Wnt5a Protein Research Articles

Abstract P3148: Wnt/β-catenin Signaling Inhibits T-type Calcium Current And Causes Sinoatrial Node Dysfunction

Background: The T-type Ca 2+ channels (I Ca,T ) are expressed in the fetal and neonatal cardiomyocytes, but in the adult heart they are exclusively expressed in the cardiac conduction system, including the sinoatrial node (SAN). In contrast, the L-type Ca 2+ channels (I Ca,L ) are expressed ubiquitously in the adult heart and are important for cardiac contraction. The Wnt/β-catenin signaling (Wnt signaling) is activated in various heart disease and we and others have recently demonstrated that Wnt signaling inhibits cardiac Na + current. In the present study, we investigated if calcium channels are also regulated by Wnt signaling. Methods and Results: Because neonatal rat ventricular myocytes (NRVMs) express both I Ca,L and I Ca,T , we investigated the effect of Wnt signaling on NRVMs by treatment with either Wnt3a protein or CHIR-99021 (CHIR) (two different activators of the Wnt signaling). PCR array and RNA sequencing studies showed that Wnt signaling did not affect the mRNA of Cacna1c (encoding the α subunit of I Ca,L , Ca v 1.2), but the mRNA of Cacna1g (encoding the α subunit of I Ca,T , Ca v 3.1) was reduced in NRVMs (p<0.05, n=3) after treatment with either Wnt3a or CHIR. These observations were verified by qRT-PCR which showed dose-dependent reductions (p<0.05, n=4) in Cacna1g mRNA after CHIR treatment. Western blot and patch-clamp also demonstrated reductions (p<0.01, n=5) in channel protein and ionic current density of T-type, but not L-type, Ca 2+ channels after treatment with Wnt3a or CHIR. These observations suggest that Wnt signaling selectively inhibits T-type Ca 2+ channels in cardiomyocytes. Consistent with a critical role of T-type Ca 2+ channels in the pacemaker activity of SAN (the primary pacemaker of the heart), the spontaneous firing rates of isolated rat and mouse SAN tissues were reduced by ML-218 (an I Ca,T blocker) in a dose-dependent manner (p<0.01, n=5). In addition, activation of Wnt signaling in adult rat SAN tissues by CHIR led to slower and irregular beating, indicative of SAN dysfunction (p<0.01, n=5). Conclusions: These observations are consistent with the conclusions that Wnt/β-catenin signaling selectively inhibits T-type calcium current and may play a role in sinoatrial node dysfunction.

Abstract P2032: Wnt5a Mediates Doxorubicin-induced Cardiomyopathy Through Induction Of Senescence

Heart failure and cancer are major causes of death in the United States. Doxorubicin (DOX) is a commonly used chemotherapeutic agent. However, its clinical use is restricted due to cardiotoxicity, which can cause cardiomyopathy as well as heart failure. Senescence is defined by irreversible cell cycle arrest, resistance to apoptosis, and a senescence-associated secretory phenotype (SASP). Elimination of senescent cells improves cardiac dysfunction, suggesting that senescent cardiomyocytes (CMs) contribute to impaired heart function. Although DOX-induced senescence has been shown to occur in CMs, little is known about the mechanisms mediating DOX-induced cardiomyopathy. Wnt5a, one of the most studied non-canonical Wnt ligands, has emerged as an important player in cardiac diseases. mRNA expression of Wnt5a is upregulated in induced pluripotent stem cells (iPSC)-derived CMs from patients with end-stage heart failure induced by DOX chemotherapy. However, the role of Wnt5a in mediating the progression of DOX-induced cardiomyopathy remains elusive. We found that Wnt5a protein expression is markedly increased in mouse hearts with DOX-induced cardiomyopathy. Upregulation of Wnt5a following DOX treatment was also observed in neonatal rat CMs (NRCMs), and the degree of upregulation was more prominent in CMs than in non-CMs. We tested whether Wnt5a induces the senescent phenotype in NRCMs. Wnt5a not only induced an increase in p16 protein levels but also increased the number of γH2AX foci per cell and SA-β-gal activity in NRCMs. Wnt5a knockdown markedly reduced DOX-induced increases in p16 protein levels in NRCMs. These results suggest that DOX may induce cardiomyopathy through Wnt5a-mediated induction of CM senescence. Wnt5a may be a novel therapeutic target for the prevention of DOX-induced cardiac dysfunction.

Reversal of dual epigenetic repression of non-canonical Wnt-5a normalises diabetic corneal epithelial wound healing and stem cells

Aims/hypothesisDiabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression.MethodsHuman LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87±20.89 years) and diabetic (71.93±9.29 years) donors. The groups were not statistically different in age. DNA was extracted from LEC for methylation analysis using Illumina Infinium 850K MethylationEPIC BeadChip and protein was extracted for Wnt phospho array analysis. Wound healing was studied using a scratch assay in LEC or 1-heptanol wounds in organ-cultured corneas. Organ-cultured corneas and LEC were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng/ml), DNA methylation inhibitor zebularine (1–20 µmol/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15–20 µmol/l).ResultsThere was significant differential DNA methylation between diabetic and non-diabetic LEC. WNT5A promoter was hypermethylated in diabetic LEC accompanied with markedly decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold (p<0.05) and 37% (p<0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca2+-dependent non-canonical pathway (phospholipase Cγ1 and protein kinase Cβ; by 1.15-fold [p<0.05] and 1.36-fold [p<0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold (p<0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p<0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% (p<0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with WNT5A siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold (p<0.01) and 23.3% (p<0.05), respectively, with increased expression of LESC and diabetic markers.Conclusions/interpretationWe provide the first account of epigenetic changes in diabetic corneas including dual inhibition of WNT5A by DNA methylation and miRNA action. Overall, Wnt-5a is a new corneal epithelial wound healing stimulator that can be targeted to improve wound healing and stem cells in the diabetic cornea.Data availabilityThe DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229328).Graphical

The antitumor effects of WNT5A against hematological malignancies.

The bone marrow (BM) microenvironment (niche) is abnormally altered in acute myeloid leukemia (AML), leading to deficient secretion of proteins, soluble factors, and cytokines by mesenchymal stromal cells (MSC) that modifies the crosstalk between MSC and hematopoietic cells. We focused on a WNT gene/protein family member, WNT5A, which is downregulated in leukemia and correlated with disease progression and poor prognosis. We demonstrated that WNT5A protein upregulated the WNT non-canonical pathway only in leukemic cells, without modulating normal cell behavior. We also introduced a novel WNT5A-mimicking compound, Foxy-5. Our results showed reduction of crucial biological functions that are upregulated in leukemia cells, including ROS generation, cell proliferation, and autophagy, as well as G0/G1 cell cycle arrest. Additionally, Foxy-5 induced early-stage macrophage cell differentiation, a crucial process during leukemia development. At a molecular level, Foxy-5 led to the downregulation of two overexpressed leukemia pathways, PI3K and MAPK, which resulted in a disarrangement of actin polymerization with consequent impairment of CXCL12-induced chemotaxis. Notably, in a novel tri-dimensional bone marrow-mimicking model, Foxy-5 led to reduced leukemia cell growth and similar results were observed in a xenograft in vivo model. Overall, our findings highlight the pivotal role of WNT5A in leukemia and demonstrate that Foxy-5 acts as a specific antineoplastic agent in leukemia, counterbalancing several leukemic oncogenic processes related to the crosstalk in the bone marrow niche, and represents a promising therapeutic option for AML. WNT5A, a WNT gene/protein family member, is naturally secreted by mesenchymal stromal cells and contributes to the maintenance of the bone marrow microenvironment. WNT5A downregulation is correlated with disease progression and poor prognosis. The treatment with Foxy-5, a WNT5A mimetizing compound, counterbalanced several leukemogenic processes that are upregulated in leukemia cells, including ROS generation, cell proliferation, and autophagy and disruption of PI3K and MAPK signaling pathways.

Synthesis and potential osteogenic applications of Wnt3a‐loaded ZIF‐8 nanoparticles

The Wnt signaling pathway plays a critical role in bone homeostasis, and the related protein therapy strategies have been reported to have great potential in osseointegration; however, they face formidable challenges such as complex external environments and unavoidable protein denaturation. In this work, we report a novel approach combining the synthesis of metal–organic frameworks (MOFs) and protein encapsulation in a one-pot process based on zeolitic imidazolate framework-8 (ZIF-8) and Wnt3a protein, with improved biomechanical behavior and enhanced protein biological response. This combination was designed to enhance the Wnt3a protein function through the improved chemical stability provided by the ZIF-8 crystals. Additionally, the zinc ions contained in the ZIF-8 crystals induced bone homeostasis, further favoring the osteogenesis. The results showed that the Wnt3a protein-loaded ZIF-8 crystals served as efficient drug delivery vehicles to promote osteogenesis, preventing protein denaturation. In particular, Wnt3a-loaded ZIF-8 nanoparticles (Wnt3a@ZIF-8 NPs) had higher efficacy on bone marrow mesenchymal stem cells (BMSCs) than ZIF-8 NPs or Wnt3a proteins, contributing to the osteogenesis through ZIF-8 crystals and intracellular Wnt3a proteins released from Wnt3a@ZIF-8 NPs. Furthermore, polymerase chain reaction (PCR) analysis showed that the osteogenic pathways were upregulated. Overall, the present one-pot process can open up new avenues to develop signaling protein-delivery systems for applications in protein therapy strategies.

Hydrogel microenvironment contributes to chemical-induced differentiation of mesenchymal stem cells: single-cell infrared microspectroscopy characterization.

Stem cell microenvironment plays vital roles in directing cell proliferation and differentiation. Due to the tiny biochemical changes in the early stage of stem cell development, technical challenges to characterize the potential effects of environmental signals remain. In this work, we have introduced synchrotron radiation-based Fourier transform infrared microspectroscopy to evaluate the synergistic effects of physical and chemical factors on stem cell differentiation at the single-cell level. By using principal component analysis and cell-cell Euclidean distance calculation, the phenotypic heterogeneity changes during stem cell osteogenesis induced by lithium chloride or Wnt5a protein loaded in the polyvinyl alcohol (PVA) hydrogel were characterized in detail. The results demonstrated that PVA hydrogel could lead to the distinct effects between low-concentration lithium and wnt5a on human mesenchymal stem cells, suggesting a vital role of niche signals in Wnt pathway. These findings highlight the importance of microenvironment to the chemical-induced effects on stem cell differentiation and also provide a label-free, noninvasive method to sensitively identify the niche function in stem cell biology.

BuOH fraction of Salix Babylonica L. extract increases pancreatic beta-cell tumor death at lower doses without harming their function

Salix babylonica L. is a species of the willow tree. Insulinoma is a tumor originating from pancreatic beta cells. This study aims to research the effect of different fractions of Salix babylonica L. leaf extract on INS-1 cells for treating pancreatic tumors. Cell death occurred at lower doses in the EtOAc fraction. The cells are functional in the BuOH fraction but not in EtOAc and H2O fractions. The EtOAc fraction has a higher percentage of necrosis and ROS. INS1, INS2, and AKT gene expressions in the H2O fraction, GLUT2, IR, HSP70 gene expressions, and WNT4 protein levels increased in the BuOH fraction. HSP90 gene expression, Beta-actin, GAPDH, insulin, HSP70, HSP90, HSF1, Beta-Catenin, and WNT7A protein levels were decreased, while IR immunolabelling intensity increased in both fractions. Ca+2, K+, Na+, and CA-19-9 in the cell, Ca+2 and K+ in secretion increased. The secondary metabolites in the EtOAc fraction cause more damage in INS-1 cells. Since the water fraction also causes the cells to die in high doses, cell function is damaged. The secondary metabolites in the BuOH fraction kill INS-1 cells with less damage. This makes the BuOH fraction of Salix babylonica L. more valuable.

Wnt5a/Ca2+ signaling regulates silica-induced ferroptosis in mouse macrophages by altering ER stress-mediated redox balance

Silicosis is a chronic pulmonary disease characterized by diffuse fibrosis of lung caused by the deposition of silica dust (SiO2). The inhaled silica-induced oxidative stress, ROS production and macrophage ferroptosis are key drivers of the pathological process of silicosis. However, mechanisms that involved in the silica-induced macrophage ferroptosis and its contributions to pathogenesis of silicosis remain elusive. In the present study, we showed that silica induced murine macrophage ferroptosis, accompanied by elevation of inflammatory responses, Wnt5a/Ca2+ signaling activation, and concurrent increase of endoplasmic reticulum (ER) stress and mitochondrial redox imbalance in vitro and vivo. Mechanistic study further demonstrated that Wnt5a/Ca2+ signaling played a key role in silica-induced macrophage ferroptosis by modulating ER stress and mitochondrial redox balance. The presence of Wnt5a/Ca2+ signaling ligand Wnt5a protein increased the silica-induced macrophage ferroptosis by activating ER-mediated immunoglobulin heavy chain binding protein (Bip)-C/EBP homology protein (Chop) signaling cascade, reducing the expression of negative regulators of ferroptosis, glutathione peroxidase 4 (Gpx4) and solute carrier family 7 member 11 (Slc7a11), subsequentially increasing lipid peroxidation. The pharmacologic inhibition of Wnt5a signaling or block of calcium flow exhibited an opposite effect to Wnt5a, resulted in the reduction of ferroptosis and the expression of Bip-Chop signaling molecules. These findings were further corroborated by the addition of ferroptosis activator Erastin or inhibitor ferrostatin-1. These results provide a mechanism by which silica activates Wnt5a/Ca2+ signaling and ER stress, sequentially leads to redox imbalance and ferroptosis in mouse macrophage cells.

Surface-based modified 3D-printed BG/GO scaffolds promote bone defect repair through bone immunomodulation

Successful bone defect repair requires a favorable immunomodulation. Macrophages are among the frontline cells activated after biomaterial implantation, and biomaterials play a role in the final determination of their fate. The surface properties of biomaterials such as roughness and hydrophilicity can activate macrophages to have an anti-inflammatory phenotype for improving tissue regeneration. Because of its known characteristics, graphene oxide (GO) is widely used in the biomaterials field. GO directly promotes stem cell proliferation and osteogenic differentiation, properties that have been extensively investigated. The aim of this study is to research the effects of the surface-based modified three-dimensional (3D) porous bioactive glass (BG)/GO scaffolds on macrophage activation and bone regeneration. In vivo, compared with the control group receiving BG with a relatively smooth surface, the group receiving the porous implant showed a decreased proinflammatory response in the skin around the implant with increasing GO density, and the 3D porous implant was covered with new bone tissue. In vitro, both Raw264.7 cells and rat bone marrow mesenchymal stem cells (rBMSCs) can adhere and stretch well on rough, hydrophilic BG/GO scaffolds. When Raw264.7 cells were cultured on scaffolds, GO significantly promoted the polarization of M0 macrophages to the M2 type to a certain extent and further promoted the secretion of osteogenic and angiogenic factors, enhancing the osteogenic differentiation of rBMSCs and the angiogenesis of endothelial cells. Further experiments showed that the rough hydrophilic surface of BG/GO might lead to the upregulation of the surface-dependent WNT-3A protein induced by the enhancement of integrin signalling and the accumulation and activation of β-catenin protein in cells. However, excessive content of GO has the opposite regulatory result. These results demonstrate that BG/GO biomaterials with surface-based modification play an important role in bone repair by adjusting the response of macrophages to biomaterials through their surface properties.

Hypercapnia alters stroma-derived Wnt production to limit β-catenin signaling and proliferation in AT2 cells.

Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with acute respiratory distress syndrome (ARDS) secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here - using a mouse model of hypercapnia exposure, cell lineage tracing, spatial transcriptomics, and 3D cultures - we show that hypercapnia limits β-catenin signaling in alveolar type II (AT2) cells, leading to their reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRα+ fibroblasts from those maintaining AT2 progenitor activity toward those that antagonize β-catenin signaling, thereby limiting progenitor function. Constitutive activation of β-catenin signaling in AT2 cells or treatment of organoid cultures with recombinant WNT3A protein bypasses the inhibitory effects of hypercapnia. Inhibition of AT2 proliferation in patients with hypercapnia may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier and increasing lung flooding, ventilator dependency, and mortality.

Wnt3a-Modified Nanofiber Scaffolds Facilitate Tendon Healing by Driving Macrophage Polarization during Repair.

Inflammation is part of the natural healing response, but persistent inflammatory events tend to contribute to pathology changes of tendon or ligament. Phenotypic switching of macrophages within the inflammatory niche is crucial for tendon healing. One viable strategy to improve the functional and biomechanical properties of ruptured tendons is to modulate the transition from inflammatory to regenerative signals during tendon regeneration at the site of injury. Here, we developed a tendon repair scaffold made of biodegradable polycaprolactone by electrospinning, which was modified to deliver Wnt3a protein and served as an implant to improve tendon healing in a rat model of Achilles tendon defect. During the in vitro study, Wnt3a protein promoted the polarization of M2 macrophages. In the in vivo experiment, Wnt3a scaffold promoted the early recruitment and counting curve of macrophages and increased the proportion of M2 macrophages. Achilles function index and mechanical properties showed that the implantation effect of the Wnt3a group was better than that of the control group. We believe that this type of scaffold can be used to repair tendon defects. This work highlights the beneficial role of local delivery of biological factors in directing inflammatory responses toward regenerative strategies in tendon healing.

Effect of glucagon-like peptide-1 receptor agonist on insulin secretion index and serum Wnt5a protein in patients with new-onset type 2 diabetes mellitus

ObjectivePrevious studies have found that wnt5a promotes β-cell insulin secretion and reduced concentrations in patients with type 2 diabetes. GLP-1RA (Glucagon-like peptide-1 receptor agonists) can regulate insulin secretion. However, the evidence that GLP-1RA affect insulin secretion through the Wnt5a is inconclusive. Therefore, this study aimed to evaluate the effect of GLP-1 RA on wnt5a levels in patients with type 2 diabetes.MethodsA total of 56 onset diabetics were selected our study, 29 of them were treated by GLP-1RAs (1.2mg subcutaneous injection once a day, liraglutide, Novo Nordisk), the rest (27 case) treated by Metformin (0.5 g twice a day, Glucophage, Merck). Individuals who were using medications to manage platelet (Aspirin) and cholesterol (Statins) were enrolled and continued treatment throughout the study.ResultsOur study found that the waist circumference and insulin secretion index in the GLP-1RA intervention group were significantly increased, and the insulin resistance index was lower than that of the control group. More interestingly, the serum Wnt5a protein level increased dramatically after the GLP-1RA intervention, and the level of Secreted frizzled-related protein 5 (Sfrp5) decreased compared with the control group. Multivariate linear regression analysis showed that the change of HOMA-β (Homeostasis model assessment- β) was significantly correlated with the changes of Wnt5a and Sfrp5, and the change of Wnt5a protein was positively correlated with HOMA-β.ConclusionOur results confirmed that GLP-1RA may improve HOMA-β in patients with type 2 diabetes by affecting the level of Wnt5a protein.

WNT5A, β‑catenin and SUFU expression patterns, and the significance of microRNA deregulation in placentas with intrauterine growth restriction.

Placental insufficiency is a common cause of intrauterine growth restriction (IUGR). It affects ~10% of pregnancies and increases fetal and neonatal morbidity and mortality. Although Wnt and Hh pathways are crucial for embryonic development and placentation, their role in the pathology of IUGR is still not sufficiently explored. The present study analyzed the expression of positive regulators of the Wnt pathway, WNT5A and β‑catenin, and the expression of the Hh pathway negative regulator suppressor of fused (SUFU). Immunohistochemical and reverse transcription‑quantitative PCR (RT‑qPCR) assays were performed on 34 IUGR and 18 placental tissue samples from physiologic singleton‑term pregnancies. Epigenetic mechanisms of SUFU gene regulation were also investigated by methylation‑specific PCR analysis of its promoter and RT‑qPCR analysis of miR‑214‑3p and miR‑378a‑5p expression. WNT5A protein expression was higher in endothelial cells of placental villi from IUGR compared with control tissues. That was also the case for β‑catenin protein expression in trophoblasts and endothelial cells and SUFU protein expression in trophoblasts from IUGR placentas. The SUFU gene promoter remained unmethylated in all tissue samples, while miR‑214‑3p and miR‑378a‑5p were downregulated in IUGR. The present results suggested altered Wnt and Hh signaling in IUGR. DNA methylation did not appear to be a mechanism of SUFU regulation in the pathogenesis of IUGR, but its expression could be regulated by miRNA targeting.

Periodontal ligaments enhance neurite outgrowth in trigeminal ganglion neurons through Wnt5a production induced by mechanical stimulation.

The peripheral sensory nerve must be maintained to perceive environmental changes. Daily physiological mechanical stimulations, like gravity, floor reaction force, and occlusal force, influence the nerve homeostasis directly or indirectly. Although the direct axonal membrane stretch enhances axon outgrowth via mechanosensitive channel activation, the indirect mechanisms remain to be elucidated. In this study, we identified the indirect pathways where Wnt5a was a molecular cue released by mechanically stimulated rat periodontal ligament (rPDL) cells. qRT-PCR and ELISA showed that mechanically stimulated rPDL cells enhanced Wnt5a expression level and Wnt5a protein in a Ca2+-dependent manner. The inhibitors of PI3K (LY294002) and MEK1/2 (U0126) suppressed the Akt/PKB and ERK1/2 phosphorylation, respectively, in Western blotting analysis and consequently abolished the increase in Wnt5a expression. Similarly, PF573228, a focal adhesion kinase inhibitor, attenuated Akt- and ERK1/2-phosphorylation and Wnt5a expression. Importantly, the culture medium of stretched PDL cells enhanced neurite elongation, sprouting, and branching in trigeminal ganglion neurons that project to PDL. Moreover, treatment with an anti-Wnt5a antibody (to neutralize Wnt5a activity), AP7677a (anti-Ryk antibody, to block Ryk receptor activity), or strictinin (Ror1 inhibitor) suppressed the morphological changes. These findings reveal the indirect mechanisms that Wnt5a, released from the connective tissues in response to mechanical stimulation, enhances the outgrowth of the peripheral nerves. Our study suggests that the peripheral connective tissues regulate peripheral nerve homeostasis and that Wnt5a signaling could be targeted for the treatment of peripheral nerve disorders.

Oncogenic Wnt3a is a promising sensitive biomarker for monitoring hepatocarcinogenesis

BackgroundThe effective treatment for hepatocellular carcinoma (HCC) depends on early diagnosis. Previously, the abnormal expression of Wnt3a as the key signaling molecule in the Wnt/β-catenin pathway was found in HCC cells and could be released into the circulation. In this study, we used rat model of hepatocarcinogenesis to dynamically investigate the alteration of oncogenic Wnt3a and to explore its early monitor value for HCC. MethodsSprague-Dawley rats (SD) were fed with diet 2-fluorenylacetamide (2-FAA, 0.05%) for inducing hepatocarcinogenesis, and grouped based on liver morphological alteration by Hematoxylin & Eosin (H&E) staining; rats fed with normal chow were used as normal control (NC). Total RNA and protein were purified from rat livers. Differently expressed genes (DEGs) or Wnt3a mRNA, cellular distribution, and Wnt3a protein levels were analyzed by whole genome microarray with signal logarithm ratio (SLR log2cy5/cy3), immunohistochemistry, and enzyme-linked immunosorbent assay, respectively. ResultsModels of rat hepatocarcinogenesis were successfully established based on liver histopathological H&E staining. Rats were divided into the cell degeneration (rDeg), precancerosis (rPre-C) and HCC (rHCC) groups. Total numbers of the up- and down-regulated DEGs with SLR ≥ 8 were 55 and 48 in the rDeg group, 268 and 57 in the rPre-C group, and 312 and 201 in the rHCC group, respectively. Significantly altered genes were involved in cell proliferation, signal transduction, tumor metastasis, and apoptosis. Compared with the NC group, Wnt3a mRNA was increased by 4.6 folds (P < 0.001) in the rDeg group, 7.4 folds (P < 0.001) in the rPre-C group, and 10.4 folds (P < 0.001) in the rHCC group; the positive rates of liver Wnt3a were 66.7% (P = 0.001) in the rDeg group, 100% (P < 0.001) in the rPre-C group, and 100% (P < 0.001) in the rHCC group, respectively. Also, there were significant differences of liver Wnt3a (P < 0.001) or serum Wnt3a (P < 0.001) among different groups. ConclusionsOverexpression of Wnt3a was associated with rat hepatocarcinogenesis and it should be expected to be a promising monitoring biomarker for HCC occurrence at early stage.

Wnt5a/Ror2 promotes vascular smooth muscle cells proliferation via activating PKC.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) can cause various vascular diseases, such as atherosclerosis, restenosis, and pulmonary hypertension. However, the effect and underlying mechanism of Wnt5a on the proliferation of VSMCs remain unclear. Our study aimed to investigate whether Wnt5a/Ror2 promotes vascular smooth muscle cell proliferation via activating protein kinase C (PKC), thereby effectively alleviating vascular proliferative diseases. The proliferation of HA-VSMC cell line was evaluated by CCK-8, EdU, and Plate clone formation assays. The Wnt5a gene knockdown and overexpression were carried out by standard methods. The interaction between Wnt5a and Ror2 was explored by co-immunoprecipitation. Western blotting and immunofluorescence were used to determine the expression levels of key proteins in VSMCs. The present study found that the expression of Wnt5a protein increased significantly in the proliferation of VSMCs stimulated by 10% serum in a time-dependent manner. Furthermore, the proliferative rate of VSMCs overexpressing Wnt5a was dramatically accelerated, whereas Wnt5a knockdown using siWnt5a reversed thisproliferative effect. Wnt5a up-regulated the expression of receptor tyrosine kinase-like orphan receptor 2 (Ror2) by binding to it. Further studies indicated that Wnt5a induces the PKC expression in VSMCs and knockdown of Wnt5a or Ror2 could inhibit PKC phosphorylation. Wnt5a could effectively promote the proliferation of VSMCs, which might be related to the binding of Wnt5a and Ror2 to activate PKC.

Chloroquine alleviates the heat-induced to injure via autophagy and apoptosis mechanisms in skin cell and mouse models.

Burns can cause cell death and irreversible tissue damage. We examined the pathway of human dermis fibroblasts cell death caused by skin burns and the roles of chloroquine in human skin keratinocytes HaCaT wound healing. Western blot assays were performed to assess expression of proteins associated with autophagy, apoptosis, and endoplasmic reticulum stress in skin cells following burns. Changes in apoptosis-related proteins were assessed using flow cytometry, and wound cell migration was examined using wound healing assays. The burn animal model was used to test whether chloroquine would promote wound healing. In human burned fibroblasts, expression of LC3B-II and Cleave-caspase-7 was increased, whereas expression of Beclin-1, p62, and Grp78 was decreased. Severe burn induced ER stress and ERK phosphorylation, but PD98059 or necrostatin-1 treatment cells did not affect expression of autophagy LC3B-II protein and can induce apoptosis. Even though added with TGF-β and FGF did not repair autophagy caused by burns. Suggesting that autophagy and apoptosis were involved in heat-injured mechanism. Recombinant Wnt3a protein can help restore expression of β-catenin which reduced following burns in keratinocytes. Wnt3a protein can promote migration of keratinocytes after burns. Interesting, chloroquine increased expression of LC3B-II protein and restored cell migration activity after 24 h of burns. Consistently, surgical dressing containing chloroquine promoted wound healing in a burn animal mode. Autophagy and Wnt/β-catenin is two signalling pathways that participate in cell repair and wound healing in human fibroblasts, keratinocytes. Surgical dressing containing chloroquine can recover wound healing in burned rats.

Zoledronic acid generates a spatiotemporal effect to attenuate osteoarthritis by inhibiting potential Wnt5a-associated abnormal subchondral bone resorption.

This study aimed to determine the effects of zoledronic acid (ZOL) on OA in rats and explored the molecular mechanism of osteoclast activation in early OA. A knee OA rat model was designed by surgically destabilizing the medial meniscus (DMM). Seventy-two male rats were randomly assigned to Sham+phosphate-buffered saline (PBS), DMM+PBS, and DMM+ZOL groups; rats were administered with 100 μg/Kg ZOL or PBS, twice weekly for 4 weeks. After 2, 4, 8, and 12 weeks of OA induction, the thickness of the hyaline and calcified cartilage layers was calculated using hematoxylin and eosin staining, degenerated cartilage stained with Safranin O-fast green staining was evaluated and scored, tartrate-resistant acid phosphatase (TRAP)-stained osteoclasts were counted, changes in subchondral bone using micro-computed tomography were analyzed, and PINP and CTX-I levels were detected using enzyme-linked immunosorbent assay. Using these results, 18 male rats were randomly assigned to three groups. Four weeks after surgery, Wnt5a, RANKL, CXCL12, and NFATc1 protein levels were measured in subchondral bone using western blotting, and mRNA levels of genes related to osteoclastogenesis in subchondral bone were measured using quantitative polymerase chain reaction. Bone marrow-derived macrophages were isolated as osteoclast precursors, and cell differentiation, migration, and adhesion were assessed by TRAP staining and Transwell assays, revealing that DMM induced knee OA in rats. Progressive cartilage loss was observed 12 weeks after OA induction. Subchondral bone remodeling was dominated by bone resorption during early OA (within 4 weeks), whereas bone formation was increased 8 weeks later. ZOL suppressed bone resorption by inhibiting Wnt5a signaling in early OA, improved the imbalance of subchondral bone remodeling, reduced cartilage degeneration, and delayed OA progression. Additionally, ZOL delayed OA progression and reduced cartilage degeneration via a spatiotemporal effect in DMM-induced OA. Osteoclast activity in early OA might be associated with Wnt5a signaling, indicating a possible novel strategy for OA treatment.

TRIM52 promotes proliferation, invasion, and migration of gastric cancer cells by regulating Wnt/β-catenin pathway

Purpose: This study aimed to reveal the role and mechanism of tripartite motif-containing 52 (TRIM52) in gastric cancer (GC) progression.Methods: The Cancer Genome Atlas (TCGA) database was utilized to analyze TRIM52 expression in GC samples and para-carcinoma tissue samples, and the results were confirmed by quantitative realtime polymerase chain reaction. Cell counting kit-8 and colony formation assays were used to evaluate cell viability. Wound healing assay was utilized to analyze cell migration, while Transwell assay was utilized to evaluate cell invasion. TRIM52, proliferating cell nuclear antigen, matrix metalloproteinase-2, Wnt5a, β-catenin, and c-Myc protein levels were measured by western blot.Results: TRIM52 was expressed more in GC tissue samples and cells compared to normal tissues and cells (p &lt; 0.001). Overexpression of TRIM52 promoted growth, migration, and invasion of HGC-27 cells, and silencing inhibited growth, migration, and invasion of HGC-27 cells (p &lt; 0.001). In addition, TRIM52 overexpression increased Wnt5a, β-catenin, and c-Myc protein expression, and silencing decreased Wnt5a, β-catenin, and c-Myc protein expression (p &lt; 0.001 or p &lt; 0.01), indicating that TRIM52 activates Wnt/β-catenin signaling pathway.Conclusion: These findings reveal that TRIM52 facilitates GC cell proliferation, migration and invasion, but activates Wnt/β-catenin signaling.

Hematopoietic stem cell regeneration through paracrine regulation of the Wnt5a/Prox1 signaling axis.

The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration. Here, we show that in mice, deletion of the Fanconi anemia (FA) genes Fanca and Fancc dampened HSC regeneration through direct effects on HSCs and indirect effects on BM niche cells. FA HSCs showed persistent upregulation of the Wnt target Prox1 in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improved long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in WT HSCs mimicked the defective repopulation phenotype of FA HSCs. WT mice but not FA mice showed significant induction by TBI of BM stromal Wnt5a protein. Mechanistically, FA proteins regulated stromal Wnt5a expression, possibly through modulating the Wnt5a transcription activator Pax2. Wnt5a treatment of irradiated FA mice enhanced HSC regeneration. Conversely, Wnt5a neutralization inhibited HSC regeneration after TBI. Wnt5a secreted by LepR+CXCL12+ BM stromal cells inhibited β-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSCs. The detrimental effect of deregulated Wnt5a/Prox1 signaling on HSC regeneration was also observed in patients with FA and aged mice. Irradiation induced upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improved HSC regeneration. Treatment of aged mice with Wnt5a enhanced hematopoietic repopulation. Collectively, these findings identified the paracrine Wnt5a/Prox1 signaling axis as a regulator of HSC regeneration under conditions of injury and aging.