow the use of high Wnt concentrations. Stabilized Wnt3a supports strongly increased self-renewal of organ and embryonic stem cells and the serum-free establishment of human organoids from healthy and diseased intestine and liver. Moreover, the lipophilicity of Wnt3a protein greatly facilitates its purification. Our findings remove a major obstacle impeding clinical applications of adult stem cells and offer advantages for all cell culture uses of Wnt3a protein.

Signal propagation from the cell membrane to a promoter can induce gene expression. To examine signal transmission through sub-cellular compartments and its effect on transcription levels in individual cells within a population, we used the Wnt/β-catenin signaling pathway as a model system. Wnt signaling orchestrates a response through nuclear accumulation of β-catenin in the cell population. However, quantitative live-cell measurements in individual cells showed variability in nuclear β-catenin accumulation, which could occur in two waves, followed by slow clearance. Nuclear accumulation dynamics were initially rapid, cell cycle independent and differed substantially from LiCl stimulation, presumed to mimic Wnt signaling. β-catenin levels increased simultaneously at adherens junctions and the centrosome, and a membrane-centrosome transport system was revealed. Correlating β-catenin nuclear dynamics to cyclin D1 transcriptional activation showed that the nuclear accumulation rate of change of the signaling factor, and not actual protein levels, correlated with the transcriptional output of the pathway., Competing Interests: The authors declare that no competing interests exist.

The Wnt signalling pathway in beta-cells has been linked to the development of type 2 diabetes. Investigating the impact of a non-canonical Wnt ligand, Wnt4, on beta-cell function we found that in INS-1 cells, Wnt4 was able to completely block Wnt3a stimulated cell growth and insulin secretion. However, despite high levels of Wnt4 protein being detected in INS-1 cells, reducing the expression of Wnt4 had no impact on cell growth or Wnt3a signalling. As such, the role of the endogenously expressed Wnt4 in beta-cells is unclear, but the data showing that Wnt4 can act as a negative regulator of canonical Wnt signalling in beta-cells suggests that this pathway could be a potential target for modulating beta-cell function., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Dishevelled (DVL) assembles Wnt signalosomes through dynamic head-to-tail polymerisation by means of its DIX domain. It thus transduces Wnt signals to cytoplasmic effectors including β-catenin, to control cell fates during normal development, tissue homeostasis and also in cancer. To date, most functional studies of Dishevelled relied on its Wnt-independent signalling activity resulting from overexpression, which is sufficient to trigger polymerisation, bypassing the requirement for Wnt signals. Here, we generate a human cell line devoid of endogenous Dishevelled (DVL1- DVL3), which lacks Wnt signal transduction to β-catenin. However, Wnt responses can be restored by DVL2 stably re-expressed at near-endogenous levels. Using this assay to test mutant DVL2, we show that its DEP domain is essential, whereas its PDZ domain is dispensable, for signalling to β-catenin. Our results imply two mutually exclusive functions of the DEP domain in Wnt signal transduction - binding to Frizzled to recruit Dishevelled to the receptor complex, and dimerising to cross-link DIX domain polymers for signalosome assembly. Our assay avoids the caveats associated with overexpressing Dishevelled, and provides a powerful tool for rigorous functional tests of this pivotal human signalling protein., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Merlin, encoded by the NF2 gene, is a tumor suppressor that acts by inhibiting mitogenic signaling and is mutated in Neurofibromatosis type II (NF2) disease, although its molecular mechanism is not fully understood. Here, we observed that Merlin inhibited Wnt/β-catenin signaling by blocking phosphorylation of LRP6, which is necessary for Wnt signal transduction, whereas mutated Merlin in NF2 patients did not. Treatment with Wnt3a enhanced phosphorylation of Ser518 in Merlin via activation of PAK1 in a PIP2-dependent manner. Phosphorylated Merlin dissociated from LRP6, allowing for phosphorylation of LRP6. Tissues from NF2 patients exhibited higher levels of β-catenin, and proliferation of RT4-D6P2T rat schwannoma cells was significantly reduced by treatment with chemical inhibitors of Wnt/β-catenin signaling. Taken together, our findings suggest that sustained activation of Wnt/β-catenin signaling due to abrogation of Merlin-mediated inhibition of LRP6 phosphorylation may be a cause of NF2 disease.

Background: Wnt proteins modulate development, stem cell fate and cancer through interactions with cell surface receptors. Wnts are cysteine-rich, glycosylated, lipid modified, two domain proteins that are prone to aggregation. The culprit responsible for this behavior is a covalently bound palmitoleoyl moiety in the N-terminal domain., Results: By combining murine Wnt3a with phospholipid and apolipoprotein A-I, ternary complexes termed nanodisks (ND) were generated. ND-associated Wnt3a is soluble in the absence of detergent micelles and gel filtration chromatography revealed that Wnt3a co-elutes with ND. In signaling assays, Wnt3a ND induced β-catenin stabilization in mouse fibroblasts as well as hematopoietic stem and progenitor cells (HSPC). Prolonged exposure of HSPC to Wnt3a ND stimulated proliferation and expansion of Lin(-) Sca-1(+) c-Kit(+) cells. Surprisingly, ND lacking Wnt3a contributed to Lin(-) Sca-1(+) c-Kit(+) cell expansion, an effect that was not mediated through β-catenin., Conclusions: The data indicate Wnt3a ND constitute a water-soluble transport vehicle capable of promoting ex vivo expansion of HSPC.

The specification of pluripotent stem cells into the bone-forming osteoblasts has been explored in a number of studies. However, the current body of literature has yet to adequately address the role of Wnt glycoproteins in the differentiation of pluripotent stem cells along the osteogenic lineage. During mouse embryonic stem cell (ESC) in vitro osteogenesis, the noncanonical WNT5a is expressed early on. Cells either sorted by their positive WNT5a expression or when supplemented with recombinant WNT5a (rWNT5a) during a 2-day window showed significantly enhanced osteogenic yield. Mechanistically, rWNT5a supplementation upregulated protein kinase C (PKC), calcium/calmodulin-dependent kinase II (CamKII) and c-Jun N-terminal kinase (JNK) activity while antagonizing the key effector of canonical Wnt signaling: β-catenin. Conversely, when recombinant WNT3a (rWNT3a) or other positive regulators of β-catenin were employed during this same time window there was a decrease in osteogenic marker expression. However, if rWNT3a was supplemented during a time window following rWNT5a treatment, osteogenic differentiation was enhanced both in murine and human ESCs. Elucidating the role of these WNT ligands in directing the early stages of osteogenesis has the potential to considerably improve tissue engineering protocols and applications for regenerative medicine.

Primary Open Angle Glaucoma (POAG) is an irreversible, vision-threatening disease that affects millions worldwide. The principal risk factor of POAG is increased intraocular pressure (IOP) due to pathological changes in the trabecular meshwork (TM). The TGFβ signaling pathway activator TGFβ2 and the Wnt signaling pathway inhibitor secreted frizzled-related protein 1 (sFRP1) are elevated in the POAG TM. In this study, we determined whether there is a crosstalk between the TGFβ/Smad pathway and the canonical Wnt pathway using luciferase reporter assays. Lentiviral luciferase reporter vectors for studying the TGFβ/Smad pathway or the canonical Wnt pathway were transduced into primary human non-glaucomatous TM (NTM) cells. Cells were treated with or without a combination of 5 μg/ml TGFβ2 and/or 100 ng/ml Wnt3a recombinant proteins, and luciferase levels were measured using a plate reader. We found that TGFβ2 inhibited Wnt3a-induced canonical Wnt pathway activation, while Wnt3a inhibited TGFβ2-induced TGFβ/Smad pathway activation (n = 6, p < 0.05) in 3 NTM cell strains. We also found that knocking down of Smad4 or β-catenin using siRNA in HTM5 cells transfected with similar luciferase reporter plasmids abolished the inhibitory effect of TGFβ2 and/or Wnt3a on the other pathway (n = 6). Our results suggest the existence of a cross-inhibition between the TGFβ/Smad and canonical Wnt pathways in the TM, and this cross-inhibition may be mediated by Smad4 and β-catenin., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Merlin, encoded by the NF2 gene, is a tumor suppressor that exerts its function via inhibiting mitogenic receptors at the plasma membrane. Although multiple mutations in Merlin have been identified in Neurofibromatosis type II (NF2) disease, its molecular mechanism is not fully understood. Here, we show that Merlin interacts with LRP6 and inhibits LRP6 phosphorylation, a critical step for the initiation of Wnt signaling. We found that treatment of Wnt3a caused phosphorylation of Merlin by PAK1, leading to detachment of Merlin from LRP6 and allowing the initiation of Wnt/β-catenin signaling. A higher level of β-catenin was found in tissues from NF2 patients. Enhanced proliferation and migration caused by knockdown of Merlin in glioblastoma cells were inhibited by suppression of β-catenin. Conclusively, these results suggest that sustained Wnt/β-catenin signaling activity induced by abrogation of Merlin-mediated inhibition of LRP6 phosphorylation might be a cause of NF2 disease. [BMB Reports 2016; 49(7): 357-358].

Aim: Duchenne muscular dystrophy (DMD) is an X-linked genetic muscular disorder with no effective treatment at present. Mesenchymal stem cell (MSC) transplantation has been used to treat DMD, but the efficiency is low. Our previous studies show that activation of Wnt3a signaling promotes myogenic differentiation of MSCs in vitro. Here we report an effective MSC transplantation therapy in mdx mice by activation of Wnt3a signaling., Methods: MSCs were isolated from mouse bone marrow, and pretreated with Wnt3a-conditioned medium (Wnt3a-CM), then transplanted into mdx mice. The recipient mice were euthanized at 4, 8, 12, 16 weeks after the transplantation, and muscle pathological changes were examined. The expression of dystrophin in muscle was detected using immunofluorescence staining, RT-PCR and Western blotting., Results: Sixteen weeks later, transplantation of Wnt3a-pretreated MSCs in mdx mice improved the characteristics of dystrophic muscles evidenced by significant reductions in centrally nucleated myofibers, the variability range of cross-sectional area (CSA) and the connective tissue area of myofibers. Furthermore, transplantation of Wnt3a-pretreated MSCs in mdx mice gradually and markedly increased the expression of dystrophin in muscle, and improved the efficiency of myogenic differentiation., Conclusion: Transplantation of Wnt3a-pretreated MSCs in mdx mice results in long-term amelioration of the dystrophic phenotype and restores dystrophin expression in muscle. The results suggest that Wnt3a may be a promising candidate for the treatment of DMD.

Wnt/β-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)--known to target Axin for proteolysis-regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly.

Intravenous administration of bone marrow derived mesenchymal stem cells (MSCs) has been shown to reduce blood brain barrier compromise and improve neurocognition following traumatic brain injury (TBI). These effects occur in the absence of engraftment and differentiation of these cells in the injured brain. Recent studies have shown that soluble factors produced by MSCs mediate a number of the therapeutic effects. In this study, we sought to determine if intravenous administration of MSCs (IV-MSCs) could enhance hippocampal neurogenesis following TBI. Our results demonstrate that IV-MSC treatment attenuates loss of neural stem cells and promotes hippocampal neurogenesis in TBI injured mice. As Wnt signaling has been implicated in neurogenesis, we measured circulating Wnt3a levels in serum following IV-MSC administration and found a significant increase in Wnt3a. Concurrent with this increase, we detected increased activation of the Wnt/β-catenin signaling pathway in hippocampal neurons. Furthermore, IV recombinant Wnt3a treatment provided neuroprotection, promoted neurogenesis, and improved neurocognitive function in TBI injured mice. Taken together, our results demonstrate a role for Wnt3a in the therapeutic potential of MSCs and identify Wnt3a as a potential stand-alone therapy or as part of a combination therapeutic strategy for the treatment of TBI. Stem Cells 2016;34:1263-1272., (© 2016 AlphaMed Press.)