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Pulmonary arterial hypertension (PAH) is a devastating disease characterized by severe pulmonary vascular wall remodeling and perivascular inflammation. Resolvin E1 (RvE1), a proresolving lipid mediator, has protective effects against various inflammatory diseases. However, the effect of RvE1 on PAH development remains to be determined. We aimed to investigate whether RvE1 has a therapeutic effect on PAH and, if so, to elucidate the molecular mechanisms underlying its effects. A hypoxia+SU5416-induced mouse model of pulmonary hypertension (PH) and an monocrotaline-induced rat model of PH were used to test therapeutic effect of RvE1. Lung tissues and plasma samples were collected from patients with PAH and rodent models to examine RvE1 production and its receptor chemerin chemokine-like receptor 1 (ChemR23) expression. We observed that RvE1 generation was reduced in the plasma of patients with idiopathic PAH and in lungs from experimental rodent models of PH. ChemR23 expression was markedly downregulated in hypoxia-exposed mouse pulmonary artery smooth muscle cells (PASMCs) and pulmonary arteries from PH rodents and patients with idiopathic PAH. RvE1 treatment alleviated experimental PH in both male and female rodents by inhibiting PASMC proliferation. Deletion of ChemR23 in vascular SMCs abolished the protective effect of RvE1 against hypoxia+SU5416-induced PAH in mice. Mechanistically, the RvE1/ChemR23 axis suppressed hypoxia-induced PASMC proliferation by inhibiting proliferative wingless-type MMTV integration site family member 7a/β-catenin signaling. Activation of ChemR23 by RvE1 diminished wingless-type MMTV integration site family member 7a expression in PASMCs by inhibiting protein kinase A-mediated Egr2 (early growth response 2) phosphorylation at Ser349. Thus, the RvE1/ChemR23 axis represses experimental PAH by modulating wingless-type MMTV integration site family member 7a/β-catenin signaling in PASMCs and may serve as a therapeutic target for the management of PAH.

Glioblastomas (GBMs) are the most frequent primary malignancies in the central nervous system. Aberrant activation of WNT/β-catenin signaling pathways is critical for GBM malignancy. However, the regulation of WNT/β-catenin signaling cascades remains unclear. Presently, we observed the increased expression of ZEB2 and decreased expression of miR-637 in GBM. The expression of miR-637 was negatively correlated with expression of ZEB2. miR-637 overexpression overcame the ZEB2-enhanced cell proliferation and G1/S phase transition. In addition, miR-637 suppressed canonical WNT/β-catenin pathways by targeting WNT7A directly. Gain- and loss-of-function experiments in U251 mice demonstrated that miR-637 inhibited cell proliferation and arrested the G1/S phase transition, leading to tumor growth suppression. The collective findings suggest that ZEB2 and WNT/β-catenin cascades merge at miR-637 and the ectopic expression of miR-637 disturbs ZEB2/WNT/β-Catenin-mediated GBM growth. The findings should inform improved β-catenin-targeted therapy against GBM.

Glioma is one of the most common and aggressive malignant primary brain tumors, with a poor 5-year survival rate. The long noncoding RNA (lncRNA) CTBP1-AS2 has been shown to be correlated with the prognosis of cancer, but the role of CTBP1-AS2 in glioma and its concrete mechanism is fully unknown. The clinical data and tissues of glioma patients were analyzed. Cell viability and migration assays were performed. Western blotting and qRT-PCR were adopted for investigation of target protein expressions. Double luciferase assay was used to investigate the interaction between different elements. The lncRNA CTBP1-AS2 had increased expression profiles in tumor tissues, which is associated with poor prognosis. In detail, CTBP1-AS2 knockdown decreased proliferation and migration phenotypes in both U87-MG and LN229 cells. Moreover, CTBP1-AS2 knockdown suppressed the key epithelial–mesenchymal transition (EMT) markers by downregulating Wnt7a-mediated signaling. Furthermore, miR-370-3p functioned as a link that could be absorbed by CTBP1-AS2, thus regulating Wnt7a expression. Lastly, the CTBP1-AS2–miR-370-3p–Wnt7a axis modulated EMT in glioma cells in vitro and in vivo. This study provides new insights that a novel lncRNA, CTBP1-AS2, regulates EMT of glioma by modulating the miR-370-3p–Wnt7a axis.

Interferon-gamma (IFN-γ) is critical for central nervous system (CNS) functions and it may be a promising treatment to stimulate CNS regeneration. However, previous studies reported inconsistent results, and the molecular mechanisms remain controversial. Here we show that IFN-γ-treated mice via intraperitoneal injection have elevated IFN-γ level in central hippocampus and superior cognitive behaviors IFN-γ could activates the level of protein expression of Wnt7a, β-catenin, and CyclinD1 in Wnt/β-catenin signaling pathway of mice hippocampus. Functional and mechanism analysis in vitro revealed that IFN-γ promoted the proliferation and differentiation in primary cultured neural stem cells (NSCs). STAT1 was accountable for IFN-γ-induced activation of the β-catenin promoter, and IFN-γ increased the binding affinity of STAT1 to β-catenin promoter based on luciferase activity and chromatin immunoprecipitation. Our results suggest that IFN-γ exerts many effects ranging from cognitive function in vivo to NSC proliferation, self-renewal, and differentiation in vitro. It does so by recruiting STAT1 to the β-catenin promoter, enhancing cis-regulation by STAT1, and ultimately activating Wnt/β-catenin signaling. In this study, we first found that STAT1 was recruited into the promoter of β-catenin to activate β-catenin expression, and this effect was regulated by IFN-γ. It is also discovered firstly that Wnt/β-catenin and JAK/STAT pathways form cross-links through STAT1. Promoting neurogenesis through immune stimulation might be a promising strategy for repairing the diseased/injured CNS. This study provides a scientific basis for immunomodulation to promote nerve regeneration and offer a new therapeutic direction for central nervous system regeneration.

Aims The canonical Wnt signaling pathway plays an essential role in blood‐brain barrier integrity and intracerebral hemorrhage in preclinical stroke models. Here, we sought to explore the association between canonical Wnt signaling and hemorrhagic transformation (HT) following intravenous thrombolysis (IVT) in acute ischemic stroke (AIS) patients as well as to determine the underlying cellular mechanisms. Methods 355 consecutive AIS patients receiving IVT were included. Blood samples were collected on admission, and HT was detected at 24 hours after IVT. 117 single‐nucleotide polymorphisms (SNPs) of 28 Wnt signaling genes and exon sequences of 4 core cerebrovascular Wnt signaling components (GPR124, RECK, FZD4, and CTNNB1) were determined using a customized sequencing chip. The impact of identified genetic variants was further studied in HEK 293T cells using cellular and biochemical assays. Results During the study period, 80 patients experienced HT with 27 parenchymal hematoma (PH). Compared to the non‐PH patients, WNT7A SNPs (rs2163910, P = .001, OR 2.727; rs1124480, P = .002, OR 2.404) and GPR124 SNPs (rs61738775, P = .012, OR 4.883; rs146016051, P A) identified in the PH patients resulted in a single amino acid alteration (p.Cys1196Tyr) in the intracellular domain of GPR124. This variant substantially reduced the activity of WNT7B‐induced canonical Wnt signaling by decreasing the ability of GPR124 to recruit cytoplasmic DVL1 to the cellular membrane. Conclusion Variants of WNT7A and GPR124 are associated with increased risk of PH in patients with AIS after intravenous thrombolysis, likely through regulating the activity of canonical Wnt signaling.

Background Non-small cell lung cancer (NSCLC) is the most important cause of lung cancer death. Wnt7a is a known tumor suppressor gene which is often downregulated in NSCLC, and restoration of Wnt7a leads to decreased NSCLC cell proliferation. However, the biological role of Wnt7a in the migration and invasion in NSCLC remains unclear. Methods We examined whether overexpression of Wnt7a transfected by pcDNA6-Wnt7a could induce the proliferation, migration and invasion of NSCLC H1650 and A549 cell lines. Wnt7a signaling pathway, such as canonical (β-catenin) or non-canonical (c-Jun N-terminal kinase, JNK) pathways, were also assessed. Results We found that re-expression of Wnt7a led to reduced cell growth in NSCLC cell lines. In spite of the antiproliferative effect, Wnt7a overexpression could affect the migration and invasion of NSCLC cells. In the Wnt7a signaling pathway, the phosphorylation of JNK (Thr-183/Tyr-185) and c-Jun (Ser-63) were increased by re-expression of Wnt7a in both H1650 and A549 cell lines. The phosphorylation of β-catenin (Thr-41/Ser-45, Ser-552, Ser-675, and Ser-45) were not altered by restoration of Wnt7a. In NSCLC cells, Wnt7a overexpression was accompanied by parallel changes in the JNK pathway but not in the β-catenin pathway. Conclusions These results help to understand that Wnt7a may play a two-sided role in NSCLC, suggesting that restoration of Wnt7a expression is not always suitable as therapeutic strategy for NSCLC.

Entorhinal cortex lesions have been established as a model for hippocampal deafferentation and have provided valuable information about the mechanisms of synapse reorganization and plasticity. Although several molecules have been proposed to contribute to these processes, the role of Wnt signaling components has not been explored, despite the critical roles that Wnt molecules play in the formation and maintenance of neuronal and synaptic structure and function in the adult brain. In this work, we assessed the reorganization process of the dentate gyrus (DG) at 1, 3, 7, and 30 days after an excitotoxic lesion in layer II of the entorhinal cortex. We found that cholinergic fibers sprouted into the outer molecular layer of the DG and revealed an increase of the developmental regulated MAP2C isoform 7 days after lesion. These structural changes were accompanied by the differential regulation of the Wnt signaling components Wnt7a, Wnt5a, Dkk1, and Sfrp1 over time. The progressive increase in the downstream Wnt-regulated elements, active-β-catenin, and cyclin D1 suggested the activation of the canonical Wnt pathway beginning on day 7 after lesion, which correlates with the structural adaptations observed in the DG. These findings suggest the important role of Wnt signaling in the reorganization processes after brain lesion and indicate the modulation of this pathway as an interesting target for neuronal tissue regeneration.

Cancer cachexia is a complex metabolic disease so far lacking effective therapy, and it accounts for approximately one third of all cancer-related deaths worldwide. The extracellular ligand Wnt7a has a dual function in skeletal muscle, inducing the anabolic AKT/mammalian target of rapamycin (mTOR) pathway in myofibers and driving muscle stem cell expansion in skeletal muscle, making it a promising candidate for treatment of muscle wasting diseases. In murine and human myotubes, Wnt7a activates the anabolic AKT/mTOR pathway, thereby preventing cachexia-induced atrophy with a single application being sufficient to prevent atrophy independently of the tumor cell type causing cachexia. Addition of Wnt7a also improved activation and differentiation of muscle stem cells in cancer cachexia, a condition under which skeletal muscle regeneration is severely impaired due to stalled muscle stem cell differentiation. Finally, we show that Wnt7a prevents cancer cachexia in an in vivo mouse model based on C26 colon carcinoma cells. Wnt7a has a dual role in cachectic skeletal muscle; that is, it effectively counteracts muscle wasting through activation of the anabolic AKT/mTOR pathway and, furthermore, reverts the loss of muscle stem cell functionality due to cancer cachexia, making Wnt7a a promising candidate for an ameliorative treatment of cancer cachexia. Keywords: Wnt7a, cancer cachexia, skeletal muscle, muscle stem cell, satellite cell, atrophy, muscle wasting

Background The WNT gene family plays an important role in the occurrence and development of malignant tumors, but its involvement has not been systematically analyzed in uterine corpus endometrial carcinoma (UCEC). This study aimed to evaluate the prognostic value of the WNT gene family in UCEC. Methods Pan-cancer transcriptome data of the UCSC Xena database and Genotype-Tissue Expression (GTEx) normal tissue data were downloaded to analyze the expression and prognosis of 19 WNT family genes in UCEC. A cohort from The Cancer Genome Atlas-Uterine Corpus Endometrial Carcinoma (TCGA-UCEC) was used to analyze the expression of the WNT gene family in different immune subtypes and clinical subgroups. The STRING database was used to analyze the interaction of the WNT gene family and its biological function. Univariate Cox regression analysis and Lasso cox analysis were used to identify the genes associated with significant prognosis and to construct multi signature prognosis model. An immunohistochemical assay was used to verify the predictive ability of the model. Risk score and the related clinical features were used to construct a nomogram. Results The expression levels of WNT2, WNT3, WNT3A, WNT5A, WNT7A, and WNT10A were significantly different among different immune subtypes and correlated with TP53 mutation. According to the WNT family genes related to the prognosis of UCEC, UCEC was classified into two subtypes (C1, C2). The prognosis of subtype C1 was significantly better than that of subtype C2. A 2-gene signature (WNT2 and WNT10A) was constructed and the two significantly prognostic groups can be divided based on median Risk score. These results were verified using real-world data, and the nomogram constructed using clinical features and Risk score had good prognostic ability. Conclusions The 2-gene signature including WNT2 and WNT10A can be used to predict the prognosis of patients with UCEC, which is important for clinical decision-making and individualized therapy for patients with UCEC.

Caducity is known to be an independent risk factor in osteoarthritis (OA), yet the molecular basis behind caducity and OA remains unclear. Klotho, an anti-caducity protein, is an endogenous antagonist of the transduction of Wnt/β-catenin signal which can stimulate the articular cartilage degradation, indicating that deficiency in Klotho may increase Wnt/β-catenin activity and consequently accelerate the development of OA. We found that expression of Klotho was markedly higher in normal mouse cartilage than in the OA model, and in this model the activity of Wnt/β-catenin and its target gene was up-regulated. Decrease in Klotho expression was closely associated with the increase of β-catenin in OA, indicating that there was a negative correlation between Klotho and Wnt signal transduction. In the vitro and in vivo experiments, Klotho was found to bind to multiple Wnt, including Wnt1, Wnt4 and Wnt7a. It was additionally found that cyclic tenisle strain (CTS) inhibited the expression of Klotho and activated β-catenin. On the contrary, over-expression of Klotho would reduce the degradation of articular cartilage induced by CTS. These results suggest that Klotho is an antagonist of endogenous Wnt/β-catenin activity. In OA cartilage, decrease in expression of Klotho can activate Wnt/β-catenin signal transduction and consequently induce cartilage injury.

Summary RECK in neural precursor cells (NPCs) was previously found to support Notch-dependent neurogenesis in mice. On the other hand, recent studies implicate RECK in endothelial cells (ECs) in WNT7-triggered canonical WNT signaling essential for brain angiogenesis. Here we report that RECK in NPCs is also critical for brain angiogenesis. When Reck is inactivated in Foxg1-positive NPCs, mice die shortly after birth with hemorrhage in the forebrain, with angiogenic sprouts stalling at the periphery and forming abnormal aggregates reminiscent of those in EC-selective Reck knockout mice and Wnt7a/b-deficient mice. The hemorrhage can be pharmacologically suppressed by lithium chloride. An effect of RECK in WNT7-producing cells to enhance canonical WNT-signaling in reporter cells is detectable in mixed culture but not with conditioned medium. Our findings suggest that NPC-expressed RECK has a non-cell-autonomous function to promote forebrain angiogenesis through contact-dependent enhancement of WNT signaling in ECs, implying possible involvement of RECK in neurovascular coupling., Graphical Abstract, Highlights • Mice lacking RECK in Foxg1-positive neural precursor cells die shortly after birth • These mice show vascular defects similar to those in mice lacking endothelial RECK • The vascular phenotype can be suppressed by LiCl, an activator of WNT signaling • RECK in WNT7-producing cell enhances contact-dependent WNT signaling in adjacent cells, Vascular Remodeling; Neuroscience; Molecular Neuroscience; Developmental Neuroscience

Background: Motor and cognitive decline as part of the normal aging process is linked to alterations in synaptic plasticity and reduction of adult neurogenesis in the dorsal striatum. Neuroinflammation, particularly in the form of microglial activation, is suggested to contribute to these age-associated changes. Objective and Methods: To explore the molecular basis of alterations in striatal function during aging we analyzed RNA-Seq data for 117 postmortem human dorsal caudate samples and 97 putamen samples acquired through GTEx. Results: Increased expression of neuroinflammatory transcripts including TREM2, MHC II molecules HLA-DMB, HLA-DQA2, HLA-DPA1, HLA-DPB1, HLA-DMA and HLA-DRA, complement genes C1QA, C1QB, CIQC and C3AR1, and MHCI molecules HLA-B and HLA-F was identified. We also identified down-regulation of transcripts involved in neurogenesis, synaptogenesis, and synaptic pruning, including DCX, CX3CL1, and CD200, and the canonical WNTs WNT7A, WNT7B, and WNT8A. The canonical WNT signaling pathway has previously been shown to mediate adult neurogenesis and synapse formation and growth. Recent findings also highlight the link between WNT/β-catenin signaling and inflammation pathways. Conclusions: These findings suggest that age-dependent attenuation of canonical WNT signaling plays a pivotal role in regulating striatal plasticity during aging. Dysregulation of WNT/β-catenin signaling via astrocyte–microglial interactions is suggested to be a novel mechanism that drives the decline of striatal neurogenesis and altered synaptic connectivity and plasticity, leading to a subsequent decrease in motor and cognitive performance with age. These findings may aid in the development of therapies targeting WNT/β-catenin signaling to combat cognitive and motor impairments associated with aging.

Long noncoding RNAs (lncRNAs) play crucial roles in the regulation of human thyroid cancer (TC), including papillary thyroid carcinoma (PTC); PTC is the most common pathological subtype of TC. To date, the expression, function, and mechanism of the lncRNA CASC15 in PTC remain unclear. The present study results showed that CASC15 was overexpressed in PTC tissues compared with normal tissues and acted as a potent oncogene to promote the proliferation and tumorigenesis of PTC cells both in vitro and in vivo. Mechanistic studies demonstrated that CASC15 could serve as an endogenous miRNA sponge to absorb and downregulate miR-7151-5p, thereby preventing the inhibition of WNT7A during PTC progression. Furthermore, the study demonstrated that CASC15 activated the WNT/β‑catenin signaling pathway by upregulating WNT7A in PTC. Taken together, our findings identified CASC15 as a potential diagnostic marker or therapeutic target for PTC progression. DATA AVAILABILITY: Please contact the corresponding author for a data request.

Type 2 diabetes mellitus (T2DM) is one of the major chronic diseases, whose prevalence is increasing dramatically worldwide and can lead to a range of serious complications. Wnt ligands (Wnts) and their activating Wnt signalling pathways are closely involved in the regulation of various processes that are important for the occurrence and progression of T2DM and related complications. However, our understanding of their roles in these diseases is quite rudimentary due to the numerous family members of Wnts and conflicting effects via activating the canonical and/or non‐canonical Wnt signalling pathways. In this review, we summarize the current findings on the expression pattern and exact role of each human Wnt in T2DM and related complications, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11 and Wnt16. Moreover, the role of main antagonists (sFRPs and WIF‐1) and coreceptor (LRP6) of Wnts in T2DM and related complications and main challenges in designing Wnt‐based therapeutic approaches for these diseases are discussed. We hope a deep understanding of the mechanistic links between Wnt signalling pathways and diabetic‐related diseases will ultimately result in a better management of these diseases.

Background: Thrombospondin (TSP) is an astrocyte-secreted protein, well-known for its function as a modulator of synaptogenesis and neurogenesis. The mechanism underlying the effects of TSP1 on synaptic activity and formation involved in MHE pathogenesis remains unclear. Methods: The present study explored the effect of TSP1 on neurodegeneration, inflammatory response and the activation of Wnt7a/CNTF signaling in the primary rat neurons and an MHE rat model. Results: When we treated neurons with TSP1, p38MAPK phosphorylation and TNFα expression was increased significantly. Also, the exposure of TSP1 increased the expression and release of Wnt7a and CNTF, upregulated spinophilin, enhanced the interaction of Wnt7a/CNTF with spinophilin, and triggered synaptic activity through p38/TNFα signaling in PC12 cells and primary neurons. The hippocampal injection of TSP1 siRNA in mice decreased the interaction of Wnt7a/CNTF with spinophilin, while injection of Wnt7a and CNTF improved learning and memory dysfunctions. MHE brains showed decreased TSP1 expression. The overexpression of TSP1 in the hippocampus of MHE rats ameliorated the disrupted synaptogenesis, learning, and memory. Conclusions: Taken together, these results indicated that TSP1 is a potential synaptic factor related to the inflammatory response and the activation of Wnt7a/CNTF signaling in MHE pathogenesis.

ObjectiveThe expression of Wnt7a in colorectal cancer tissues and cell lines was analyzed, and the effect of Wnt7a on the proliferation of colorectal cancer cells was studied, so as to confirm the relationship between Wnt7a and the occurrence and development of colorectal cancer.Methods(1) Immunohistochemical method was used to compare the expression of Wnt7a in different tissues and its relationship with the clinicopathology of colorectal adenocarcinoma. (2) The expression levels of Wnt7a in colorectal cancer cell lines HT-29 and HCT 116 were detected by qRT-PCR. (3) The down-regulated Wnt7A expression vector was constructed, and the down-regulated Wnt7A expression cell line was established. The regeneration ability of cancer cells was detected by stem cell ball formation assay, and the influence of plate cloning assay on the proliferation ability of colorectal cancer cells was detected.Results(1) The positive rates of Wnt7a in normal colorectal mucosa, colorectal adenoma and colorectal adenocarcinoma tissues gradually increased,Wnt7a are closely related to the degree of colorectal adenocarcinoma differentiation, lymph node metastasis and Duke stage. (2) The expression level of Wnt7a in colorectal cancer cells was higher than that in normal colorectal epithelial cells. (3) The down-regulation of Wnt7A reduced the proliferation ability of colorectal cancer cells.ConclusionsWnt7a promotes the occurrence and development of colorectal adenocarcinoma.

Preterm infant brain injury is a leading cause of morbidity and disability in survivors of preterm infants. Unfortunately, the effective treatment remains absent. Recent evidence suggests that GSK-3β inhibitor TWS119 has a neuroprotective role in adult brain injury by activation of Wnt/β-catenin signaling pathway. However, the role on neonatal brain injury is not yet explored. The study aims to evaluate the effect of TWS119 at 7 d after hypoxic-ischemic brain damage and investigate the mechanism that it regulates Wnt and Notch signaling pathways at 24 h after hypoxic-ischemic brain damage in neonatal rats. Three-day-old rats were randomly divided into 3 groups: sham group, HI group and TWS119 group. The neonatal rats were subjected to left carotid artery ligation followed by 2 h of hypoxia (8.0% O2). A single dose of TWS119 (30 mg/kg) was intraperitoneally injected 20 min prior to hypoxia-ischemia (HI). At 7 d after HI, TWS119 improved the tissue structure, reduced cell apoptosis, up-regulated bcl-2 expression, up-regulated the expression of PSD-95 and Synapsin-1. At 24 h after HI, it activated Wnt/β-catenin signaling pathway by up-regulation of β-catenin protein expression and wnt3a/wnt5a/wnt7a mRNA expression. Simultaneously, it suppressed Notch signaling pathway by down-regulation of Notch1 and HES-1 proteins expression. Our study suggested that TWS119 performed a neuroprotective function at 7 d after hypoxic-ischemic brain damage via a crosstalk with Wnt/β-catenin and Notch signaling pathways at 24 h after hypoxic-ischemic brain damage in neonatal rats.

Background Wnt signaling plays key roles in cellular and physiological processes, including cell proliferation, differentiation and migration during development and tissue homeostasis in adults. This pathway can be defined as Wnt/β-catenin-dependent or β-catenin-independent or “non-canonical”, both signaling are involved in neurite and synapse development/maintenance. Porcupine (PORCN), an acylase that o-acylates Wnt ligands, a major modification in secretion and interaction with its receptors. We use Wnt-C59, a specific PORCN inhibitor, to block the secretion of endogenous Wnts in embryonic hippocampal neurons (DIV 4). Under these conditions, the activity of exogenous Wnt ligands on the complexity of the dendritic tree and axonal polarity were evaluated Methods Cultured primary embryonic hippocampal neurons obtained from Sprague–Dawley rat fetuses (E18), were cultured until day in vitro (DIV) 4 (according to Banker´s protocol) and treated with Wnt-C59 for 24 h, Wnt ligands were added to the cultures on DIV 3 for 24 h. Dendritic arbors and neurites were analysis by fluorescence microscopy. Transfection with Lipofectamine 2000 on DIV 2 of plasmid expressing eGFP and KIF5-Cherry was carried out to evaluate neuronal polarity. Immunostaining was performed with MAP1B and Tau protein. Immunoblot analysis was carried out with Wnt3a, β-catenin and GSK-3β (p-Ser9). Quantitative analysis of dendrite morphology was carried out with ImageJ (NIH) software with Neuron J Plugin. Results We report, here, that Wnt-C59 treatment changed the morphology of the dendritic arbors and neurites of embryonic hippocampal neurons, with decreases β-catenin and Wnt3a and an apparent increase in GSK-3β (p-Ser9) levels. No effect was observed on axonal polarity. In sister cultures, addition of exogenous Wnt3a, 5a and 7a ligands rescued the changes in neuronal morphology. Wnt3a restored the length of neurites to near that of the control, but Wnt7a increased the neurite length beyond that of the control. Wnt5a also restored the length of neurites relative to Wnt concentrations. Conclusions Results indicated that Wnt ligands, added exogenously, restored dendritic arbor complexity in embryonic hippocampal neurons, previously treated with a high affinity specific Porcupine inhibitor. We proposed that PORCN is an emerging molecular target of interest in the search for preclinical options to study and treat Wnt-related diseases.

Rationale: Idiopathic Pulmonary Fibrosis (IPF) is a progressive pulmonary disorder usually associated with epithelial damage and fibroblast activation and involves numerous molecular signalling cascades governing activation, differentiation, proliferation and matrix remodelling. Canonical Wingless/integrase-1 (WNT) / s-catenin signalling pathway operates via phosphorylation of its co-receptor LRP5 upon Wnt ligand binding and positively regulates proliferation and cell survival in many types of cells. Moreover, WNT signalling is dysregulated in IPF and has been linked to pulmonary disease pathogenesis and progression. However, the direct role of LRP5 is not fully understood. Hypothesis: Decrease in Wnt/s-catenin pathway activity and stabilization of LRP5 plays a role in fibroblast activation and differentiation in pulmonary fibrosis. Methods and Results: Our study focused mainly on the contribution of Wnt ligand stimulation (Wnt1, 3A and 7A) on normal human lung fibroblast (NHLF) activity. Stimulation of NHLF (2 donors) with Wnt ligands for 24h induced fibroblast to myofibroblast transition as assessed by phase-contrast microscopy and by proliferation. We found a dramatic increase in aSMA expression and cell proliferation, with Wnt7A showing the most prominent effect. LRP5 knockdown followed by treatment with WNT ligands and/or TGF- s1 stimulation for 24h partially abrogated the WNT and TGF-s1 induced changes. Conclusions: Our findings suggest that elevation of aSMA expression and cell proliferation are modulated by LRP5 with a key role in fibroblast activation, differentiation and proliferation.

Colorectal cancer (CRC) is the fourth leading cause of cancer death worldwide, and constitutive activation of the Wnt signaling pathway is universal in most CRC cases. Wnt ligands (Wnts) are secreted glycoproteins and fundamentally essential for the transduction of Wnt signaling pathway. However, the 19 members of Wnts in humans imply a daunting complexity of Wnt signaling and biological effects, and our understanding of their roles in CRC tumorigenesis is still quite rudimentary. This review will give an overview of the structural characteristics and maturation process of Wnts. The expression pattern of all human Wnts in CRC tissues, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16, and their relationship with the tumorigenesis and the progression of CRC will be specifically summarized separately. Despite certain challenges, Wnt-based therapeutics for CRC emerge continuously and some are now in clinical trials. In conclusion, a deep understanding of Wnts is very helpful for a better management of this disease.

Wnt signaling constitutes a fundamental cellular and molecular pathway, necessary from proper embryogenesis to function-maintenance of fully developed complex organisms. In this regard, Wnt pathway plays a crucial role in both the development of the central nervous system and in maintaining the structure and function of the neuronal circuits, and it has been suggested that its dysregulation is critical in the onset of several pathologies including cancer and neurodegenerative disorders, such as Alzheimer’s disease (AD). Due to its relevance in the maintenance of the neuronal activity and its involvement in the outbreak of devastating diseases, we explored the age-related changes in the expression of Wnt key components in the cortex and hippocampus of 7 to 72-months-old Octodon degus (O. degus), a Chilean long-living endemic rodent that has been proposed and used as a natural model for AD. We found a down-regulation in the expression of different Wnt ligands (Wnt3a, Wnt7a, and Wnt5a), as well as in the Wnt co-receptor LRP6. We also observed an increase in the activity of GSK-3β related to the down-regulation of Wnt activity, a fact that was confirmed by a decreased expression of Wnt target genes. Relevantly, an important increase was found in secreted endogenous Wnt inhibitors, including the secreted-frizzled-related protein 1 and 2 (SFRP-1 and SFRP-2) and Dickkopf-1 (Dkk-1), all them antagonists at the cell surface. Furthermore, treatment with Andrographolide, a labdane diterpene obtained from Andrographis paniculata, prevents Wnt signaling loss in aging degus. Taken together, these results suggest that during the aging process Wnt signaling activity decreases in the brain of O. degus.

The Wingless-type protein 7a (Wnt7a) plays an antiproliferative role in non-small-cell lung cancer (NSCLC). Previous studies have indicated that Wnt7a expression was downregulated in radiation-resistant NSCLC cells. However, little is known about its biological functions and molecular mechanisms in radiosensitivity of NSCLC. Thus, NSCLC cell proliferation and apoptosis in response to Wnt7a overexpression and/or radiation were determined by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl-tertazolium bromide (MTT) assay and flow cytometry, respectively. The activation of the Wnt/cJun N-terminal kinase (JNK) and Wnt/β-catenin signaling pathways were further examined by western blot in NSCLC cell lines H1650 and A549. Wnt7a overexpression combined with radiation-inhibited cell proliferation and induced apoptosis in NSCLC cell lines compared to Wnt7a overexpression or radiotherapy alone. In addition, the phosphorylation of JNK, but not β-catenin, was congruent with the changes in Wnt7a overexpression and/or radiation. Moreover, the Wnt/JNK pathway could induce the apoptosis of NSCLC cells through the mitochondrial pathway. Inhibition of the Wnt/JNK signaling pathway by SP600125, a JNK inhibitor, contributed to proliferation induction in NSCLC cells. Taken together, these results showed that Wnt7a overexpression sensitized NSCLC cell lines to radiotherapy through the Wnt/JNK signaling pathway., Summary: The radiosensitizing effect of Wnt7a in non-small-cell lung cancer (NSCLC).

Lung squamous cell carcinoma (LUSC) is the most common histologic type of smoking-related non-small cell lung cancer (NSCLC). However, there are no identified potential biomarkers for smoking-related LUSC diagnosis and prognosis. Especially, the characteristics of genetic alteration and tumor microenvironment induced by cigarette smoking remain unknown. Here, we performed integrative analysis of 463 LUSC with smoking history information from The Cancer Genome Atlas (TCGA). Non-smokers had the best prognosis, and current reformed smokers had better overall survival (OS) than current smokers in all and stage I-II cohort. Then, pathway enrichment analysis might suggest that smoking may play a role in regulating tumor metabolism and invasion and metastasis via those pathways. We constructed an eight-gene signature and identified WNT7A, Solute carrier-7A5 (SLC7A5) and Brain‑type glycogen phosphorylase (PYGB), which may be served as biomarkers related to the smoking. Notably, the single copy deletion of WNT7A and SLC17A5 and the low-level amplification of PYGB may be related to the epigenetic mechanism of smoking on tumorigenesis. We also estimated the relative proportion of 24 immune cell subtypes within tumor microenvironment in different smoking status. Interestingly, we found NK cells activated, NK cells resting and endothelial cells might play an important role in immunologic dysfunction and harmful tumor microenvironment induced by cigarette smoking. Our research has identified potential biomarkers for smoking-related LUSC diagnosis and prognosis, which would help to further understand the pathogenesis of LUSC.

In the adult hippocampus of many mammals, a particular microenvironment in the neurogenic niche regulates the proliferation, self-renewal, and differentiation of neuronal stem cells. In this proliferative niche, a variety of molecules provide a finely regulated molecular signaling that controls stem cell properties. During development, Wnt signaling has been implicated in cell fate determination and proliferation, in the establishment of cell polarity, as well as a cue for axonal growth and dendrite orientation. In the adult brain, this pathway also participates in the stem cell self-renewal and neuronal differentiation. However, the effects of the chronic Wnt signaling modulation in the adult hippocampus, through the infusion of Wnt7a, Wnt5a, and Dkk-1, on the rate of neurogenesis and on the induction of neurite arborization have not been studied. In this study, we show that Wnt7a and Wn5a further increased the rate of newly generated neurons. However, Wnt5a exerted additional effects by promoting neurite growth and neurite misorientation in the dentate gyrus of adult rats. The chronic exposure to Dkk-1 also generated aberrant location of growing neurites. These results suggest that the interplay of canonical and non-canonical Wnt ligands participates in neuronal stem cell proliferation and in the establishment of proper neurite maturation. Anat Rec, 302:1647-1657, 2019. © 2019 American Association for Anatomy.

Background: Colorectal cancer is one of the most common malignant tumors in China, and the number of new cases and the number of cases of deaths has increased annually. However, its pathogenesis is still unclear. Wnt7a is a member of the wingless-type MMTV integration site family, and it plays an important role in tumorigenesis and development by controlling cell proliferation and differentiation as a secreted glycoprotein. Whether Wnt7a has the properties of an oncogene or not is an important focus for future research as this target has diverse roles in different tumors.Methods: Wnt7a protein expression in normal colorectal mucosa and colorectal tumors was detected via immunohistochemistry and Western blot analysis. Univariate and multivariate analyses were used to explore the associations between Wnt7a staining score and various clinical parameters.Results: Wnt7a was strongly expressed in colorectal cancer tissues but weakly expressed in adjacent normal mucosa and colorectal adenomas. The level of Wnt7a expression was correlated with lymph node involvement (P < 0.001), Duke stage (P < 0.001), and cell differentiation (P < 0.001). Knockdown of Wnt7a inhibits proliferation of colon cancer cells and inhibits the ability of both colon cancer cell lines to migrate.Conclusions: Collectively, our results present evidence that Wnt7a is associated with an unfavorable prognosis of colorectal cancer.

Mesenchymal stem cells (MSCs) have the ability of differentiating into osteoblasts. Elucidating the molecular mechanisms of MSC differentiation into osteoblasts may provide novel therapeutic strategies for bone‑related diseases. Increasing evidence has confirmed that Wnt signaling plays the key role in osteoblast differentiation; however, the role of individual Wnt proteins in osteogenesis needs to be investigated. The present study thus aimed to explore the role of Wnt7a in bone formation. For this purpose, human bone‑derived MSCs were identified by flow cytometry and the cell differentiation potential, including osteogenic and adipogenic differentiation was examined. In order to explore the role of Wnt7a in MSC osteogenic differentiation, Wnt7a expression was measured at the mRNA and protein level following treatment with the osteogenic inducer, bone morphogenetic protein (BMP)4/7, and following the induction of osteogenic or adipogenic differentiation. The ectopic expression of Wnt7a in MSCs was confirmed and its influence on MSC osteogenic differentiation was detected using osteocyte markers and by Alizarin Red S staining. Mechanistically, the influence of Wnt7a on Runt‑related transcription factor 2 (RUNX2) expression was examined at the mRNA and protein level. The regulatory effects of Wnt7a on RUNX2 promoter activities were examined by promoter reporter assay, and by examining the binding of TCF1, a downstream target of Wnt, to the RUNX2 promoter by ChIP assay. The results revealed that the knockdown of Wnt7a in MSCs decreased the expression of osteocyte markers and inhibited osteogenic differentiation. In accordance, the overexpression of Wnt7a in MSCs increased the expression of osteocyte markers and promoted osteogenic differentiation. Mechanistically, the knockdown of Wnt7a in MSCs reduced RUNX2 expression and the overexpression of Wnt7a in MSCs promoted RUNX2 expression. Furthermore, it was confirmed that Wnt7a regulated RUNX2 promoter activities by promoter report assay, and by examining the binding of TCF1 to the RUNX2 promoter by ChIP assay. On the whole, the present study demonstrates that Wnt7a plays a key role in MSC differentiation into osteoblasts and the findings presented herein may provide a promising therapy target for bone‑related diseases.

In a previous work, we detected that postnatal exposure to a glyphosate-based herbicide (GBH) alters uterine development in prepubertal rats causing endometrial hyperplasia and increasing cell proliferation. Our goal was to determine whether exposure to low dose of a GBH during postnatal development might enhance the sensitivity of the uterus to an estrogenic treatment. Female Wistar pups were subcutaneously injected with saline solution (control) or GBH using the reference dose (2 mg/kg/day, EPA) on postnatal days (PND) 1, 3, 5 and 7. At weaning (PND21), female rats were bilaterally ovariectomized and treated with silastic capsules containing 17β-estradiol (E2, 1 mg/mL) until they were 2 months of age. On PND60, uterine samples were removed and processed for histology, immunohistochemistry and mRNA extraction to evaluate: (i) uterine morphology, (ii) uterine cell proliferation by the detection of Ki67, (iii) the expression of the estrogen receptors alpha (ESR1) and beta (ESR2) and (iv) the expression of WNT7A and CTNNB1. GBH-exposed animals showed increased luminal epithelial height and stromal nuclei density. The luminal and glandular epithelium were markedly hyperplastic in 43% of GBH-exposed animals. GBH exposure caused an increase in E2-induced cell proliferation in association with an induction of both ESR1 and ESR2. GBH treatment decreased membranous and cytoplasmic expression of CTNNB1 in luminal and glandular epithelial cells and increased WNT7A expression in the luminal epithelium. These results suggest that early postnatal exposure to a GBH enhances the sensitivity of the rat uterus to estradiol and induces histomorphological and molecular changes associated with uterine hyperplasia. Fil: Guerrero Schimpf, Marlise Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Milesi, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Luque, Enrique Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Varayoud, Jorgelina Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina

To investigate the role of Wnt/β-catenin signaling pathway in the restoration of induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) after laser photocoagulation. After differentiation of RPE cells from hiPSCs, laser photocoagulation was performed. Activation of Wnt/β-catenin signaling at days 1 and 5 after laser photocoagulation was evaluated by expression of β-catenin. Cell proliferation and alteration in cell-to-cell contact at day 5 after laser photocoagulation with or without Dickkopf-1 (Dkk-1) treatment were studied using ethynyl-2'-deoxyuridine (EdU) assay and zonula occludens-1 (ZO-1) expression analysis, respectively. The mRNA levels of Wnt genes at day 5 after laser photocoagulation were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Activation of Wnt/β-catenin signaling at days 1 and 5 after laser photocoagulation was confirmed by β-catenin accumulation in the cytoplasm and nucleus of hiPSC-RPE. Many EdU-positive cells also expressed β-catenin, and the number of EdU-positive cells was decreased at day 5 after laser photocoagulation after Dkk-1 treatment, indicating that Wnt/β-catenin signaling mediated hiPSC-RPE proliferation. ZO-1 expression was not decreased with Dkk-1 treatment at day 5 after laser photocoagulation, indicating that Wnt/β-catenin signaling mediated hiPSC-RPE restoration. At day 5, after laser photocoagulation, mRNA levels of Wnt2b, Wnt3, Wnt5a, Wnt7a, and Wnt10b were increased. Wnt/β-catenin signaling has a crucial role in restoration of hiPSC-RPE proliferation after laser photocoagulation. Manipulation of Wnt/β-catenin signaling while elucidating the underlying mechanisms of RPE restoration might have a therapeutic potential in retinal degenerative diseases.

Paracrine Wnt signals are critical regulators of cell proliferation, specification, and differentiation during embryogenesis. Consistent with the discovery that Wnt ligands are post-translationally modified with palmitoleate (a 16 carbon mono-unsaturated fatty acid), our studies show that the vast majority of bioavailable chick WNT1 (cWNT1) produced in stably transfected L cells is cell-associated. Thus, it seems unlikely that the WNT1 signal is propagated by diffusion alone. Unfortunately, the production and transport of vertebrate Wnt proteins has been exceedingly difficult to study as few antibodies are able to detect endogenous Wnt proteins and fixation is known to disrupt the architecture of cells and tissues. Furthermore, vertebrate Wnts have been extraordinarily refractory to tagging. To help overcome these obstacles, we have generated a number of tools that permit the detection of WNT1 in palmitoylation assays and the visualization of chick and zebrafish WNT1 in live cells and tissues. Consistent with previous studies in fixed cells, live imaging of cells and tissues with overexpressed cWNT1-moxGFP shows predominant localization of the protein to a reticulated network that is likely to be the endoplasmic reticulum. As PORCN and WLS are important upstream regulators of Wnt gradient formation, we also undertook the generation of mCherry-tagged variants of both proteins. While co-expression of PORCN-mCherry had no discernible effect on the localization of WNT1-moxGFP, co-expression of WLS-mCherry caused a marked redistribution of WNT1-moxGFP to the cell surface and cellular projections in cultured cells as well as in neural crest and surface ectoderm cells in developing chick embryos. Our studies further establish that the levels of WLS, and not PORCN, are rate limiting with respect to WNT1 trafficking.

Wnt signaling plays an important role in the adult brain function and its dysregulation has been implicated in some neurodegenerative pathways. Despite the functional role of the Wnt signaling in adult neural circuits, there is currently no evidence regarding the relationships between exogenously Wnt signaling activation or inhibition and hippocampal structural changes in vivo. Thus, we analyzed the effect of the chronic infusion of Wnt agonists, Wnt7a and Wnt5a, and antagonist, Dkk-1, on different markers of plasticity such as neuronal MAP-2, Tau, synapse number and morphology, and behavioral changes. We observed that Wnt7a and Wnt5a increased the number of perforated synapses and the content of pre-and postsynaptic proteins associated with synapse assembly compared to control and Dkk-1 infusion. These two Wnt agonists also reduced anxiety-like behavior. Conversely, the canonical antagonist, Dkk-1, increased anxiety and inhibited spatial memory recall. Therefore, the present study elucidates the potential participation of Wnt signaling in the remodeling of hippocampal circuits underlying plasticity events in vivo, and provides evidence of the potential benefits of Wnt agonist infusion for the treatment of some neurodegenerative conditions.

Polycystic ovarian syndrome (PCOS) is a common endocrinologic disorder in women of reproductive age characterized by polycystic ovaries, oligo/anovulation, and hyperandrogenism. Not only anovulation but also endometrial dysfunction can reduce fertility in PCOS patients. Wnt pathway is responsible for endometrial proliferation which be strongly regulated by estradiol. To determine the effects of clomiphene citrate (CC) and letrozole, we measured the expression of some main ligands of Wnt/β-catenin signaling including Wnt7a, Wnt3, and Wnt8b in the endometrial samples taken from PCOS women on day 12 of the menses who received 100 mg CC or 5 mg letrozole as well as from women without treatment. Significantly, the mean estrogen and progesterone concentration were lower and higher, respectively, in letrozole than CC. The mean endometrial thickness (ET) was significantly greater in letrozole compared to CC. Assessment of the mRNA and protein expression of Wnt7a, Wnt3, and Wnt8b showed significantly lower expression in CC than the letrozole and control groups. Collectively, letrozole provided a better molecular response in the endometrium of PCOS patients during the proliferative phase, similar to natural cycles, compared to CC. CC decreased the ligands expression of Wnt3, Wnt7a, and Wnt8b, resulting in endometrial dysfunction.

Objectives The cyclical changes in proliferation and differentiation of endometrial cells are regulated by estrogen and progesterone via modulating Wnt/β-catenin signaling. Imbalance in the expression of estrogen and progesterone receptors causes progesterone resistance in endometriosis patients. The aim of this study was to investigate the expression of some main components of Wnt/β-catenin signaling including WNT7a, DKK-1, β-catenin, and GSK-3β in eutopic endometrium and peritoneal endometriotic lesions of endometriosis patients compared to healthy endometrium in the mid-secretory phase of menstrual cycle. Study Design This prospective study was performed, during a 12 months period from December 2015 to November 2016, on healthy women as the control group (n = 14) and endometriosis patients (n = 34). We used real-time polymerase chain reaction and Western blot techniques. Results Protein and mRNA expression of DKK-1 were significantly down-regulated in both endometriotic lesions and eutopic endometrium of endometriosis group. We also demonstrated that the expression of non-phosphorylated β-catenin (active form) and phosphorylated GSK-3β (inactive form) were up-regulated in endometriosis patients. The mRNA levels of β-catenin, GSK-3β, and WNT7a, as well as the protein levels of total β-catenin, total GSK-3β, and WNT7a in endometriosis group, were not significantly different with those in control group. The patterns of mRNA and protein expression of all interested factors in the lesions were similar to those in the eutopic endometrium of same patients. Conclusions It seems that the aberrant activation of Wnt/β-catenin signaling in the secretory phase of the menstrual cycle in endometriosis has two essential elements: excessive inactivation of GSK-3β and suppression of the expression of Wnt signaling inhibitor DKK-1. Interventions in this signaling pathway may allow for the exploration of potential new targets for the control of development and progression of endometriosis.

The signaling pathways that sustain the disease process of chronic rhinosinusitis with nasal polyps (CRSwNP) remain poorly understood. We sought to determine the expression levels of Wnt signaling genes in CRSwNP and to study the role of the Wnt pathway in inflammation and epithelial remodeling in the nasal mucosa. Microarrays and real time-quantitative polymerase chain reaction comparing gene expression in matched NPs and inferior turbinates revealed that WNT2B, WNT3A, WNT4, WNT7A, WNT7B, and FZD2 were up-regulated and that FZD1, LRP5, LRP6, and WIF1 were down-regulated in NPs. Immunolabeling showed robust expression of Wnt ligands, nuclear β-catenin, and Axin-2 in NP tissue, suggesting that Wnt/β-catenin signaling is activated in NPs. We used primary human nasal epithelial cell (HNEpC) cultures to test the functional consequences of Wnt pathway activation. Monolayer HNEpCs treated with recombinant human WNT (rhWNT) 3A, but not with rhWNT4, had altered epithelial morphology and decreased adhesion, without loss of viability. We found that neither rhWNT3A nor rhWNT4 treatment induced proliferation. The expression and release of inflammatory cytokines IL-6 and granulocyte-macrophage colony-stimulating factor were increased after rhWNT3A exposure of HNEpCs. When differentiated at an air-liquid interface, rhWNT3A- and WNT agonist-, but not rhWNT4-treated HNEpCs, had abnormal epithelial architecture, failed to undergo motile ciliogenesis, and had defective noncanonical Wnt (planar cell polarity) signaling. On the basis of these results, we propose a model in which Wnt/β-catenin signaling sustains mucosal inflammation and leads to a spectrum of changes consistent with those seen during epithelial remodeling in NPs.

Uterine gland development occurs after birth in cattle and other mammals. The timeline of gland development has been described in various species, but little is known about how postnatal diet influences uterine gland development. This is especially concerning in dairy heifers, where a variety of milk replacer and whole milk nutrition options exist. Little work also exists in cattle to describe how early exposure to steroids influences reproductive tract and uterine gland development. The objective of this work was to determine the effects of early postnatal plane of nutrition and estrogen supplementation on uterine gland development in calves. In both studies, Holstein heifer calves were assigned to restricted milk replacer (R-MR) or enhanced milk replacer (EH-MR) diets. In study 1, calves (R-MR, n = 6; EH-MR, n = 5) were euthanized at 8 wk. In study 2, calves were weaned at 8 wk and administered estradiol (R-MR, n = 6; EH-MR, n = 6) or placebo (R-MR, n = 6; EH-MR, n = 5) for an additional 14 d before euthanasia. Average daily gain and final body weight was greater in both studies in heifers fed the enhanced diet. At 8 wk, EH-MR calves had a greater number of glands and a smaller average gland size, but total gland area was not different from the R-MR group. At 10 wk, uterine gland number and size were not affected by diet or estrogen. Expression profiles of several paracrine mediators of gland development were examined. Increases in transcript abundance for IGF1 and IGFBP3 and a decrease in abundance of WNT7A were detected in calves fed the enhanced diet at 8 wk of age. Plane of nutrition did not affect transcript profiles at 10 wk of age, but estradiol supplementation decreased MET and WNT7A transcript abundance. To conclude, heifer calves on a restricted diet exhibited a uterine morphology and transcript profile suggestive of delayed uterine gland development. These changes appear to be corrected by wk 10 of life. Also, this work provides evidence supporting the contention that early estradiol exposure has detrimental effects on uterine gene expression.

The exposure to endocrine-disrupting compounds (EDCs), such as glyphosate-based herbicides (GBHs), during early life might alter female fertility. The aim of the present study was to evaluate the effects of neonatal exposure to a GBH on sheep uterine development. To achieve this, Friesian ewe lambs were exposed to GBH (2 mg/kg of body weight/day; n = 12) or vehicle (controls; n = 10) through s.c. injections, from postnatal day (PND) 1 to PND14; on PND45, the uteri were obtained to evaluate histomorphological and molecular parameters. Morphological parameters were determined by picrosirius-hematoxylin staining. Protein expression of Ki67 (as a cell proliferation marker), p27, and molecules involved in uterine organogenetic differentiation was measured by immunohistochemistry. We also determined the mRNA expression of the IGF molecular pathway by RT-PCR. Although histomorphology was not modified, the uteri of GBH-exposed ewe lambs showed lower cell proliferation, together with higher p27 protein expression. In addition, the uteri of GBH-exposed ewe lambs showed increased gene expression of insulin-like growth factor binding protein 3 (IGFBP-3), decreased expression of ERα in the luminal (LE) and glandular (GE) epithelia and in the subepithelial stroma (SS), and lower PR expression in the LE but higher in the GE and SS. In addition, GBH treatment decreased the uterine expression of Wnt5a in the GE, of Wnt7a in the SS, of β-catenin in the LE and GE, of Hoxa10 in the SS, and of Foxa2 in the GE as compared with controls. In conclusion, neonatal exposure to GBH decreased cell proliferation and altered the expression of molecules that control proliferation and development in the uterus. All these changes might have adverse consequences on uterine differentiation and functionality, affecting the female reproductive health of sheep. GBH may be responsible for uterine subfertility, acting as an EDC. Fil: Alarcón, Ramiro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Rivera, Oscar Edgardo. Universidad Nacional de Lomas de Zamora. Facultad de Cs.agrarias. Instituto de Investigacion En Produccion Agropecuaria Ambiente y Salud. - Comision de Investigaciones Cientificas de la Provincia de Buenos Aires. Instituto de Investigacion En Produccion Agropecuaria Ambiente y Salud.; Argentina Fil: Ingaramo, Paola Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Tschopp, María Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Dioguardi, Gisela Haydeé. Universidad Nacional de Lomas de Zamora. Facultad de Cs.agrarias. Instituto de Investigacion En Produccion Agropecuaria Ambiente y Salud. - Comision de Investigaciones Cientificas de la Provincia de Buenos Aires. Instituto de Investigacion En Produccion Agropecuaria Ambiente y Salud.; Argentina Fil: Milesi, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Muñoz de Toro, Monica Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina Fil: Luque, Enrique Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Salud y Ambiente del Litoral. Universidad Nacional del Litoral. Instituto de Salud y Ambiente del Litoral; Argentina

Background Bone marrow mesenchymal stem cells (BMMSCs) can be used for bone regeneration in the specified condition. Osteogenic differentiation of BMMSCs is controlled by microRNAs (miRNAs) and other factors. This study was aimed to identify the role and mechanism of miR-889 in regulating the osteogenic differentiation of BMMSCs. Methods Osteoporosis patients and normal control bone tissues were collected and used PCR techniques to identify the change of miR-889 and WNT7A. Moreover, the dynamic change of miR-889 and WNT7A during osteogenic differentiation of BMMSCs was also measured. Bioinformatic analysis was performed to identify the target genes and potential pathways of miR-889. Then, we constructed miR-889 mimic and inhibitor, ALP staining, ARS, osteoblastic-related protein, and Wnt β-catenin signaling pathway-related protein were also measured. WNT7A siRNA was also used to verify the function of miR-889. Results In the present study, we showed that miR-889 expression was upregulated in osteoporosis patients than healthy control. However, the miR-889 expression was downregulated during osteogenic differentiation. Bioinformatics analysis found that miR-889 targets 666 genes and mainly through Wnt β-catenin signaling pathway. Administrated miR-889 mimic, the ALP activity, and calcium deposition were decreased than the control group, while miR-889 inhibitor shown the opposite trend. And miR-889 could bind the 3′UTR of WNT7A. We further used WNT7A siRNA to explore the function of miR-889, and the results revealed that co-cultured with miR-889 inhibitor and WNT7A siRNA was associated with a reduction of ALP activity and calcium deposition and osteoblastic-related proteins than miR-889 inhibitor alone. Conclusion Our results revealed that miR-889 plays a negative role in inducing osteogenic differentiation of BMSCs through Wnt β-catenin signaling pathway.

Once urinary bladder cancer (UBC) develops into muscle-invasive bladder cancer, its mortality rate increases dramatically. However, the molecular mechanisms of UBC invasion and metastasis remain largely unknown. Herein, using 5637 UBC cells, we generated two sublines with low (5637 NMI) and high (5637 HMI) invasive capabilities. Mass spectrum analyses revealed that the Wnt family protein Wnt7a is more highly expressed in 5637 HMI cells than in 5637 NMI cells. We also found that increased Wnt7a expression is associated with UBC metastasis and predicted worse clinical outcome in UBC patients. Wnt7a depletion in 5637 HMI and T24 cells reduced UBC cell invasion and decreased levels of active β-catenin and its downstream target genes involved in the epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) degradation. Consistently, treating 5637 NMI and J82 cells with recombinant Wnt7a induced cell invasion, EMT, and expression of ECM degradation–associated genes. Moreover, TOP/FOPflash luciferase assays indicated that Wnt7a activated canonical β-catenin signaling in UBC cells, and increased Wnt7a expression was associated with nuclear β-catenin in UBC samples. Wnt7a ablation suppressed matrix metalloproteinase 10 (MMP10) expression, and Wnt7a overexpression increased MMP10 promoter activity through two TCF/LEF promoter sites, confirming that Wnt7a-mediated MMP10 activation is mediated by the canonical Wnt/β-catenin pathway. Of note, the microRNA miR-370-3p directly repressed Wnt7a expression and thereby suppressed UBC cell invasion, which was partially restored by Wnt7a overexpression. Our results have identified an miR-370-3p/Wnt7a axis that controls UBC invasion through canonical Wnt/β-catenin signaling, which may offer prognostic and therapeutic opportunities.

Wnt pathway activation maintains the cancer stem cell (CSC) phenotype and promotes tumor progression, making it an attractive target for anti-cancer therapy. Wnt signaling at the tumor and tumor microenvironment (TME) front have not been investigated in depth in head and neck squamous cell carcinoma (HNSCC). In a cohort of 48 HNSCCs, increased Wnt signaling, including Wnt genes (AXIN2, LGR6, WISP1) and stem cell factors (RET, SOX5, KIT), were associated with a more advanced clinical stage. Key Wnt pathway proteins were most abundant at the cancer epithelial-stromal boundary. To investigate these observations, we generated three pairs of cancer-cancer associated fibroblast (CAF) cell lines derived from the same HNSCC patients. 3D co-culture of cancer spheres and CAFs mimicked these in vivo interactions, and using these we observed increased expression of Wnt genes (e.g. WNT3A, WNT7A, WNT16) in both compartments. Of these Wnt ligands, we found Wnt3a, and less consistently Wnt16, activated Wnt signaling in both cancer cells and CAFs. Wnt activation increased CSC characteristics like sphere formation and invasiveness, which was further regulated by the presence of CAFs. Time lapse microscopy also revealed preferential Wnt activation of cancer cells. Wnt inhibitors, OMP-18R5 and OMP-54F28, significantly reduced growth of HNSCC patient-derived xenografts and suppressed Wnt activation at the tumor epithelial-stromal boundary. Taken together, our findings suggest that Wnt signaling is initiated in cancer cells which then activate CAFs, and in turn perpetuate a paracrine signaling loop. This suggests that targeting Wnt signaling in the TME is essential.

Temperature stress is one of the main environmental stressors affecting the welfare, health and productivity of livestock. Temperature changes can modify cell membrane components, disrupting the crosstalk between the cell and its surroundings by affecting signaling pathways including Wnt signaling pathway, which subsequently disrupts cell energy metabolism. The present study aims to understand the effect of temperature stress on the expression of genes involved in Wnt signaling pathways, and their interaction with energy metabolism in C2C12 myoblasts cells. The C2C12 cells were exposed to cold stress (35 °C), mild heat stress (39 °C) and severe heat stress (41 °C), whereas 37 °C was used as control temperature. Transcript levels of important genes involved in Wnt signaling including Axin2, Tnks2, Sfrp1, Dkk1, Dact1, Cby1, Wnt5a, Wnt7a, Wnt11, Porcn, Ror2, Daam1, and Ppp3ca were significantly altered under severe heat stress (41 °C), whereas eight Wnt signaling-related transcripts (Daam1, Ppp3ca, Fzd7, Wnt5a, Porcn, Tnks2, Lrp6, and Aes) were significantly altered under cold stress (35 °C) compared to control. Under heat stress transcripts of the Wnt/β-catenin inhibitors (Sfrp1, Dkk1, and Cby1) and negative regulators (Dact1 and Axin2) are activated. A positive correlation between oxidative phosphorylation and Wnt-related transcripts was found under high temperatures. Transcripts of the cell membrane receptors, including Lrp6 and Fzd7, and the members of Wnt/Ca+2 signaling pathway, including Ppp3ca and Porcn were downregulated under cold stress. Many Wnt signaling-related transcripts were positively correlated with glycolysis under cold stress. These findings indicate a cross-talk between Wnt signaling and energy metabolism under thermal stress.

The role of astrocytes in dysregulation of blood-brain barrier (BBB) function following ischemic stroke is not well understood. Here, we investigate the effects of restoring the repair properties of astrocytes on the BBB after ischemic stroke. Mice deficient for NHE1, a pH-sensitive Na(+)/H(+) exchanger 1, in astrocytes have reduced BBB permeability after ischemic stroke, increased angiogenesis and cerebral blood flow perfusion, in contrast to wild-type mice. Bulk RNA-sequencing transcriptome analysis of purified astrocytes revealed that ~177 genes were differentially upregulated in mutant astrocytes, with Wnt7a mRNA among the top genes. Using a Wnt reporter line, we confirmed that the pathway was upregulated in cerebral vessels of mutant mice after ischemic stroke. However, administration of the Wnt/β-catenin inhibitor, XAV-939, blocked the reparative effects of Nhel-deficient astrocytes. Thus, astrocytes lacking pH-sensitive NHE1 protein are transformed from injurious to “protective” by inducing Wnt production to promote BBB repair after ischemic stroke.

The aim of this study is to evaluate the effect of repeated controlled ovarian hyperstimulation (COH) on the structure and function of the uterus and mammary gland. Three adult female rhesus monkeys were superovulated up to four times, and three spontaneously ovulating monkeys were used as controls. After a 5-year period, the uterus and mammary gland tissue samples were collected for examination of their structure and function. Further, the expression of certain tumor markers was examined to assess the cancer risk for each organ. Expression of Wnt7a (associated with the functional/developmental status of the uterus) was significantly decreased in the uterus of superovulated monkeys, and decreased expression of proliferation marker PCNA was found in uterine cells. Meanwhile, abnormal Golgi-derived secretory vesicles with an irregular shape were observed in the mammary glands of the superovulated monkeys, and decreased PCNA expression together with increased expression of caspase-3 (an apoptosis marker) was indicated in the mammary cells. The expression of tumor molecular markers of the uterus and mammary gland was not significantly different between the two groups. Repeated COH affects the expression of the uterine development-related gene several years later, and uterine cells exhibited a low proliferation status. The ultrastructure of the mammary gland epithelial cells was abnormal, and the cells exhibited both low proliferation and high apoptosis status. Cancer risk for these organs was not observed. Given that primates are the closest relatives of humans, the results obtained from this study provide more intuitive information for optimization of clinical COH.

Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Furthermore, if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa, as shown by various fate-tracing studies. However, the molecular bases of reprogramming remain elusive. Using two models of biliary injury where repair occurs through cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify up-regulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt coreceptors low-density lipoprotein-related protein 5/6, transgenic mice expressing stable β-catenin, and in vitro studies, we show a role of Wnt signaling through β-catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a β-catenin-independent manner. Conclusion: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both β-catenin-dependent and -independent manners. (Hepatology 2016;64:1652-1666)

The endometrium consists of stromal and epithelial compartments (luminal and glandular) with distinct functions in the regulation of uterine homeostasis. Ovarian sex steroids, namely 17β-estradiol and progesterone, play essential roles in modulating uterine cell proliferation, stromal-epithelial cross-talk and differentiation in preparation for pregnancy. The effect of androgens on uterine function remains poorly understood. The current study investigated the effect of the non-aromatizable androgen dihydrotestosterone (DHT) on mouse endometrial function. Ovx female mice were given a single sc injection (short treatment) or 7 daily injections (long treatment) of vehicle alone (5% ethanol, 0.4% methylcellulose) or vehicle with the addition of 0.2 mg DHT (n=8/group) and a single injection of bromodeoxyuridine 2 hours prior to tissue recovery. Treatment with DHT increased uterine weight, the area of the endometrial compartment and immunoexpression of the androgen receptor in the luminal and glandular epithelium. Treatment-dependent proliferation of epithelial cells was identified by immunostaining for MKi67 and bromodeoxyuridine. Real-time PCR identified significant DHT-dependent changes in the concentrations of mRNAs encoded by genes implicated in the regulation of the cell cycle (Wee1, Ccnd1, Rb1) and stromal-epithelial interactions (Wnt4, Wnt5a, Wnt7a, Cdh1, Vcl, Igf1, Prl8, Prlr) as well as a striking effect on the number of endometrial glands. This study has revealed a novel role for androgens in regulating uterine function with an effect on the glandular compartment of the endometrium. This previously unrecognized role for androgens has implications for our understanding of the role of androgens in regulation of endometrial function and fertility in women.

The transformation suppressor gene RECK was isolated by cDNA expression cloning (1998), and GPR124/TEM5 was detected as a tumor endothelial marker by differential screening (2000). The importance of Wnt7a/b and Gpr124 in brain angiogenesis was demonstrated by reverse genetics in mice (2008-2010). A series of recent studies using genetically engineered mice and zebrafish as well as luciferase reporter assays in cultured cells led to the discovery of functional interactions among Reck, Gpr124, and Wnt7a/b in triggering canonical Wnt signaling with relevance to embryonic brain angiogenesis and blood-brain barrier formation.

Neonatal exposure to a low dose of endosulfan may disrupt the expression of Wnt7a and β-catenin during uterine development leading to the failure of uterine functional differentiation during implantation. New-born female Wistar rats were treated with vehicle, endosulfan (600 μg/kg/d, E600) or diethylstilbestrol (0.2 μg/kg/d, DES) on postnatal days (PNDs) 1, 3, 5 and 7. Subsequently, uterine histomorphology and the protein expression of Wnt7a and β-catenin were evaluated on PND8, PND21 and gestational day (GD) 5 (pre-implantation period). In the E600 rats, Wnt7a and β-catenin protein expression was increased in the epithelium on PND8, and Wnt7a expression was decreased in the endometrial glands on PND21. On GD5, the number of uterine glands was decreased in the E600-and DES-treated rats. In addition, Wnt7a expression was decreased in all uterine compartments, and β-catenin expression was increased in the luminal and glandular epithelia of the E600-and DES-treated rats. Disruption of Wnt7a and β-catenin uterine expression in the prepubertal and adult females altered the uterine preparation for embryo implantation, which could be associated with the subfertility triggered by endosulfan.

Five small protein domains, the CC-domains, at the N terminus of the RECK protein, play essential roles in signaling by WNT7A and WNT7B in the context of central nervous system angiogenesis and blood-brain barrier formation and maintenance. We have determined the structure of CC domain 4 (CC4) at 1.65-Å resolution and find that it folds into a compact four-helix bundle with three disulfide bonds. The CC4 structure, together with homology modeling of CC1, reveals the surface locations of critical residues that were shown in previous mutagenesis studies to mediate GPR124 binding and WNT7A/WNT7B recognition and signaling. Surprisingly, sequence and structural homology searches reveal no other cell-surface or secreted domains in vertebrates that resemble the CC domain, a pattern that is in striking contrast to other ancient and similarly sized domains, such as Epidermal Growth Factor, Fibronectin Type 3, Immunoglobulin, and Thrombospondin type 1 domains, which are collectively present in hundreds of proteins.

Prostate cancer (PCa) is the most common non-cutaneous cancer among American men. In 2019, 174,650 new patients will be diagnosed from this disease. Most patients with localized PCa could be treated successfully. However, when PCa develops into castrate-resistant prostate cancer (CRPCa), the five-year survival rate drops below 30%. Neuroendocrine prostate cancer (NEPCa) is an aggressive subtype of PCa observed in 20%-30% CRPCa patients. Understanding how NEPCa progressed from prostate adenocarcinoma could reveal novel therapeutic targets. Previous studies indicate that activation of Wnt/beta-Catenin signaling promotes the progression to CRPCa with an increased NE differentiation. Additionally, animal studies suggest that Wnt/beta-Catenin signaling is active in NEPCa. Here we explored the mechanisms that promote Wnt/beta-Catenin signaling in NEPCa. Using TRAMP mice, which develop NEPCa after castration, we assessed the expression of Wnt-related genes and found that after androgen deprivation, the expression of Wnt7A is increased in prostate derived from both wild type and TRAMP mice but the expression of DKK1, a Wnt inhibitor, is decreased in wild type prostate but not in TRAMP prostate. The elevated expression of Wnt7a in combination with the lack of induction of Dkk1 in TRAMP tumors provides a mechanism to activate Wnt/beta-Catenin signaling in these tumors. Furthermore, we found the expression of YES-associated Protein (YAP1), a key transcriptional coactivator of Hippo pathway, is lost in NEPCa, in contrast to the elevated expression of YAP1 in high-grade prostate adenocarcinoma. YAP1 knockdown activated Wnt/beta-Catenin activity in PCa PC3 and 22Rv1 cells, indicating that the loss of YAP1 expression provides another mechanism to activate Wnt/beta-Catenin signaling, promoting NEPCa phenotype in PCa cells. Funding: This study is supported by: FWCC/Foundation Legacy Funds, DOD New Investigator Award, The Office of Research SEED awards, NIH R01 AND R03. Citation Format: Siyuan Cheng, Shu Yang, Nestor Prieto-Dominguez, Zachary M. Connelly, Yingli Shi, Xiuping Yu. Loss of YAP1 activates Wnt/beta-Catenin signaling in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4861.