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Oxidative stress, endoplasmic reticulum (ER) stress, and neuronal cell apoptosis have been considered as the main pathogenesis factors of brain injury after intracerebral hemorrhage (ICH). Chrysophanol (CHR) has been proved to have neuroprotective effects, but the role and underlying mechanisms of CHR in ICH remain unclear. HT22 cells were dealt with hemin to mimic an in vitro ICH model and then subjected to treatment with or without CHR. The cell viability, apoptosis, ER stress, and oxidative stress were evaluated by conducting the cell counting kit-8 (CCK-8), TdT-mediated dUTP nick end labeling (TUNEL) staining assays, western blot, and corresponding kit, respectively. Further, microRNA-sequencing, bioinformatic analysis, dual-luciferase reporter method, and rescue experiments were conducted to explore the molecular mechanisms of CHR alleviating hemin-induced ER in HT22 cell. Our data revealed that CHR increased cells viability, antiapoptosis, anti-ER stress, and antioxidative stress under conditions of hemin-induced HT22 cell injury. Mechanically, it was observed that Wnt3a was competitively sponged by miR-320-5p, and CHR activated β-catenin pathway by regulating miR-320-5p/Wnt3a molecular axis. Finally, results from the rescue experiment suggested that CHR inhibited hemin-induced cells apoptosis, ER stress, and oxidative stress through regulating the miR-320-5p/Wnt3a axis in HT22 cells. In conclusion, CHR prevented hemin-induced apoptosis, ER stress, and oxidative stress via inhibiting the miR-320-5p/Wnt3a/β-catenin pathway in HT22 cells. Our results certified that CHR could be served as a promising treatment for brain damage following ICH.

Imatinib is a nontoxic tyrosine kinase inhibitor, used in the treatment of advanced renal cell carcinoma. However, some patients with renal cell carcinoma develop resistance to imatinib. Protein disulfide isomerase family 6 (PDIA6) was involved in the chemo-resistance of lung adenocarcinoma. In this study, the effect of PDIA6 on imatinib-resistance of renal cell carcinoma was investigated. First, PDIA6 was found to be up-regulated in the imatinib-resistant renal cell carcinoma tissues and cells. Functional assays showed that knockdown of PDIA6 sensitized imatinib-resistant renal cell carcinoma cells to imatinib through decreasing the half-maximal inhibitory concentration (IC50) of imatinib-resistant renal cell carcinoma cells. Secondly, cell proliferation of imatinib-resistant renal cell carcinoma cells was suppressed by PDIA6 silencing, and the apoptosis was promoted with reduced Bcl-2, enhanced Bax and cleaved caspase-3. Moreover, the interference of PDIA6 increased phosphorylation of H2A histone family member X (γH2AX), while decreased Rad51 and phosphorylated DNA-dependent protein kinase (DNA-PK) (p-DNA-PK) in imatinib-resistant renal cell carcinoma cells. Lastly, protein expression levels of Wnt3a and Frizzled1 (FZD1) in imatinib-resistant renal cell carcinoma cells were down-regulated by silencing of PDIA6. Over-expression of FZD1 attenuated PDIA6 silencing-induced increase in cell apoptosis and decrease in cell proliferation in imatinib-resistant renal cell carcinoma cells. In conclusion, knockdown of PDIA6 sensitized imatinib-resistant renal cell carcinoma cells into imatinib through inactivation of Wnt3a-FZD1 axis.

Background We aimed to investigate the functions and underlying mechanism of lncRNA SNHG1 in bone differentiation and angiogenesis in the development of osteoporosis. Methods The differential gene or proteins expressions were measured by qPCR or western blot assays, respectively. The targeted relationships among molecular were confirmed through luciferase reporter, RIP and ChIP assays, respectively. Alkaline phosphatase (ALP), alizarin red S (ARS) and TRAP staining were performed to measure the osteoblast/osteoclast differentiation of BMSCs. The viability, migration and angiogenesis in BM-EPCs were validated by CCK-8, clone formation, transwell and tube formation assays, respectively. Western blot and immunofluorescence detected the cytosolic/nuclear localization of β-catenin. Ovariectomized (OVX) mice were established to confirm the findings in vitro. Results SNHG1 was enhanced and miR-181c-5p was decreased in serum and femoral tissue from OVX mice. SNHG1 directly inhibited miR-181c-5p to activate Wnt3a/β-catenin signaling by upregulating SFRP1. In addition, knockdown of SNHG1 promoted the osteogenic differentiation of BMSCs by increasing miR-181c-5p. In contrast, SNHG1 overexpression advanced the osteoclast differentiation of BMSCs and inhibited the angiogenesis of BM-EPCs, whereas these effects were all reversed by miR-181c-5p overexpression. In vivo experiments indicated that SNHG1 silencing alleviated osteoporosis through stimulating osteoblastogenesis and inhibiting osteoclastogenesis by modulating miR-181c-5p. Importantly, SNHG1 could be induced by SP1 in BMSCs. Conclusions Collectively, SP1-induced SNHG1 modulated SFRP1/Wnt/β-catenin signaling pathway via sponging miR-181c-5p, thereby inhibiting osteoblast differentiation and angiogenesis while promoting osteoclast formation. Further, SNHG1 silence might provide a potential treatment for osteoporosis. Graphic abstract

Background Stearoyl-coenzyme A desaturase 1 (SCD1) is required for de novo synthesis of fatty acids. Through the fatty acid acylation process, this enzyme orchestrates post-translational modifications to proteins involved in cell development and differentiation. In this study, we used biochemical methods, immunostaining, and covalent labeling to evaluate whether a small molecule modulating unsaturated fatty acids can influence the early endodermal differentiation of human-induced pluripotent stem cells (iPSCs). Methods The hiPSCs were cultured in an endoderm-inducing medium containing activin A and defined fetal bovine serum in the presence of an SCD1 inhibitor at different time points. The cell cycles and the yields of the three germ layers (endoderm, mesoderm, and ectoderm) were assessed using flow cytometry. The expression of endoderm and pluripotency markers and the expressions of Wnt signaling pathway proteins were assessed using western blotting and RT-PCR. Total protein acylation was evaluated using a click chemistry reaction. Results When SCD1 was inhibited on the first day, the population of cells with endodermal features decreased at the end of differentiation. Moreover, early SCD1 inhibition preserved the properties of hiPSCs, preventing their shift toward mesodermal or ectodermal lineage. Also, first-day-only treatment of cells with the SCD1 inhibitor decreased β-catenin gene expression and the intensity of fluorescent emission in the click chemistry assay. The cells were effectively rescued from these effects by cotreatment with oleate. Late treatment with the inhibitor in the two subsequent days of endoderm induction did not have any significant effects on endoderm-specific markers or fluorescent intensity. Reproducible results were also obtained with human embryonic stem cells. Conclusion The small molecule SCD1 inhibitor attenuates the Wnt/β-catenin signaling pathway, conferring the maintenance of hiPSCs by opposing the initiation of endoderm differentiation. The immediate requirement for SCD1 activity in the endoderm commitment of pluripotent stem cells may be of importance in disorders of endoderm-derived organs and dysregulated metabolism. The schematic representation of the study design and main results. Activin A induces endoderm features through Smad2/3/4 and increases the expression of SCD1. SCD1 can produce MUFAs and subsequently modify the Wnt molecules. MUFA acylated/activated Wnts are secreted to interact with corresponding receptors on the target cells. β-catenin accumulates in the cytoplasm and is translocated into the nucleus after the interaction of Wnt with the receptor. Then, β-catenin increases the expression of the endoderm markers Sox17 and CXCR4.

Adipocyte-rich omentum offers "good soil" for disseminating ovarian cancer (OvCa), contributing to therapeutic difficulty. However, little is understood about the association between adipocytes and tumor growth at peritoneal dissemination site. Herein, we report the induction of adipocyte dedifferentiation by OvCa cells and pro-tumorigenic effects of resulted adipocyte-derived fibroblasts. We confirmed that malignant ascites promoted the dedifferentiation of the primary human adipocytes obtained from surgical omental specimen into omental adipocyte-derived fibroblast (O-ADF) that possess both mesenchymal stem cell and myofibroblast-like features. This promotion of dedifferentiation by malignant ascites was blocked by addition of Wnt signaling inhibitor. The effects of dedifferentiated adipocytes in proliferation and migration of OvCa cells were analyzed with in vitro co-culturing experimental models and in vivo mice model, and we demonstrated that OvCa cell lines showed enhanced proliferative characteristics, as well as increased migratory abilities upon co-culturing with O-ADF. Additionally, exogenous transforming growth factor-β1 augmented desmoplastic morphological change of O-ADF, leading to higher proliferative ability. Our results suggest that OvCa cells promote dedifferentiation of peritoneal adipocytes by activating Wnt/β-catenin signaling, and generated O-ADFs exhibit pro-tumoral hallmarks. This article is protected by copyright. All rights reserved.

Primary cilia, antenna-like cellular sensor structures, are generated from the mother centriole in the G0/G1 cell-cycle phase under control by cellular signaling pathways involving Wnt, hedgehog, and platelet-derived growth factor. Although primary ciliary dynamics have been reported to be closely related to ciliopathy and tumorigenesis, the molecular basis for the role of primary cilia in human disease is lacking. To clarify how Wnt3a affects primary ciliogenesis in anticancer drug-resistant cells, we derived specific drug-resistant subcell lines from A549 human lung cancer cells using anticancer drugs doxorubicin, dasatinib, and paclitaxel (A549/Dox, A549/Das, and A549/Pac, respectively). The primary cilia-containing cell population and primary cilia length increased in the A549/Dox and A549/Pac subcell lines under increased MDR1 expression, when compared to those in the parental A549 cells. In the A549/Das subcell line, primary cilia length increased but the cell population was not affected. In addition, Wnt3a increased primary cilia-containing cell population and primary cilia length in A549/Dox, A549/Das, and A549/Pac cells, without change of cell growth. Abnormal shapes of primary cilia were frequently observed by anticancer drug resistance and Wnt3a stimulation. Taken together, our results indicate that anticancer drug resistance and Wnt3a affect primary ciliogenesis synergistically, suggesting a potential new strategy for overcoming anticancer drug resistance.

Wntless (WLS), an evolutionarily conserved multi-pass transmembrane protein, is essential for secretion of Wnt proteins. Wnt-triggered signaling pathways control many crucial life events, whereas aberrant Wnt signaling is tightly associated with many human diseases including cancers. Here, we report the cryo-EM structure of human WLS in complex with Wnt3a, the most widely studied Wnt, at 2.2 Å resolution. The transmembrane domain of WLS bears a GPCR fold, with a conserved core cavity and a lateral opening. Wnt3a interacts with WLS at multiple interfaces, with the lipid moiety on Wnt3a traversing a hydrophobic tunnel of WLS transmembrane domain and inserting into membrane. A β-hairpin of Wnt3a containing the conserved palmitoleoylation site interacts with WLS extensively, which is crucial for WLS-mediated Wnt secretion. The flexibility of the Wnt3a loop/hairpin regions involved in the multiple binding sites indicates induced fit might happen when Wnts are bound to different binding partners. Our findings provide important insights into the molecular mechanism of Wnt palmitoleoylation, secretion and signaling., The transmembrane protein Wntless (WLS) is essential for the secretion of Wnt proteins, which are signaling molecules of the Wnt signaling pathways. Here, the authors present the 2.2 Å cryo-EM structure of human WLS in complex with Wnt3a and discuss mechanistic implications for the palmitoleoylation of Wnt3a that is required for WLS mediated Wnt secretion.

Conventional methods for obtaining pancreatic β cells are based on simulating the embryonic development phase of endocrine cells via hierarchical differentiation of pluripotent stem cells (PSCs). Accordingly, we attempted to modify the protocols for obtaining insulin-secreting cells (ISCs) by sequential differentiation of a human embryonic stem cell (hESC), using the HS181 cell line. Furthermore, we hypothesize that actual pancreatic endocrine cells may arise from trans-differentiation of mature ductal cells after the embryonic developmental stage and throughout the rest of life. According to the hypothesis, ductal cells are trans-differentiated into endocrine and exocrine cells, undergoing a partial epithelial to mesenchymal transition (EMT). To address this issue, we developed two new protocols based on hESC differentiation to obtain ductal cells and then induce EMT in cells to obtain hormone-secreting islet-like cells (HSCs). The ductal (pre-EMT exocrine) cells were then induced to undergo partial EMT by treating with Wnt3a and activin A, in hypoxia. The cell derived from the latter method significantly expressed the main endocrine cell-specific markers and also β cells, in particular. These experiments not only support our hypothetical model but also offer a promising approach to develop new methods to compensate β cell depletion in patients with type 1 diabetes mellitus (T1DM). Although this protocol of generating islet-like cells from ductal cells has a potential to treat T1DM, this strategy may be exploited to optimize the function of these cells in an animal model and future clinical applications.

Both hair follicle stem cells (HFSC) and dermal papilla cells (DPC) are essential for hair follicle growth and proliferation. In this study, HFSCs and DPCs that made signature proteins like KRT14, KRT15, KRT19

Background Prostate cancer occurs through multiple steps until advanced metastasis. Signaling pathways studies can result in the identification of targets to interrupt cancer progression. Glypicans are cell surface proteoglycans linked to the membrane through glycosylphosphatidylinositol. Their interaction with specific ligands has been reported to trigger diverse signaling, including Wnt. In this study, prostate cancer cell lines PC-3, DU-145, and LNCaP were compared to normal prostate RWPE-1 cell line to investigate glypican family members and the activation of the Wnt signaling pathway. Results Glypican-1 (GPC1) was highly expressed in all the examined cell lines, except for LNCaP, which expressed glypican-5 (GPC5). The subcellular localization of GPC1 was detected on the cell surface of RWPE-1, PC-3, and DU-145 cell lines, while GPC5 suggested cytoplasm localization in LNCaP cells. Besides glypican, flow cytometry analysis in these prostate cell lines confirmed the expression of Wnt-3a and unphosphorylated β-catenin. The co-immunoprecipitation assay revealed increased levels of binding between Wnt-3a and glypicans in cancer cells, suggesting a relationship between these proteoglycans in this pathway. A marked increase in nuclear β-catenin was observed in tumor cells. However, only PC-3 cells demonstrated activation of canonical Wnt signaling, according to the TOPFLASH assay. Conclusions GPC1 was the majorly expressed gene in all the studied cell lines, except for LNCaP, which expressed GPC5. We assessed by co-immunoprecipitation that these GPCs could interact with Wnt-3a. However, even though nuclear β-catenin was found increased in the prostate cancer cells (i.e., PC-3, DU-145 and LNCaP), activation of Wnt pathway was only found in PC-3 cells. In these PC-3 cells, GPC1 and Wnt-3a revealed high levels of colocalization, as assessed by confocal microscopy studies. This suggests a localization at the cellular surface, where Frizzled receptor is required for downstream activation. The interaction of Wnt-3a with GPCs in DU-145 and LNCaP cells, which occurs in absence of Wnt signaling activation, requires further studies. Once non-TCF-LEF proteins can also bind β-catenin, another signaling pathway may be involved in these cells with regulatory function.

Background:. Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1β)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/β-catenin signaling pathway. Methods:. Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1β and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1β. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1β, the translocation of β-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/β-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3β) plasmids were assessed for their effects on HDAC4 levels using Western blotting. Results:. IL-1β downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1β-induced increases in MMP3 and MMP13. IL-1β upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3β rescued IL-1β-induced downregulation of HDAC4 in SW1353 cells. Conclusion:. HDAC4 exerted an inhibitory effect on IL-1β-induced extracellular matrix degradation and was regulated partially by the WNT3A/β-catenin signaling pathway.

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid-liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFβ and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2-a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis.

There is increasing evidence that the study of normal human enteroids duplicates many known aspects of human intestinal physiology. However, this epithelial cell-only model lacks the many nonepithelial intestinal cells present in the gastrointestinal tract and exposure to the mechanical forces to which the intestine is exposed. We tested the hypothesis that physical shear forces produced by luminal and blood flow would provide an intestinal model more closely resembling normal human jejunum. Jejunal enteroid monolayers were studied in the Emulate, Inc. Intestine-Chip under conditions of constant luminal and basolateral flow that was designed to mimic normal intestinal fluid flow, with human umbilical vein endothelial cells (HUVECs) on the basolateral surface and with Wnt3A, R-spondin, and Noggin only on the luminal surface. The jejunal enteroids formed monolayers that remained confluent for 6–8 days, began differentiating at least as early as day 2 post plating, and demonstrated continuing differentiation over the entire time of the study, as shown by quantitative real-time polymerase chain reaction and Western blot analysis. Differentiation impacted villus genes and proteins differently with early expression of regenerating family member 1α (REG1A), early reduction to a low but constant level of expression of Na(+)-K(+)-2Cl(−) cotransporter 1 (NKCC1), and increasing expression of sucrase-isomaltase (SI) and downregulated in adenoma (DRA). These results were consistent with continual differentiation, as was shown to occur in mouse villus enterocytes. Compared with differentiated enteroid monolayers grown on Transwell inserts, enteroids exposed to flow were more differentiated but exhibited increased apoptosis and reduced carbohydrate metabolism, as shown by proteomic analysis. This study of human jejunal enteroids-on-chip suggests that luminal and basolateral flow produce a model of continual differentiation over time and NaCl absorption that mimics normal intestine and should provide new insights in intestinal physiology. NEW & NOTEWORTHY This study showed that polarized enteroid models in which there is no basolateral Wnt3a, are differentiated, regardless of the Wnt3a status of the apical media. The study supports the concept that in the human intestine villus differentiation is not an all or none phenomenon, demonstrating that at different days after lack of basolateral Wnt exposure, clusters of genes and proteins exist geographically along the villus with different domains having different functions.

Rationale: Idiopathic pulmonary fibrosis (IPF) is one of the most aggressive forms of idiopathic interstitial pneumonia. Some miRNAs may be associated with IPF and may affect the occurrence and development of IPF in various pathways. Many miRNAs and genes that may be involved in the development of IPF have been discovered using chip and high throughput technologies. Methods: We analyzed one miRNA and four mRNA databases. We identified hub genes and pathways related to IPF using GO, KEGG enrichment analysis, gene set variation analysis (GSVA), PPI network construction, and hub gene analysis. A comprehensive analysis of differentially expressed miRNAs (DEMs), predicted miRNA target genes, and differentially expressed genes (DEGs) led to the creation of a miRNA-mRNA regulatory network in IPF. Results: We found 203 DEGs and 165 DEMs that were associated with IPF. The findings of enrichment analyses showed that these DEGs were mainly involved in antimicrobial humoral response, antimicrobial humoral immune response mediated by antimicrobial peptide, extracellular matrix organization, cell killing, and organ or tissue specific immune response. The VEGFA, CDH5, and WNT3A genes overlapped between hub genes and the miRNA-mRNA regulatory network. The miRNAs including miR-199b-5p, miR-140-5p, miR-199a-5p, miR-125A-5p, and miR-107 that we predicted would regulate the VEGFA, CDH5, and WNT3A genes, which were also associated with IPF or other fibrosis-related diseases. GSVA indicated that metabolic processes of UTP and IMP, immune response, regulation of Th2 cell cytokine production, and positive regulation of NK cell-mediated immunity are associated with the pathogenesis and treatment of IPF. These pathways also interact with VEGFA, CDH5, and WNT3A. Conclusion: These findings provide a new research direction for the diagnosis and treatment of IPF.

Activation of hepatic stellate cells (HSC) is associated with hepatic fibrogenesis, which is one of complications of diabetes mellitus. Captopril possesses potent anti-inflammation, oxidative stress and fibrosis effects. However, the specific molecular mechanism of captopril in high glucose (HG)-induced hepatic stellate cells has not been elucidated. Following the treatment of HG or captopril treatment for rat hepatic stellate cells (HSC-T6), cell activities were detected by Cell Counting Kit-8 (CCK8) assay. Reactive oxygen species (ROS) levels were determined by ROS staining. The expression of inflammation-related proteins (Interleukin (IL)-1β, IL-6 and IL-8) and fibrosis-related proteins (fibronectin (FN), collagen I, collagen III, collagen IV, matrix metallopeptidase (MMP-2 and MMP-9) were determined by Western blot. Captopril significantly decreased HSC-T6 cell viability induced by HG in a dose-dependent manner, as well as decreased levels of malondialdehyde (MDA), ROS, pro-inflammatory markers and fibrosis-related proteins, while upregulated superoxide dismutase (SOD) activities. We further found that captopril decreased the ratio of p-IκBα/IκBα and the ratio of p-p65/p65. Intriguing, phorbol myristate acetate (PMA) or LiCl was able to significantly reverse the captopril-induced alteration of oxidative stress-, inflammation- and fibrosis-marker levels. In conclusion, in HG-stimulated HSC-T6 cells, captopril displayed a potent ability to inhibit oxidative stress, inflammation and hepatic fibrogenesis via NF-kappaB or wnt3α/β-catenin. These results demonstrated the mechanism of captopril as well as the role of the NF-kappaB or wnt3α/β-catenin on HSC-T6 activation induced by HG.

Background Thyroid carcinoma (TC) has been a global issue for its rapid increasing incidence worldwide. Although most TC was not so aggressive with a good prognosis, treatment against anaplastic TC was relatively limited and the mechanisms are not well elucidated yet. Methods TC cell lines (IHH4 and TPC-1) were used. Flow cytometry was used to identify the surface marker of M2-like tumor-associated macrophages (TAMs) from cell culture. Quantitative real-time polymerase chain reaction, western blot analysis, immunostaining, and immunohistochemistry were used to detect the expression of Wnt1, Wnt3a, components of Wnt/β-catenin pathway, and proliferation/epithelial-mesenchymal transition (EMT)-related proteins. Alkaline phosphatase activity assay, colony formation assay, and transwell assay were used to examine the roles of Wnt1, Wnt3a, and β-catenin pathway in cell dedifferentiation, proliferation, migration, and invasion of TC cells, respectively. Subcutaneous tumor growth was monitored in nude mice. Results Coculture with M2-like TAMs facilitated dedifferentiation, proliferation, migration, and invasion in TC cells. EMT and proliferation-related proteins were also promoted in cocultured TC cells. The level of Wnt1 and Wnt3a was increased in the coculture system. Block of Wnt1 or Wnt3a suppressed malignant behaviors in cocultured tumor cells. Furthermore, Wnt1 or Wnt3a knockdown inhibited Wnt/β-catenin signaling pathway, and suppressed EMT and proliferation-related signals in cocultured tumor cells. Knockdown of Wnt1 or Wnt3a inhibited tumor growth in xenograft model. Conclusion M2-like TAMs promoted dedifferentiation, proliferation, and metastasis of TC by Wnt1 and Wnt3a secretion and ensuing β-catenin activation.

Type 2 diabetes mellitus (T2DM) patients have compromised mandibular bone architecture/quality, which markedly increase the risks of tooth loosening, tooth loss, and failure of dental implantation. However, it remains lacks effective and safe countermeasures against T2DM-related mandibular bone deterioration. Herein, we studied the effects of pulsed electromagnetic fields (PEMF) on mandibular bone microstructure/quality and relevant regulatory mechanisms in T2DM db/db mice. PEMF exposure (20 Gs, 15 Hz) for 12 weeks preserved trabecular bone architecture, increased cortical bone thickness, improved material properties and stimulated bone anabolism in mandibles of db/db mice. PEMF also upregulated the expression of canonical Wnt3a ligand (but not Wnt1 or Wnt5a) and its downstream β-catenin. PEMF improved the viability and differentiation of primary osteoblasts isolated from the db/db mouse mandible, and stimulated the specific activation of Wnt3a/β-catenin signaling. These positive effects of PEMF on mandibular osteoblasts of db/db mice were almost totally abolished after Wnt3a silencing in vitro, which were equivalent to the effects following blockade of canonical Wnt signaling using the broad-spectrum antagonist DKK1. Injection with Wnt3a siRNA abrogated the therapeutic effects of PEMF on mandibular bone quantity/quality and bone anabolism in db/db mice. Our study indicates that PEMF might become a non-invasive and safe treatment alternative resisting mandibular bone deterioration in T2DM patients, which is helpful for protecting teeth from loosening/loss and securing the dental implant stability.

Abnormal accumulation of lymphoblasts in the blood and bone marrow is the main characteristic of acute lymphoblastic leukaemia (ALL). Glucocorticoids are effective drugs for ALL, while glucocorticoid resistance is an obstacle to ALL therapy. MicroRNAs (miRNAs) are implicated in the drug resistance and modulate the response of ALL to glucocorticoids. The role of miR-503 in glucocorticoid sensitivity of ALL was investigated in this study. Firstly, T-leukaemic cells were isolated from patients with ALL. The human ALL cell line (CCRF/CEM) was incubated with dexamethasone to establish a glucocorticoid- resistant ALL cell line (CCRF/CEM-R). Data from MTT showed that IC

Arrested alveolar development is the main pathological characteristic of neonatal bronchopulmonary dysplasia (BPD); however, a number of studies aiming to improve alveolarization have focused on alveolar epithelial cell damage and impairment. Previously, the authors reported that the Wnt signaling plays a key role in alveolar injury and repair by regulating alveolar epithelial type II cell (AECII) proliferation and differentiation. In the present study, the authors wished to investigate whether Yes‑associated protein (YAP), a transcriptional coactivator in the Hippo signaling pathway, affects AECII proliferation and differentiation via the Wnt/β‑catenin pathway in BPD. It was found that YAP regulated AECII proliferation and differentiation. A decreased expression of YAP, Wnt3a and nuclear β‑catenin was observed in lung tissues affected by BPD. In vitro, YAP and Wnt3a overexpression in BPD promoted AECII proliferation and differentiation into AECIs by increasing the nuclear transfer of β‑catenin and vice versa. The effects of a decreased Wnt3a expression in primary AECIIs in BPD were compensated by YAP overexpression, as were the effects of Wnt3a knockdown in primary AECIIs. On the whole, the findings of the present study demonstrate that YAP and Wnt3a independently promote AECII proliferation and differentiation in experimental BPD by increasing the nuclear β‑catenin levels. Therefore, Wnt3a or YAP may be candidate regulatory targets for improving the outcomes of BPD.

Although chemotherapy is a key cancer treatment, many chemotherapeutic drugs produce chronic neuropathic pain, called chemotherapy-induced neuropathic pain (CINP), which is a dose-limiting adverse effect. To date, there is no medicine that prevents CINP in cancer patients and survivors. We determined whether blockers of the canonical Wnt signaling pathway prevent CINP. Neuropathic pain was induced by intraperitoneal injection of paclitaxel (PAC) on four alternate days in male Sprague-Dawley rats or male Axin2-LacZ knock-in mice. XAV-939, LGK-974, and iCRT14, Wnt/β-catenin blockers, were administered intraperitoneally as a single or multiple doses before or after injury. Mechanical allodynia, phosphoproteome profiling, Wnt ligands, and inflammatory mediators were measured by von Frey filament, phosphoproteomics, reverse transcription-polymerase chain reaction, and Western blot analysis. Localization of β-catenin was determined by immunohistochemical analysis in the dorsal root ganglia (DRGs) in rats and human. Our phosphoproteome profiling of CINP rats revealed significant phosphorylation changes in Wnt signaling components. Importantly, repeated systemic injections of XAV-939 or LGK-974 prevented the development of CINP in rats. In addition, XAV-939, LGK-974, and iCRT14 ameliorated CINP. PAC increased Wnt3a and Wnt10a, activated β-catenin in DRG, and increased monocyte chemoattractant protein-1 and interleukin-1β in DRG. PAC also upregulated rAxin2 in mice. Furthermore, β-catenin was expressed in neurons, including calcitonin gene–related protein-expressing neurons and satellite cells in rat and human DRG. In conclusion, chemotherapy increases Wnt3a, Wnt10a, and β-catenin in DRG and their pharmacological blockers prevent and ameliorate CINP, suggesting a target for the prevention and treatment of CINP. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13311-020-00956-w.

Wnt signaling maintains homeostasis in the bone marrow cavity: if Wnt signaling is inhibited then bone volume and density would decline. In this study, we identified a population of Wnt-responsive cells as osteoprogenitor in the intact trabecular bone region, which were responsible for bone development and turnover. If an implant was placed into the long bone, this Wnt-responsive population and their progeny contributed to osseointegration. We employed Axin2CreCreERT2/+;R26mTmG/+ transgenic mouse strain in which Axin2-positive, Wnt-responsive cells, and their progeny are permanently labeled by GFP upon exposure to tamoxifen. Each mouse received femoral implants placed into a site prepared solely by drilling, and a single-dose liposomal WNT3A protein was used in the treatment group. A lineage tracing strategy design allowed us to identify cells actively expressing Axin2 in response to Wnt signaling pathway. These tools demonstrated that Wnt-responsive cells and their progeny comprise a quiescent population residing in the trabecular region. In response to an implant placed, this population becomes mitotically active: cells migrated into the peri-implant region, up-regulated the expression of osteogenic proteins. Ultimately, those cells gave rise to osteoblasts that produced significantly more new bone in the peri-implant region. Wnt-responsive cells directly contributed to implant osseointegration. Using a liposomal WNT3A protein therapeutic, we showed that a single application at the time of implant placed was sufficient to accelerate osseointegration. The Wnt-responsive cell population in trabecular bone, activated by injury, ultimately contributes to implant osseointegration. Liposomal WNT3A protein therapeutic accelerates implant osseointegration in the long bone.

Supplemental Digital Content is available in the text., 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 ROSAmT/mG::Nanogfl/+::Cdh5CreERT2 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.

Retinoblastoma (RB) is a common intraocular malignant tumor. The growing evidence has reported that circular RNAs (circRNAs) play critical roles in RB development. Therefore, the purpose of the study is to investigate the regulatory mechanism of circDHDDS in RB. The real-time quantitative polymerase chain reaction (RT-qPCR) assay was used to quantify the expression levels of circDHDDS, miR-361-3p, and WNT3A in RB tissues and cells (RPCs, Y-79, and WERI-Rb-1). The proliferation and cell cycle of RB cells were assessed by colony formation assay and flow cytometry assays, respectively. The migration and invasion of RB cells were measured by transwell assay. The protein expression levels of Nectin-3 (CD113), SOX2, Nanog, and WNT3A were measured by Western blot assay. The functional targets of circDHDDS and miR-361-3p were predicted by bioinformatics databases, and the dual-luciferase reporter assay was used to confirm the interaction relationship between miR-361-3p and circDHDDS or WNT3A. The functional role of circDHDDS silencing in vivo was evaluated by xenograft experiment. We found that circDHDDS was overexpressed in RB tissues and cells compared with normal retinas tissues and retinal pigment epithelial cells, correspondingly. Furthermore, silencing of circDHDDS impeded proliferation, migration, invasion, and induced cell cycle arrest in vitro, which were abolished by knockdown of miR-361-3p. The in vivo experiments also suggested that tumor growth was inhibited by knockdown of circDHDDS. Moreover, we also found that miR-361-3p specifically bound to WNT3A, and overexpression of miR-361-3p suppressed RB development by decreasing WNT3A expression. Summarily, circDHDDS, a molecule sponge of miR-361-3p, regulated the expression of WNT3A. Therefore, circDHDDS/miR-361-3p/WNT3A axis stimulated the development of RB by regulation of proliferation, cell cycle program, migration, and invasion of RB cells.

Wnt signaling represents a highly versatile signaling system, which plays critical roles in developmental morphogenesis as well as synaptic physiology in adult life and is implicated in a variety of neural disorders. Recently, we demonstrated that Wnt3a is able to recruit multiple non-canonical signaling pathways to alter peripheral sensory neuron function in a nociceptive modality-specific manner. Furthermore, several studies recently reported an important role for Wnt5a acting via canonical and non-canonical signaling in spinal processing of nociception in a number of pathological pain disorders. Here, using diverse molecular, genetic, and behavioral approaches in mouse models of pain in vivo, we report a novel role for Wnt5a signaling in nociceptive modulation at the structural level. In models of chronic pain, using male and female mice, we found that Wnt5a is released spinally from peripheral sensory neurons, where it recruits the tyrosine kinase receptors Ror2 and Ryk to modulate dendritic spine rearrangement. Blocking the Wnt5a-Ryk/Ror2 axis in spinal dorsal horn neurons prevented activity-dependent dendritic spine remodeling and significantly reduced mechanical hypersensitivity induced by peripheral injury as well as inflammation. Moreover, we observed that peripheral Wnt3a signaling triggers the release of Wnt5a in the spinal cord, and inhibition of spinal Wnt5a signaling attenuates the functional impact of peripheral Wnt3a on nociceptive sensitivity. In conclusion, this study reports a novel role for the Wnt signaling axis in coordinating peripheral and spinal sensitization and shows that targeting Wnt5a-Ryk/ROR2 signaling alleviates both structural and functional mechanisms of nociceptive hypersensitivity in models of chronic pain in vivo. SIGNIFICANT STATEMENT There is a major need to elucidate molecular mechanisms underlying chronic pain disorders in order to develop novel therapeutic approaches. Wnt signaling represents a highly versatile signaling system, which plays critical roles during development and adult physiology, and it was implicated in several diseases, included chronic pain conditions. Using mouse models, our study identifies a novel role for Wnt5a signaling in nociceptive modulation at the spinal cord level. We observed that Wnt5a recruits Ror2 and Ryk receptors to enhance dendritic spine density, leading to nociceptive sensitization. Blocking the Wnt5a-Ryk/Ror2 interaction in the spinal dorsal horn prevented spine remodeling and significantly reduced inflammatory and neuropathic hypersensitivity. These findings provide proof-of-concept for targeting spinal Wnt signaling for alleviating nociceptive hypersensitivity in vivo.

The Wnt signaling pathway has critical roles in dysregulated inflammation during sepsis; however, its impacts on clinical outcomes remain uncertain. This prospective observational study investigated the association between the Wnt pathway and clinical outcomes in patients with urosepsis. The study included 38 patients with urosepsis and 20 healthy individuals. Wnt3a and Wnt5a levels were measured at admission. The primary outcome was the occurrence of major adverse kidney events (MAKE), defined as new renal replacement therapy, stage 3 acute kidney injury, or death. Both Wnt3a and Wnt5a levels were higher in the patient group than in the control (P = 0.001 and P

Significance Canonical Wnt signaling is one of the most important and widely distributed pathways in metazoan development. Dishevelled is thought to serve as an essential bridge between the membrane receptors and downstream signaling components, which has the tendency to aggregate in vitro and to form large aggregates of dubious significance in vivo, when overexpressed. To obtain a molecular understanding of the role of Dvl in Wnt signaling, while circumventing these aggregation problems, we have expressed a fluorescent-tagged Dishevelled in cells at its physiological concentration and quantified the size distribution of Dishevelled before and after Wnt treatment. We found that limited oligomerization in response to the Wnt ligand is very dynamic and provides a key step in signal transduction., Dvl (Dishevelled) is one of several essential nonenzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits the destruction complex, eventually leading to inactivation of β-catenin degradation. Although this model is widespread, direct evidence for the individual steps is lacking. In this study, we tagged mEGFP to C terminus of dishevelled2 gene using CRISPR/Cas9-induced homologous recombination and observed its dynamics directly at the single-molecule level with total internal reflection fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and what are the dynamic features of membrane-bound Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane-bound Dvl2 is predominantly monomer in the absence of Wnt (observed mean size 1.1). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.12/μm2 to 0.54/μm2. Wnt3a also leads to increased oligomerization which raises the weighted mean size of Dvl2 complexes to 1.5, with 56.1% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of membrane Dvl2 caused by increased affinity of Dvl2 for Fzd, which is independent of LRP5/6. The oligomerized Dvl2 complexes have increased dwell time, 2 ∼ 3 min, compared to less than 1 s for monomeric Dvl2. These properties make Dvl a unique scaffold, dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.

ObjectiveCalcification of cartilage with basic calcium phosphate (BCP) crystals is a common phenomenon during osteoarthritis (OA). It is directly linked to the severity of the disease and known to be associated to hypertrophic differentiation of chondrocytes. One morphogen regulating hypertrophic chondrocyte differentiation is Wnt3a.MethodsCalcification and sulfation of extracellular matrix of the cartilage was analysed over a time course from 6 to 22 weeks in mice and different OA grades of human cartilage. Wnt3a and ß-catenin was stained in human and murine cartilage. Expression of sulfation modulating enzymes (HS2St1, HS6St1) was analysed using quantitative reverse transcription PCR (RT-PCR). The influence of BCP crystals on the chondrocyte phenotype was investigated using quantitative RT-PCR for the marker genes Axin2, Sox9, Col2, MMP13, ColX and Aggrecan. Using western blot for β-catenin and pLRP6 we investigated the activation of Wnt signalling. The binding capacity of BCP for Wnt3a was analysed using immunohistochemical staining and western blot.ResultsHere, we report that pericellular matrix sulfation is increased in human and murine OA. Wnt3a co-localised with heparan sulfate proteoglycans in the pericellular matrix of chondrocytes in OA cartilage, in which canonical Wnt signalling was activated. In vitro, BCP crystals physically bound to Wnt3a. Interestingly, BCP crystals were sufficient to induce canonical Wnt signalling as assessed by phosphorylation of LRP6 and stabilisation of β-catenin, and to induce a hypertrophic shift of the chondrocyte phenotype.ConclusionConsequently, our data identify BCP crystals as a concentrating factor for Wnt3a in the pericellular matrix and an inducer of chondrocyte hypertrophy.

Background Accumulating evidence suggests a regulatory role of Wnt proteins in innate immune responses. However, the effects of Wnt3a signaling on TLR4-mediated inflammatory responses are controversial and the signaling crosstalk between TLR4 and Wnt3a remains uncertain. Methods Gain- and Loss- of function approaches were utilized to determine the function of Wnt3a signaling in TLR4-mediated inflammatory responses. Cytokine production at protein and mRNA levels and phosphorylation of signaling molecules were measured by ELISA, qRT-PCR, and Western Blot, respectively. Endotoxemia mouse model was employed to assess the effect of Wnt3a on systemic inflammatory cytokine levels and neutrophil infiltration. Results LPS stimulation leads to an increase of Wnt3a expression and its downstream molecule, Dvl3, in primary monocytes. Inhibition or silence of Wnt3a or Dvl3 significantly increases the production of pro-inflammatory cytokines (IL-12, IL-6, TNFα), robustly reduces β-catenin accumulation, and enhances the phosphorylation of NF-κB P65 and its DNA binding activity. These results were confirmed by multiple gain- and loss- of function approaches including specific siRNA and ectopic expression of Dvl3, GSK3β, and β-catenin in monocytes. Moreover, in vivo relevance was established in a murine endotoxin model, in which Wnt3a inhibition enhances the inflammatory responses by augmenting the systemic pro-inflammatory cytokine levels and neutrophil infiltration. Conclusions TLR4 activation promotes Wnt3a-Dvl3 signaling, which acts as rheostats to restrain the intensity of inflammation through regulating GSK3β-β-catenin signaling and NF-κB activity. General significance Wnt3a-Dvl3-β-catenin signaling axis could be a potential interventional target for manipulating the direction and intensity of inflammatory responses.

The canonical Wnt signalling pathway is a critical pathway involved in the proliferation of cells. It has been well‐established that it plays the central role during colorectal carcinogenesis and development. Yet the exact molecular mechanism of how the canonical Wnt pathway is fine‐tuned remains elusive. We found that SLC35C1, a GDP‐fucose transporter, negatively regulates the Wnt signalling pathway. We show here that SLC35C1 is reduced in all colon cancer by both immunohistochemistry images and TCGA data, whereas β‐catenin is increased. Down‐regulation of SLC35C1 is also detected by real‐time PCR in stage 3 and stage 4 colorectal cancer tissues. Moreover, analysing the TCGA database with cBioPortal reveals the negative correlation of SLC35C1 mRNA level to the expression of β‐catenin. Reduced SLC35C1 significantly promotes cell proliferation and colony formation of HEK293 cells. Meanwhile, in HEK293 cells silencing SLC35C1 activates canonical Wnt pathway, whereas overexpressing SLC35C1 inhibits it. Consistently, the reduction of SLC35C1 in HEK293 cells also elevated the mRNA level of Wnt target genes C‐myc, Axin2 and Cyclin D1, as well as the secretion of Wnt3a. In conclusion, we identified SLC35C1 as a negative regulator of the Wnt signalling pathway in colon cancer. Decreased SLC35C1 may cause over‐activation of Wnt signalling in colorectal cancer.

The mechanism of inflammatory pain involves the central nervous system (CNS) and the immune system. It is reported that immunopotentiator thymosin alpha-1 (Tα1) can reduce inflammation, protect neurons and strengthen the immune function. However, the roles of Tα1 in inflammatory pain still remain unclear. In this study, we found Tα1 can attenuate the complete Freund's adjuvant (CFA)-induced mechanical allodynia and heat hyperalgesia. Meanwhile, it reduced the upregulation of CFA-induced inflammatory mediators (interferon (IFN)-γ, tumor necrosis factor-α and brain-derived neurotrophic factor). In addition, we found the Wnt3a/β-catenin pathway was activated in spinal cord after the injection of CFA, paralleling with pain hypersensitivity. However, Tα1 reversed this status. In summary, Tα1 could attenuate inflammatory pain by modulating the Wnt3a/β-catenin pathway. It might be related to the downregulation of inflammatory mediators.

Background: Hypoxic-ischaemic encephalopathy (HIE) is a prevailing severe brain damage disease in newborns, and caused by perinatal asphyxia cerebral ischaemia and reperfusion. Here, we investigate...