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High dietary sugar (HDS), a contemporary dietary concern due to excessive intake of added sugars and carbohydrates, escalates the risk of metabolic disorders and concomitant cancers. However, the molecular mechanisms underlying HDS-induced cancer progression are not completely understood. We found that phosphoenolpyruvate carboxykinase 1 (PEPCK1), a pivotal enzyme in gluconeogenesis, is paradoxically upregulated in tumors by HDS, but not by normal dietary sugar (NDS), during tumor progression. Targeted knockdown of pepck1, but not pepck2, specifically in tumor tissue in Drosophila in vivo, not only attenuates HDS-induced tumor growth but also significantly improves the survival of Ras/Src tumor-bearing animals fed HDS. Interestingly, HP1a-mediated heterochromatin interacts directly with the pepck1 gene and downregulates pepck1 gene expression in wild-type Drosophila. Mechanistically, we demonstrated that, under HDS conditions, pepck1 knockdown reduces both wingless and TOR signaling, decreases evasion of apoptosis, reduces genome instability, and suppresses glucose uptake and trehalose levels in tumor cells in vivo. Moreover, rational pharmacological inhibition of PEPCK1, using hydrazinium sulfate, greatly improves the survival of tumor-bearing animals with pepck1 knockdown under HDS. This study is the first to show that elevated levels of dietary sugar induce aberrant upregulation of PEPCK1, which promotes tumor progression through altered cell signaling, evasion of apoptosis, genome instability, and reprogramming of carbohydrate metabolism. These findings contribute to our understanding of the complex relationship between diet and cancer at the molecular, cellular, and organismal levels and reveal PEPCK1 as a potential target for the prevention and treatment of cancers associated with metabolic disorders., (© 2024. The Author(s).)

Aim of the Study: Research on the mechanism of Huangqin Qingre Chubi Capsules (HQC) in improving rheumatoid arthritis accompanied depression (RA-dep) model rats., Methods: We employed real-time qPCR (RT-qPCR), western blotting (WB), confocal microscopy, bioinformatics, and other methods to investigate the anti-RA-dep effects of HQC and its underlying mechanisms., Results: HQC alleviated the pathological indexes of inflammation and depression in RA-dep model rats, decreased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, increased the levels of norepinephrine(NE) and serotonin(5-HT), and improved the injury of hippocampus. The analysis of network pharmacology suggests that HQC may target the Wnt/β-catenin pathway in the treatment of RA-dep. Furthermore, molecular dynamics simulations revealed a strong affinity between HQC and the Wnt1 molecule. RT-qPCR and Western Blot (WB) experiments confirmed the critical role of the Wnt1/β-catenin signaling pathway in the treatment of RA-dep model rats with HQC. In vitro, the HQC drug-containing serum (HQC-serum) activates the Wnt1/β-catenin signaling pathway in hippocampal cells and, in conjunction with Wnt1, ameliorates RA-dep. In summary, HQC exerts its anti-inflammatory and antidepressant effects in the treatment of RA-dep by binding to Wnt1 and regulating the Wnt1/β-catenin signaling pathway., Conclusions: HQC improved the inflammatory reaction and depression-like behavior of RA-dep model rats by activating Wnt1/β-catenin signal pathway. This study revealed a new pathogenesis of RA-dep and contributes to the clinical promotion of HQC in the treatment of RA-dep., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The immunomodulatory oligodeoxynucleotide (ODN) IMT504 might harbor antifibrotic properties within the liver., Methods: Fibrosis models were induced in mice through thioacetamide (TAA) administration and bile-duct ligation. Cre-loxP mice were utilized to identify GLAST + Wnt1 + bone marrow stromal progenitors (BMSPs) and to examine their contribution with cells in the liver. In vivo and in vitro assays; flow-cytometry, immunohistochemistry, and qPCR were conducted., Results: IMT504 demonstrated significant inhibition of liver fibrogenesis progression and reversal of established fibrosis. Early responses to IMT504 involved the suppression of profibrogenic and proinflammatory markers, coupled with an augmentation of hepatocyte proliferation. Additionally, this ODN stimulated the proliferation and mobilization of GLAST + Wnt1 + BMSPs, likely amplifying their contribution with endothelial- and hepatocytes-like cells. Moreover, IMT504 significantly modulated the expression levels of Wnt ligands and signaling pathway/target genes specifically within GLAST + Wnt1 + BMSPs, with minimal impact on other BMSPs. Intriguingly, both IMT504 and conditioned media from IMT504-pre-treated GLAST + Wnt1 + BMSPs shifted the phenotype of fibrotic macrophages, hepatic stellate cells, and hepatocytes, consistent with the potent antifibrotic effects observed., Conclusion: In summary, our findings identify IMT504 as a promising candidate molecule with potent antifibrotic properties, operating through both direct and indirect mechanisms, including the activation of GLAST + Wnt1 + BMSPs., (© 2024. The Author(s).)

Osteogenesis imperfecta (OI) is a group of severe genetic bone disorders characterized by congenital low bone mass, deformity, and frequent fractures. Type XV OI is a moderate to severe form of skeletal dysplasia caused by WNT1 variants. In this cohort study from southern China, we summarized the clinical phenotypes of patients with WNT1 variants and found that the proportion of type XV patients was around 10.3% (25 out of 243) with a diverse spectrum of phenotypes. Functional assays indicated that variants of WNT1 significantly impaired its secretion and effective activity, leading to moderate to severe clinical manifestations, porous bone structure, and enhanced osteoclastic activities. Analysis of proteomic data from human skeleton indicated that the expression of SOST (sclerostin) was dramatically reduced in type XV patients compared to patients with COL1A1 quantitative variants. Single-cell transcriptome data generated from human tibia samples of patients diagnosed with type XV OI and leg-length discrepancy, respectively, revealed aberrant differentiation trajectories of skeletal progenitors and impaired maturation of osteocytes with loss of WNT1, resulting in excessive CXCL12+ progenitors, fewer mature osteocytes, and the existence of abnormal cell populations with adipogenic characteristics. The integration of multi-omics data from human skeleton delineates how WNT1 regulates the differentiation and maturation of skeletal progenitors, which will provide a new direction for the treatment strategy of type XV OI and relative low bone mass diseases such as early onset osteoporosis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

Signaling pathways activated by secreted Wnt ligands play an essential role in tissue development and the progression of diseases, like cancer. Secretion of the lipid-modified Wnt proteins is tightly regulated by a repertoire of intracellular factors. For instance, a membrane protein, Evi, interacts with the Wnt ligand in the ER, and it is essential for its further trafficking and release in the extracellular space. After dissociating from the Wnt, the Wnt-unbound Evi is recycled back to the ER via Golgi. However, where in this trafficking path Wnt proteins dissociate from Evi remains unclear. Here, we have used the Drosophila wing epithelium to trace the route of the Evi-Wg (Wnt homolog) complex leading up to their separation. In these polarized cells, Wg is first trafficked to the apical surface; however, the secretion of Wg is believed to occurs post-internalization via recycling. Our results show that the Evi-Wg complex is internalized from the apical surface and transported to the retromer-positive endosomes. Furthermore, using antibodies that specifically label the Wnt-unbound Evi, we show that Evi and Wg separation occurs post-internalization in the acidic endosomes. These results refine our understanding of the polarized trafficking of Wg and highlight the importance of Wg endocytosis in its secondary secretion., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Using a transgenic zebrafish line harboring a heat-inducible dominant-interference pou5f3 gene (en-pou5f3), we reported that this PouV gene is involved in isthmus development at the midbrain-hindbrain boundary (MHB), which patterns the midbrain and cerebellum. Importantly, the functions of pou5f3 reportedly differ before and after the end of gastrulation. In the present study, we examined in detail the effects of en-pou5f3 induction on isthmus development during embryogenesis. When en-pou5f3 was induced around the end of gastrulation (bud stage), the isthmus was abrogated or deformed by the end of somitogenesis (24 hours post-fertilization). At this stage, the expression of MHB markers -- such as pax2a, fgf8a, wnt1, and gbx2 -- was absent in embryos lacking the isthmus structure, whereas it was present, although severely distorted, in embryos with a deformed isthmus. We further found that, after en-pou5f3 induction at late gastrulation, pax2a, fgf8a, and wnt1 were immediately and irreversibly downregulated, whereas the expression of en2a and gbx2 was reduced only weakly and slowly. Induction of en-pou5f3 at early somite stages also immediately downregulated MHB genes, particularly pax2a, but their expression was restored later. Overall, the data suggested that pou5f3 directly upregulates at least pax2a and possibly fgf8a and wnt1, which function in parallel in establishing the MHB, and that the role of pou5f3 dynamically changes around the end of gastrulation. We next examined the transcriptional regulation of pax2a using both in vitro and in vivo reporter analyses; the results showed that two upstream 1.0-kb regions with sequences conserved among vertebrates specifically drove transcription at the MHB. These reporter analyses confirmed that development of the isthmic organizer is regulated by PouV through direct regulation of pax2/pax2a in vertebrate embryos., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Previous studies have suggested that perioperative anesthesia could have direct impacts on cancer cell biology. The present study investigated the effects of ropivacaine administration on lung adenocarcinoma cells., Methods: Ropivacaine was administered to A549 cells at concentrations of 0.1, 1, and 6 mM for 2 h. Angiotensin-converting enzyme 2 (ACE2) small interfering RNA (siRNA) transfection was performed 6 h prior to ropivacaine administration. Cell proliferation and migration were assessed with cell counting kit 8 (CCK-8) and a wound healing assay at 0 and 24 h after anesthesia exposure. PCR arrays were performed, followed by PCR validation., Results: Ropivacaine administration inhibited A549 cell proliferation and migration in a concentration-dependent manner, with ACE2 upregulation and HIF1α (hypoxia-inducible factor 1α) downregulation. The anticancer effect of ropivacaine was canceled out via ACE2 siRNA transfection. PCR arrays showed specific gene change patterns in the ropivacaine and respective ACE2-knockdown groups. EGFR (epidermal growth factor receptor), BAX (Bcl-2-associated X protein) and BCL2 (B-cell/CLL lymphoma 2) were suppressed with ropivacaine administration; these effects were reversed via ACE2 siRNA induction., Conclusion: Ropivacaine administration inhibited A549 cell biology in conjunction with ACE2 upregulation via the inhibition of the Wnt1 (wingless/Integrated 1) pathway.

During development, unique combinations of transcription factors and signaling pathways carve the nascent eye-antennal disc of the fruit fly Drosophila melanogaster into several territories that will eventually develop into the compound eye, ocelli, head epidermis, bristles, antenna and maxillary palpus of the adult head. Juxtaposed patterns of Hedgehog (Hh) and Decapentaplegic (Dpp) initiate compound eye development, while reciprocal domains of Dpp and Wingless (Wg) induce formation of the antennal and maxillary palp fields. Hh and Wg signaling, but not Dpp, contribute to the patterning of the dorsal head vertex. Here, we show that combinatorial reductions of the Pax6 transcription factor Twin of Eyeless and either the Wg pathway or the Mirror (Mirr) transcription factor trigger a transformation of the ocelli into a compound eye and the neighboring head epidermis into an antenna. These changes in fate are accompanied by the ectopic expression of Dpp, which might be expected to trigger these changes in fate. However, the transformation of the field cannot be replicated by increasing Dpp levels alone despite the recreation of adjacent Hh-Dpp and Wg-Dpp domains. As such, the emergence of these ectopic organs occurs through a unique regulatory path., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Objective. Myocardial fibrosis is a devastating condition causing millions of deaths yearly. Several factors, such as aging, cause myocardial fibrosis. The Wnt/β-catenin pathway is one of the critical intracellular signaling for the development of cardiac fibrosis. Molecular and cellular mechanism of myocardial fibrosis induced by intensive exercise is not well-understood. The current study evaluates the effects of short- and long-term intensive exercise on the Wnt1 gene expression in a heart left ventricle in an animal model. Methods. Twenty-one male Wistar rats (mean weight 250±50 g) were divided into three groups (n=7): 1) control group (C); 2) short-term regular intensive exercise group (S-RIE, high-intensity exercise for one month six days weekly for 60 min with speed of 35 m/min), and 3) long-term regular intensive exercise group (L-RIE, high-intensity exercise for six months six days daily for 60 min with speed of 35 m/min). The heart left ventricle was isolated at the end of the experiment, and the relative gene expression of the Wnt1 gene was measured by the Real-Time PCR. Results. The L-RIE group showed a significant increase in the Wnt1 expression compared to the S-RIE and the control group. Although no difference was observed in the Wnt1 mRNA level in the S-RIE group compared to the control group, Wnt1 mRNA level increased in the L-RIE group compared to the S-RIE group. Conclusion. The exercise duration was of a great importance in the Wnt1 gene expression. Regular intensive exercise may be involved in the formation of the myocardial fibrosis by increasing the expression of the Wnt1 gene., (© 2024 Farzam Sheikhzadeh et al., published by Sciendo.)

Skeletal growth, modeling, and remodeling are regulated by various molecules, one of them being the recently identified osteoanabolic factor WNT1. We have previously reported that WNT1 transcriptionally activates the expression of Omd, encoding Osteomodulin (OMD), in a murine mesenchymal cell line, which potentially explained the skeletal fragility of mice with mutational WNT1 inactivation, since OMD has been shown to regulate type I collagen fibril formation in vitro. In this study we confirmed the strong induction of Omd expression in a genome-wide expression analysis of transfected cells, and we obtained further evidence for Omd being a direct target gene of WNT1. To assess the in vivo relevance of this regulation, we crossed Omd-deficient mice with a mouse line harboring an inducible, osteoblast-specific Wnt1 transgene. After induction of Wnt1 expression for 1 or 3 weeks, the osteoanabolic potency of WNT1 was not impaired despite the Omd deficiency. Since current knowledge regarding the in vivo physiological function of OMD is limited, we next focused on skeletal phenotyping of wild-type and Omd-deficient littermates, in the absence of a Wnt1 transgene. Here we did not observe an impact of Omd deficiency on trabecular bone parameters by histomorphometry and μCT either. Importantly, however, male and female Omd-deficient mice at the ages of 12 and 24 weeks displayed a slender bone phenotype with significantly smaller long bones in the transversal dimension, while the longitudinal bone growth remained unaffected. Although mechanical testing revealed no significant changes explained by impaired bone material properties, atomic force microscopy of the femoral bone surface of Omd-deficient mice revealed moderate changes at the nanostructural level, indicating altered regulation of collagen fibril formation and aggregation. Taken together, our data demonstrate that, although OMD is dispensable for the osteoanabolic effect of WNT1, its deficiency in mice specifically modulates transversal cortical bone morphology., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

Objectives: This study aimed to explore the heterogeneity and gene ontology of Wnt1-Cre-marked and Pax2-Cre-marked first branchial arch cranial neural crest cells (CNCs) in mice., Methods: The embryos of Wnt1-Cre;R26R mTmG and Pax2-Cre;R26R mTmG at embryonic day (E)8.0-E9.25 were collected for histological observation. We performed immunostaining to compare green fluorescent protein (GFP)-positive CNCs in Pax2-Cre;R26R Ai9 and Wnt1-Cre;R26R Ai9 mice at E15.5. Single-cell RNA sequencing (scRNA-seq) was used to analyze the first branchial arch GFP-positive CNCs from Wnt1-Cre;R26R mTmG and Pax2-cre;R26R mTmG mice at E10.5. Real time fluorescence quantitative polymerase chain reaction (q-PCR) was performed to validate the differential genes., Results: Wnt1-Cre-marked and Pax2-Cre-marked CNCs migrated from the neural plateto first and second branchial arches and to the first branchial arch, respectively, at E8.0. Although Wnt1-Cre-marked and Pax2-Cre - marked CNCs were found mostly in cranial-facial tissues, the former had higher expression in palate and tongue. The results of scRNA-seq showed that Pax2-Cre-marked CNCs specifically contributed to osteoblast differentiation and ossification, while Wnt1-Cre-marked CNCs participated in limb development, cell migration, and ossification. The q-PCR data also confirmed the results of gene ontology analysis., Conclusions: Pax2-Cre mice are perfect experimental animal models for research on first branchial arch CNCs and derivatives in osteoblast differentiation and ossification.

Despite the deep conservation of the DNA damage response (DDR) pathway, cells in different contexts vary widely in their susceptibility to DNA damage and their propensity to undergo apoptosis as a result of genomic lesions. One of the cell signaling pathways implicated in modulating the DDR is the highly conserved Wnt pathway, which is known to promote resistance to DNA damage caused by ionizing radiation in a variety of human cancers. However, the mechanisms linking Wnt signal transduction to the DDR remain unclear. Here, we use a genetically encoded system in Drosophila to reliably induce consistent levels of DNA damage in vivo, and demonstrate that canonical Wnt signaling in the wing imaginal disc buffers cells against apoptosis in the face of DNA double-strand breaks. We show that Wg, the primary Wnt ligand in Drosophila, activates epidermal growth factor receptor (EGFR) signaling via the ligand-processing protease Rhomboid, which, in turn, modulates the DDR in a Chk2-, p53-, and E2F1-dependent manner. These studies provide mechanistic insight into the modulation of the DDR by the Wnt and EGFR pathways in vivo in a highly proliferative tissue. Furthermore, they reveal how the growth and patterning functions of Wnt signaling are coupled with prosurvival, antiapoptotic activities, thereby facilitating developmental robustness in the face of genomic damage., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ewen-Campen, Perrimon. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Glypicans are closely associated with organ development and tumorigenesis in animals. Dally-like (Dlp), a membrane-bound glypican, plays pivotal roles in various biological processes in Drosophila . In this study, we observed that an excess of Dlp led to the malformation of legs, particularly affecting the distal part. Accordingly, the leg disc was shrunken and frequently exhibited aberrant morphology. In addition, elevated Dlp levels induced ectopic cell death with no apparent cell proliferation changes. Furthermore, Dlp overexpression in the posterior compartment significantly altered Wingless (Wg) distribution. We observed a marked expansion of Wg distribution within the posterior compartment, accompanied by a corresponding decrease in the anterior compartment. It appears that excess Dlp guides Wg to diffuse to cells with higher Dlp levels. In addition, the distal-less ( dll ) gene, which is crucial for leg patterning, was up-regulated significantly. Notably, dachshund ( dac ) and homothorax ( hth ) expression, also essential for leg patterning and development, only appeared to be negligibly affected. Based on these findings, we speculate that excess Dlp may contribute to malformations of the distal leg region of Drosophila , possibly through its influence on Wg distribution, dll expression and induced cell death. Our research advances the understanding of Dlp function in Drosophila leg development.

The Wnt/Wingless signaling pathway plays critical roles in metazoan development and energy metabolism, but its role in regulating lipid homeostasis remains not fully understood. Here, we report that the activation of canonical Wnt/Wg signaling promotes lipolysis while concurrently inhibiting lipogenesis and fatty acid β-oxidation in both larval and adult adipocytes, as well as cultured S2R+ cells, in Drosophila . Using RNA-sequencing and CUT&RUN (Cleavage Under Targets & Release Using Nuclease) assays, we identified a set of Wnt target genes responsible for intracellular lipid homeostasis. Notably, active Wnt signaling directly represses the transcription of these genes, resulting in decreased de novo lipogenesis and fatty acid β-oxidation, but increased lipolysis. These changes lead to elevated free fatty acids and reduced triglyceride (TG) accumulation in adipocytes with active Wnt signaling. Conversely, downregulation of Wnt signaling in the fat body promotes TG accumulation in both larval and adult adipocytes. The attenuation of Wnt signaling also increases the expression of specific lipid metabolism-related genes in larval adipocytes, wing discs, and adult intestines. Taken together, these findings suggest that Wnt signaling-induced transcriptional repression plays an important role in regulating lipid homeostasis by enhancing lipolysis while simultaneously suppressing lipogenesis and fatty acid β-oxidation., Competing Interests: Competing interests statement:The authors declare no competing interest.

LIM homeodomain transcription factor 1-alpha (LMX1a) is a neuronal lineage-specific transcription activator that plays an essential role during the development of midbrain dopaminergic (mDA) neurons. LMX1a induces the expression of multiple key genes, which ultimately determine the morphology, physiology, and functional identity of mDA neurons. This function of LMX1a is dependent on its homeobox domain. Here, we determined the structures of the LMX1a homeobox domain in complex with the promoter sequences of the Wnt family member 1 (WNT1) or paired like homeodomain 3 (Pitx3) gene, respectively. The complex structures revealed that the LMX1a homeobox domain employed its α3 helix and an N-terminal loop to achieve specific target recognition. The N-terminal loop (loop1) interacted with the minor groove of the double-stranded DNA (dsDNA), whereas the third α-helix (α3) was tightly packed into the major groove of the dsDNA. Structure-based mutations in the α3 helix of the homeobox domain significantly reduced the binding affinity of LMX1a to dsDNA. Moreover, we identified a nonsyndromic hearing loss (NSHL)-related mutation, R199, which yielded a more flexible loop and disturbed the recognition in the minor groove of dsDNA, consistent with the molecular dynamics (MD) simulations. Furthermore, overexpression of Lmx1a promoted the differentiation of SH-SY5Y cells and upregulated the transcription of WNT1 and PITX3 genes. Hence, our work provides a detailed elucidation of the specific recognition between the LMX1a homeobox domain and its specific dsDNA targets, which represents valuable information for future investigations of the functional pathways that are controlled by LMX1a during mDA neuron development., (© 2024 Federation of European Biochemical Societies.)

Heart failure (HF) prognosis depends on various regulatory factors; microRNA-128 (miR-128) is identified as a regulator of cardiac fibrosis, contributing to HF. MyoD family inhibitor (MDFI), which is reported to be related with Wnt/β-catenin pathway, is supposed to be regulated by miR-128. This study investigates the interaction between miR-128 and MDFI in cardiomyocyte development and elucidates its role in heart injury. Gene expression profiling assessed miR-128's effect on MDFI expression in HF using qPCR and Western blot analysis. Luciferase assays studied the direct interaction between miR-128 and MDFI. MTT, transwell, and immunohistochemistry evaluated the effects of miR-128 and MDFI on myocardial cells in mice HF. Genescan and luciferase assays validated the interaction between miR-128 and MDFI sequences. miR-128 mimics significantly reduced MDFI expression at mRNA and protein levels with decrease rate of 55%. Overexpression of miR-128 promoted apoptosis with the increase rate 65% and attenuated cardiomyocyte proliferation, while MDFI upregulation significantly enhanced proliferation. Elevated miR-128 levels upregulated Wnt1 and β-catenin expression, whereas increased MDFI levels inhibited these expressions. Histological analysis with haematoxylin and eosin staining revealed that miR-128 absorption reduced MDFI expression, hindering cell proliferation and cardiac repair, with echocardiography showing corresponding improvements in cardiac function. Our findings suggest miR-128 interacts with MDFI, playing a crucial role in HF management by modulating the Wnt1/β-catenin pathway. Suppression of miR-128 could promote cardiomyocyte proliferation, highlighting the potential value of the miR-128/MDFI interplay in HF treatment., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Objective: This study aimed to investigate the effect of the soluble Klotho (sKlotho)/Wnt/β-catenin signaling pathway on vascular calcification in rat models of chronic kidney disease (CKD) and the intervention effect of Shenyuan granules., Methods: Rats with 5/6 nephrectomy and high phosphorus feeding were used to establish the vascular calcification model. The rats were given gradient doses of Shenyuan granules aqueous solution and calcitriol solution by gavage for 8 weeks, which were divided into experimental group and positive control group., Results: The 5/6 nephrectomy combined with high phosphorus feeding induced thoracic aortic calcification in rats. Shenyuan granules intervention increased the serum sKlotho level, inhibited the mRNA and protein expression of Wnt1, β-catenin, and Runx2 in the thoracic aorta, and alleviated thoracic aortic media calcification in rats., Conclusion: Shenyuan granules may partially regulate the Wnt/β-catenin signaling pathway via serum sKl to interfere with the expression of Runx2, thereby improving vascular calcification in CKD.

Wnt/Wingless (Wg) signaling is critical in development and disease, including cancer. Canonical Wnt signaling is mediated by β-catenin/Armadillo (Arm in Drosophila) transducing signals to the nucleus, with IFT-A/Kinesin 2 complexes promoting nuclear translocation of β-catenin/Arm. Here, we demonstrate that a conserved small N-terminal Arm 34 - 87 /β-catenin peptide binds to IFT140, acting as a dominant interference tool to attenuate Wg/Wnt signaling in vivo. Arm 34 - 87 expression antagonizes endogenous Wnt/Wg signaling, resulting in the reduction of its target expression. Arm 34 - 87 inhibits Wg/Wnt signaling by interfering with nuclear translocation of endogenous Arm/β-catenin, and this can be modulated by levels of wild-type β-catenin or IFT140, with the Arm 34 - 87 effect being enhanced or suppressed. Importantly, this mechanism is conserved in mammals with the equivalent β-catenin 24 - 79 peptide blocking nuclear translocation and pathway activation, including in cancer cells. Our work indicates that Wnt signaling can be regulated by a defined N-terminal β-catenin peptide and thus might serve as an entry point for therapeutic applications to attenuate Wnt/β-catenin signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Wnt/Wg pathway controls myriads of biological phenomena throughout the development and adult life of all organisms across the phyla. Thus, an aberrant Wnt signaling is associated with a wide range of pathologies in humans. Tight regulation of Wnt/Wg signaling is required to maintain proper cellular homeostasis. Here, we report a novel role of E3 ubiquitin ligase Deltex in Wg signaling regulation. Drosophila dx genetically interacts with wg a nd its pathway components. Furthermore, Dx LOF results in a reduced spreading of Wg while its over-expression expands the diffusion gradient of the morphogen. We attribute this change in Wg gradient to the endocytosis of Wg through Dx which directly affects the short- and long-range Wg targets. We also demonstrate the role of Dx in regulating Wg effector Armadillo where Dx down-regulates Arm through proteasomal degradation. We also showed the conservation of Dx function in the mammalian system where DTX1 is shown to bind with β-catenin and facilitates its proteolytic degradation, spotlighting a novel step that potentially modulates Wnt/Wg signaling cascade., Competing Interests: VS, NS, BS, MM, AM No competing interests declared, (© 2023, Sharma et al.)

Wnt/β-catenin signaling dysregulation is associated with the pathogenesis of many human diseases, including hypertension and heart disease. The aim of this study was to immunohistochemically evaluate and compare the expression of the Fzd8, WNT1, GSK-3β, and β-catenin genes in the hearts of rats with spontaneous hypertension (SHRs) and deoxycorticosterone acetate (DOCA)-salt-induced hypertension. The myocardial expression of Fzd8, WNT1, GSK-3β, and β-catenin was detected by immunohistochemistry, and the gene expression was assessed with a real-time PCR method. In SHRs, the immunoreactivity of Fzd8, WNT1, GSK-3β, and β-catenin was attenuated in comparison to that in normotensive animals. In DOCA-salt-induced hypertension, the immunoreactivity of Fzd8, WNT1, GSK-3β, and β-catenin was enhanced. In SHRs, decreases in the expression of the genes encoding Fzd8, WNT1, GSK-3β, and β-catenin were observed compared to the control group. Increased expression of the genes encoding Fzd8, WNT1, GSK-3β, and β-catenin was demonstrated in the hearts of rats with DOCA-salt-induced hypertension. Wnt signaling may play an essential role in the pathogenesis of arterial hypertension and the accompanying heart damage. The obtained results may constitute the basis for further research aimed at better understanding the role of the Wnt/β-catenin pathway in the functioning of the heart.

Aim: Assessment of WNT1, WNT3a, and LRP6 concentrations in patients with ischemic heart disease (IHD) and obstructive and non-obstructive coronary artery (CA) disease., Material and Methods: This cross-sectional observational study included 50 IHD patients (verified by coronary angiography, CAG), of which 25 (50%) were men, mean age 64.9±8.1 years; 20 patients had non-obstructive CA disease (stenosis <50%), and 30 patients had hemodynamically significant stenosis. Concentrations of WNT1, WNT3a and LRP6 were measured in all patients., Results: The concentrations of WNT1 and WNT3a proteins were significantly higher in patients with IHD and obstructive CA disease (p < 0.001), while the concentration of LRP6 was higher in the group with non-obstructive CA disease (p = 0.016). Data analysis of the group with obstructive CA disease showed a moderate correlation between WNT1 and LRP6 (ρ=0.374; p=0.042). Correlation analysis of all groups of patients with CA disease revealed a moderate association between the concentrations of WNT1 and uric acid (ρ=0.416; p=0.007). Regression analysis showed that risk factors for the development of IHD, such as increased body mass index, age, smoking, dyslipidemia, and hypertension, did not significantly influence the type of CA disease in IHD patients. According to ROC analysis, the obstructive form of IHD was predicted by a WNT3a concentration higher than 0.155 ng/ml and a LRP6 concentration lower than 12.94 ng/ml., Conclusion: IHD patients with non-obstructive CA disease had the greatest increase in LRP6, while patients with obstructive CA disease had significantly higher concentrations of the canonical WNT cascade proteins, WNT1 and WNT3a. According to the ROC analysis, a WNT3a concentration >0.155 ng/ml can serve as a predictor for the presence of hemodynamically significant CA stenosis in IHD patients (sensitivity 96.7%; specificity 70%), whereas a LRP6 concentration >12.94 ng/ml can predict the development of non-obstructive CA disease (sensitivity 76.7%; specificity 65%).

Despite the conservation of genetic machinery involved in eye development, there is a strong diversity in the placement of eyes on the head of animals. Morphogen gradients of signaling molecules are vital to patterning cues. During Drosophila eye development, Wingless (Wg), a ligand of Wnt/Wg signaling, is expressed anterolaterally to form a morphogen gradient to determine the eye- versus head-specific cell fate. The underlying mechanisms that regulate this process are yet to be fully understood. We characterized defective proventriculus (dve) ( Drosophila ortholog of human SATB1), a K50 homeodomain transcription factor, as a dorsal eye gene, which regulates Wg signaling to determine eye versus head fate. Across Drosophila species, Dve is expressed in the dorsal head vertex region where it regulates wg transcription. Second, Dve suppresses eye fate by down-regulating retinal determination genes. Third, the dve -expressing dorsal head vertex region is important for Wg-mediated inhibition of retinal cell fate, as eliminating the Dve-expressing cells or preventing Wg transport from these dve -expressing cells leads to a dramatic expansion of the eye field. Together, these findings suggest that Dve regulates Wg expression in the dorsal head vertex, which is critical for determining eye versus head fate. Gain-of-function of SATB1 exhibits an eye fate suppression phenotype similar to Dve. Our data demonstrate a conserved role for Dve/SATB1 in the positioning of eyes on the head and the interocular distance by regulating Wg. This study provides evidence that dysregulation of the Wg morphogen gradient results in developmental defects such as hypertelorism in humans where disproportionate interocular distance and facial anomalies are reported., Competing Interests: Competing interests statement:The authors declare no competing interest.

The control of Wnt receptor abundance is critical for animal development and to prevent tumorigenesis, but the mechanisms that mediate receptor stabilization remain uncertain. We demonstrate that stabilization of the essential Wingless/Wnt receptor Arrow/LRP6 by the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex controls signaling strength in Drosophila. By reducing Arrow ubiquitylation and turnover, the Usp46 complex increases cell surface levels of Arrow and enhances the sensitivity of target cells to stimulation by the Wingless morphogen, thereby increasing the amplitude and spatial range of signaling responses. Usp46 inactivation in Wingless-responding cells destabilizes Arrow, reduces cytoplasmic accumulation of the transcriptional coactivator Armadillo/β-catenin, and attenuates or abolishes Wingless target gene activation, which prevents the concentration-dependent regulation of signaling strength. Consequently, Wingless-dependent developmental patterning and tissue homeostasis are disrupted. These results reveal an evolutionarily conserved mechanism that mediates Wnt/Wingless receptor stabilization and underlies the precise activation of signaling throughout the spatial range of the morphogen gradient., (© 2023. Springer Nature Limited.)

Cell recruitment is a process by which a differentiated cell induces neighboring cells to adopt its same cell fate. In Drosophila, cells expressing the protein encoded by the wing selector gene, vestigial (vg), drive a feed-forward recruitment signal that expands the Vg pattern as a wave front. However, previous studies on Vg pattern formation do not reveal these dynamics. Here, we use live imaging to show that multiple cells at the periphery of the wing disc simultaneously activate a fluorescent reporter of the recruitment signal, suggesting that cells may be recruited without the need for their contact neighbors be recruited in advance. In support of this observation, when Vg expression is inhibited either at the dorsal-ventral boundary or away from it, the activation of the recruitment signal still occurs at a distance, suggesting that Vg expression is not absolutely required to send or propagate the recruitment signal. However, the strength and extent of the recruitment signal is clearly compromised. We conclude that a feed-forward, contact-dependent cell recruitment process is not essential for Vg patterning, but it is necessary for robustness. Overall, our findings reveal a previously unidentified role of cell recruitment as a robustness-conferring cell differentiation mechanism., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Endometrial cancer remains a common cancer affecting the female reproductive system. There is still a need for more efficient ways of determining the degree of malignancy and optimizing treatment. WNT and mTOR are components of signaling pathways within tumor cells, and dysfunction of either protein is associated with the pathogenesis of neoplasms. Therefore, the aim of our study was to assess the impact of subcellular WNT-1 and mTOR levels on the clinical course of endometrial cancer. WNT-1 and mTOR levels in the plasma membrane, nucleus, and cytoplasm were evaluated using immunohistochemical staining in a group of 64 patients with endometrial cancer of grades 1-3 and FIGO stages I-IV. We discovered that the levels of WNT-1 and mTOR expression in the cellular compartments were associated with tumor grade and staging. Membranous WNT-1 was negatively associated, whereas cytoplasmic WNT-1 and nuclear mTOR were positively associated with higher grading of endometrial cancer. Furthermore, nuclear mTOR was positively associated with FIGO stages IB-IV. To conclude, we found that the assessment of WNT-1 in the cell membrane may be useful for exclusion of grade 3 neoplasms, whereas cytoplasmic WNT-1 and nuclear mTOR may be used as indicators for confirmation of grade 3 neoplasms.

Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Wnt1 is the first mammalian Wnt gene, which is discovered as proto-oncogene and in human the gene is located on the chromosome 12q13. Mutations in Wnt1 are reported to be associated with various cancers and other human diseases. The structural and functional consequences of most of the non-synonymous SNPs (nsSNPs), present in the human Wnt1 gene, are not known. In the present work, extensive bioinformatics analyses are used to screen 292 nsSNPs of Wnt1 for predicting pathogenic and harmless polymorphisms. We have identified 10 highly deleterious nsSNPs among which 7 are located within the highly conserved areas. These 10 nsSNPs are also predicted to affect the post-translational modifications of Wnt1. Further, structure based stability analyses of these 10 highly deleterious nsSNPs revealed 8 variants as highly destabilizing. These 8 highly destabilizing variants were shown to have high BC score and high RMSIP score from normal mode analyses. Based on the deformation energies, obtained from the normal mode analyses, variants like G169A, G169S, G331R and G331S were found to be unstable. Molecular Dynamics (MD) simulations revealed structural stability and fluctuation of WT Wnt1 and its prioritized variants. RMSD remained fluctuating mostly between 4 and 5 Å and occasionally between 3.5 and 5.5 Å ranges. RMSF in the CTD region (residues 330-360) of the binding pocket were lower compared to that of WT. Studying the impacts of nsSNPs on the binding interface of Wnt1 and seven Frizzled receptors have predicted substitutions which can stabilize or destabilize the binding interface. We have found that Wnt1 and FZD8-CRD is the best docked complex in our study. MD simulation based analyses of wild type Wnt1-FZD8-CRD complex and the 8 prioritized variants revealed that RMSF was higher in the unstructured regions and RMSD remained fluctuating in the region of 5 Å ± 1 Å. We have also observed differential Wnt1 gene expression pattern in normal, tumor and metastatic conditions across different tissues. Wnt1 gene expression was significantly higher in metastatic tissues of lungs, colon and skin; and was significantly lower in metastatic tissues of breast, esophagus and kidney. We have also found that Wnt1 deregulation is associated with survival outcome in patients with gastric and breast cancer. Furthermore, these computationally screened highly deleterious nsSNPs of Wnt1 can be analyzed in population based genetic studies and may help understand the Wnt1 associated diseases., (© 2022. The Author(s).)

Objective To explore the effect and mechanism of paeoniflorin (PAE) on apoptosis of fibroblast synovial cells derived from rheumatoid arthritis tissues. Methods Rheumatoid arthritis fibroblast-like synovial cells (RA-FLSs) were cultured under different concentrations of PAE (20, 40, 80 μmol/L). Survival rate of cells was determined at different incubation time-points (24, 48, 72 hours) by MTT assay. Flow cytometry was performed to determine apoptosis rate of cells. Cells were transfected with 3 small interfering RNAs(siRNAs) fragments of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1(lncRNA MALAT1) for 48 hours. Real-time quantitative PCR was performed to determine if MALAT1 knockdown was successful. Cells were assigned into control group, PAE group, si-NC group, si-MALAT1 group and PAE combined with si-MALAT1 group. Cell apoptosis rate was determined by flow cytometry after knockdown of MALAT1 expression. mRNA expression levels of Wnt1, β-catenin, caspase-3, caspase-9, B-cell lymphoma 2(Bcl2) and Bcl2-associated X protein (BAX) were determined using real-time quantitative PCR. Western blot analysis was performed to determine protein expression levels of Wnt1, β-catenin, caspase-3, caspase-9, Bcl2 and BAX in each group. Results The findings showed that incubation of RA-FLSs with PAE at the tested concentration decreased their viability in a time-dependent manner. Expression level of lncRNA MALAT1 was lower in RA-FLSs group compared with the level in NC-FLSs group. Analysis showed that apoptosis rate was higher in cells treated with PAE. si-MALAT1 group had the lowest apoptosis rate, whereas the PAE combined with si-MALAT1 group showed a significantly higher apoptosis rate compared with si-MALAT1 group. mRNA levels of Bcl2, Wnt1 and β-catenin were lower in PAE group compared with the level in control group, whereas mRNA expression levels of caspase-3, caspase-9 and BAX were higher in PAE group compared with the levels in control group. Expression level of Bcl2 was significantly higher in si-MALAT1 group compared with the level in the control group. Expression level of BAX, caspase-3 and caspase-9 were higher in PAE combined with si-MALAT1 group compared with the level in the control group. Conclusion PAE promotes apoptosis and inhibits the Wnt1/β-catenin pathway in RA-FLSs by upregulating expression of lncRNA MALAT1.

Intrauterine infection induces inflammation-mediated microglial activation and brain injury. This study aimed to explore the regulatory mechanism of Wnt family member 1 (Wnt1) in intrauterine infection-mediated microglial polarization. The cell counting kit-8 (CCK-8) assay was used to determine the viability of microglia, and cytokine expression levels were determined using enzyme linked immunosorbent assay (ELISA) kits and real-time quantitative PCR (RT-qPCR). The number of CD206 + and CD16/32 + cells was determined by flow cytometry. Wnt1 expression was analyzed using western blotting and immunofluorescence. Moreover, an in vivo assay was performed to verify the role of WNT1 in inflammation-sensitized brain injury in newborn mice. Lipopolysaccharide (LPS) exposure resulted in a decrease in microglial cell viability while increasing the expression levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β), simultaneously promoting M1-type microglial conversion. However, these effects were rescued by overexpression of Wnt1, which was expressed less in microglia exposed to LPS in vitro and in vivo . Here, we found that Wnt1 activated the LKB1-AMPK pathway, and the inhibition of LKB1 attenuated the rescue effects of Wnt1. In addition, LPS exposure reduced the autophagy of microglia, and Wnt1 overexpression enhanced the autophagy, but this effect was reversed by treatment with an LKB1 inhibitor. Wnt1 activated LKB1 to suppress inflammation-mediated activation of microglia, promote M2-type microglia conversion via the AMPK pathway, and alleviate inflammation-sensitized neonatal brain injuries. This provides a potential avenue for the treatment of neonatal brain injuries.

Emerging evidences suggest abundant expression of Carboxy terminus of Hsc70 Interacting Protein or CHIP (alias STIP1 Homology and U-box Containing Protein 1 or STUB1) in colorectal carcinoma, but the mechanistic detail of this augmented expression pattern is unclear. The signature driver of canonical Wnt pathway, β-catenin, and its co-activator RNA helicase p68, are also overexpressed in colorectal carcinoma. In this study, we describe a novel mechanism of Wnt/β-catenin and p68 mediated transcriptional activation of CHIP gene leading to enhanced proliferation of colorectal carcinoma cells. Bioinformatic analyses reconfirmed an elevated CHIP expression level in colorectal carcinoma datasets. Wnt3A treatment and pharmacological activation of canonical Wnt signaling pathway resulted in increased nuclear translocation of β-catenin, augmenting CHIP expression. Likewise, immunoblotting and Real time PCR following overexpression and knockdown of β-catenin and p68 demonstrated upregulated and downregulated CHIP expression, respectively, at both mRNA and protein levels. p68 along with β-catenin were found to occupy Transcription Factor 4 (TCF4) binding sites on endogenous CHIP promoter and regulate its transcription. After cloning CHIP promoter, the increased and decreased promoter activities of CHIP induced by overexpression and knockdown of either β-catenin or p68 further confirmed transcriptional regulation of CHIP gene by Wnt/β-catenin signaling cascade. Finally, enhanced cellular propagation and migration of colorectal carcinoma cells induced by 'Wnt/β-catenin-p68-CHIP' axis established the significance of this pathway in oncogenesis. To the best of our knowledge, this is the first report elucidating the mechanistic details of transcriptional regulation of CHIP (STUB1) gene expression., (Copyright © 2021 Elsevier B.V. All rights reserved.)

C‑terminal‑binding protein 2 (CtBP2), a transcriptional co‑repressor, plays a main role in tumorigenesis and in the development of multiple tumors. Transforming growth interacting factor (TGIF) is involved in a number of cellular signal transduction pathways and is related to tumor occurrence and development. In the present study, the proteins interacting with CtBP2 were identified and the mechanisms underlying the biological activity of CtBP2 in esophageal squamous cell carcinoma (ESCC) were investigated. The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to search for known proteins interacting with CtBP2, and co‑immunoprecipitation (Co‑IP) assay was performed to validate the interactions. Reverse transcription‑quantitative PCR (RT‑qPCR), immunohistochemistry (IHC) and western blot analysis were performed to examine the expression levels of CtBP2 and TGIF in ESCC. The correlation between CtBP2 and TGIF was analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) by Pearson's correlation analysis, and the co‑localization of CtBP2 with TGIF in the ECA109 cells was identified using immunofluorescence staining. XAV939 treatment, CCK‑8, 5‑ethynyl‑2'‑deoxyuridine (EdU) staining, wound healing and Transwell assays were performed to investigate the signaling pathways involved in the biological activity of CtBP2 in ECA109 cells. According to the results obtained from STRING and Co‑IP analysis, an interaction between CtBP2 and TGIF was indicated, and these proteins were co‑localized in the nucleus. CtBP2 and TGIF mRNA and protein expression levels were robustly and simultaneously increased in both ESCC tissues and cell lines. There was a direct correlation between CtBP2 and TGIF expression levels in ESCC tissues, and both were significantly associated with metastasis and survival. The TGIF and CtBP2 expression levels were significantly increased or decreased simultaneously, in ECA109 cells transfected with LV‑CtBP2 or sh‑CtBP2, and vice versa. According to the results of CCK‑8 assay, EdU staining and Transwell assay, CtBP2 promoted the proliferation, migration and invasion of ECA109 cells through the Wnt/β‑catenin pathway. On the whole, the present study demonstrates that CtBP2 interacts with TGIF and promotes the malignant progression of ESCC through the Wnt/β‑catenin pathway.

In Drosophila melanogaster , loss of regenerative capacity in wing imaginal discs coincides with an increase in systemic levels of the steroid hormone ecdysone, a key coordinator of their developmental progression. Regenerating discs release the relaxin hormone Dilp8 ( Drosophila insulin-like peptide 8) to limit ecdysone synthesis and extend the regenerative period. Here, we describe how regenerating tissues produce a biphasic response to ecdysone levels: lower concentrations of ecdysone promote local and systemic regenerative signaling, whereas higher concentrations suppress regeneration through the expression of broad splice isoforms. Ecdysone also promotes the expression of wingless during both regeneration and normal development through a distinct regulatory pathway. This dual role for ecdysone explains how regeneration can still be completed successfully in dilp8 - mutant larvae: higher ecdysone levels increase the regenerative activity of tissues, allowing regeneration to reach completion in a shorter time. From these observations, we propose that ecdysone hormone signaling functions to coordinate regeneration with developmental progression., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Aims: Angiotensin (Ang) II signalling has been suggested to promote cardiac fibrosis in inflammatory heart diseases; however, the underlying mechanisms remain obscure. Using Agtr1a-/- mice with genetic deletion of angiotensin receptor type 1 (ATR1) and the experimental autoimmune myocarditis (EAM) model, we aimed to elucidate the role of Ang II-ATR1 pathway in development of heart-specific autoimmunity and post-inflammatory fibrosis., Methods and Results: EAM was induced in wild-type (WT) and Agtr1a-/- mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund's adjuvant. Agtr1a-/- mice developed myocarditis to a similar extent as WT controls at day 21 but showed reduced fibrosis and better systolic function at day 40. Crisscross bone marrow chimaera experiments proved that ATR1 signalling in the bone marrow compartment was critical for cardiac fibrosis. Heart infiltrating, bone-marrow-derived cells produced Ang II, but lack of ATR1 in these cells reduced transforming growth factor beta (TGF-β)-mediated fibrotic responses. At the molecular level, Agtr1a-/- heart-inflammatory cells showed impaired TGF-β-mediated phosphorylation of Smad2 and TAK1. In WT cells, TGF-β induced formation of RhoA-GTP and RhoA-A-kinase anchoring protein-Lbc (AKAP-Lbc) complex. In Agtr1a-/- cells, stabilization of RhoA-GTP and interaction of RhoA with AKAP-Lbc were largely impaired. Furthermore, in contrast to WT cells, Agtr1a-/- cells stimulated with TGF-β failed to activate canonical Wnt pathway indicated by suppressed activity of glycogen synthase kinase-3 (GSK-3)β and nuclear β-catenin translocation and showed reduced expression of Wnts. In line with these in vitro findings, β-catenin was detected in inflammatory regions of hearts of WT, but not Agtr1a-/- mice and expression of canonical Wnt1 and Wnt10b were lower in Agtr1a-/- hearts., Conclusion: Ang II-ATR1 signalling is critical for development of post-inflammatory fibrotic remodelling and dilated cardiomyopathy. Our data underpin the importance of Ang II-ATR1 in effective TGF-β downstream signalling response including activation of profibrotic Wnt/β-catenin pathway., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Paranasal sinus tumors are a rare type of cancer. Most of these tumors are of epithelial origin and 80% of them are maxillary sinus squamous cell carcinoma. The WNT signaling pathway is an essential embryonic regulatory pathway known to play an important role in many cancers, including head and neck cancers. However, the effect of this pathway in maxillary sinus tumors has not been studied before. The aim of the study was to determine the changes in the regulatory genes of the WNT signaling pathway in maxillary sinus tumors. For this purpose, total RNA was isolated from the pathological preparations of 85 patients who had previously been operated on for squamous cell maxillary sinus tumor, and gene expression changes were evaluated by real-time RT-qPCR. The interactions among proteins encoded by genes, whose expression levels were found to be decreased and increased, were determined by protein-protein interaction (PPI) network analysis using string database, and signaling pathways that they are involved in were examined by Reactome database. A significant decrease in the expression of 28 genes compared to the control (fold change < 2.00 and p-value < 0.05) and a significant increase in the expression of 23 genes (fold change < 2.00 and p-value < 0.05) were detected. According to in silico analysis results, Signal Transduction (REACTOME:R-HSA-162582) and Signaling by WNT (REACTOME:R-HSA-195721) pathways were determined as most regulated pathways and FZD4-LRP5 and BCL9-CTNNB1 were determined as the strongest interactions. The current study contributes to illuminating the genetic regulation of maxillary sinus carcinoma in which genetic knowledge is limited. Our findings take attention to the dysregulations of the WNT signaling pathway that may support maxillary sinus carcinogenesis. The results will pave the way for further studies that investigate the therapy target potential of the WNT signaling pathway in this rare cancer., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

De novo truncations in Interferon Regulatory Factor 2 Binding Protein Like ( IRF2BPL ) lead to severe childhood-onset neurodegenerative disorders. To determine how loss of IRF2BPL causes neural dysfunction, we examined its function in Drosophila and zebrafish. Overexpression of either IRF2BPL or Pits , the Drosophila ortholog, represses Wnt transcription in flies. In contrast, neuronal depletion of Pits leads to increased wingless ( wg ) levels in the brain and is associated with axonal loss, whereas inhibition of Wg signaling is neuroprotective. Moreover, increased neuronal expression of wg in flies is sufficient to cause age-dependent axonal loss, similar to reduction of Pits. Loss of irf2bpl in zebrafish also causes neurological defects with an associated increase in wnt1 transcription and downstream signaling. WNT1 is also increased in patient-derived astrocytes, and pharmacological inhibition of Wnt suppresses the neurological phenotypes. Last, IRF2BPL and the Wnt antagonist, CKIα, physically and genetically interact, showing that IRF2BPL and CkIα antagonize Wnt transcription and signaling.

Objective: To explore the role of WNT family member 1 (WNT1) in the development of dysplasia of the hip (DDH) and the molecular mechanism involved in this process. Methods : Si-WNT1, pcDNA3.1-WNT1 or corresponding negative controls were transfected into human osteoblast hFOB1.19 and human chondrocyte C28/I2, respectively. The proliferation of cells was measured by EdU assay. The relative expressions of human noggin gene (NOG), growth differentiating factor 5 (GDF5), WNT1, and WNT1-inducible-signaling pathway protein 2 (WISP2) were determined by immunofluorescence analysis. The protein expressions of RNA-binding protein of multiple splice forms 2 (RBPMS2), NOG, bone morphogenetic protein 2 (BMP2), BMP4, WNT1 and WISP2 were determined by western blot. Animal experiment was also performed and the morphological development of hip joint was observed. Results : Overexpression of WNT1 promoted osteoblast proliferation and inhibited chondrocyte proliferation, while knockdown of WNT1 inhibited osteoblast proliferation. In chondrocytes, knockdown of WNT1 upregulated NOG expression, while overexpression of WNT1 downregulated its expression. In osteoblasts and chondrocytes, overexpression of WNT1 increased BMP2, BMP4, WNT1, and WISP2 expression. RBPMS2 and NOG were slightly expressed in each group. Conclusion : Overexpression of WNT1 promoted osteoblast proliferation, inhibited chondrocyte proliferation, and increased the expressions of BMP2, BMP4, WNT1, and WISP2. Therefore, WNT1 may be a new therapeutic target for DDH.

Objective: To investigate the regulatory effect of long non-coding RNA (lncRNA) SUMO1P3 on invasion, migration and cell cycle of gastric cancer (GC) cells through Wnt/β-catenin signaling pathway., Methods: Tumor tissues and adjacent normal tissues from the GC patients were collected, and human normal gastric epithelial cells GES1 and GC cells SGC-7901, MKN45, HGC-27 and AGS were selected for study. The expression of SUMO1P3 in GC tissues and cells were detected by RT-qPCR. The effects of SUMO1P3 on the proliferation, invasion and migration of SGC-7901 and MKN45 cells were detected by CCK-8, transwell and wound healing assay respectively, and the effects of SUMO1P3 on apoptosis and cycle progression of SGC-7901 and MKN45 cells were detected by flow cytometry. The expressions of Wnt/β-catenin pathway-related and cell cycle-related proteins were detected by Western blot., Results: The expression of SUMO1P3 was significantly upregulated in GC tissues and cell lines. Downregulation of SUMO1P3 significantly inhibited the SGC-7901 and MKN45 cell proliferation, invasion, migration, and cycle progression and promoted the cell apoptosis, while overexpression of SUMO1P3 showed the opposite effect. Further study showed that downregulation of SUMO1P3 significantly reduced the expressions of Wnt1, β-catenin, c-myc, and Cyclin D1 in SGC-7901 and MKN45 cells., Conclusion: SUMO1P3 may promote invasion, migration, and cycle progression of SGC-7901 and MKN45 cells by enhancing the Wnt/β-catenin pathway.

Wnt signalling is a core pathway involved in a wide range of developmental processes throughout the metazoa. In vitro studies have suggested that the small GTP binding protein Arf6 regulates upstream steps of Wnt transduction, by promoting the phosphorylation of the Wnt co-receptor, LRP6, and the release of β-catenin from the adherens junctions. To assess the relevance of these previous findings in vivo, we analysed the consequence of the absence of Arf6 activity on Drosophila wing patterning, a developmental model of Wnt/Wingless signalling. We observed a dominant loss of wing margin bristles and Senseless expression in Arf6 mutant flies, phenotypes characteristic of a defect in high level Wingless signalling. In contrast to previous findings, we show that Arf6 is required downstream of Armadillo/β-catenin stabilisation in Wingless signal transduction. Our data suggest that Arf6 modulates the activity of a downstream nuclear regulator of Pangolin activity in order to control the induction of high level Wingless signalling. Our findings represent a novel regulatory role for Arf6 in Wingless signalling., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Epithelial-mesenchymal transition (EMT) refers to the acquisition of mesenchymal properties in cells participating in tumor progression. One hallmark of EMT is the increased level of active β-catenin, which can trigger the transcription of Wnt-specific genes responsible for the control of cell fate. We investigated how Monocyte Chemotactic Protein-1-Induced Protein-1 (MCPIP1), a negative regulator of inflammatory processes, affects EMT in a clear cell renal cell carcinoma (ccRCC) cell line, patient tumor tissues and a xenotransplant model. We showed that MCPIP1 degrades miRNAs via its RNase activity and thus protects the mRNA transcripts of negative regulators of the Wnt/β-catenin pathway from degradation, which in turn prevents EMT. Mechanistically, the loss of MCPIP1 RNase activity led to the upregulation of miRNA-519a-3p, miRNA-519b-3p, and miRNA-520c-3p, which inhibited the expression of Wnt pathway inhibitors (SFRP4, KREMEN1, CXXC4, CSNK1A1 and ZNFR3). Thus, the level of active nuclear β-catenin was increased, leading to increased levels of EMT inducers (SNAI1, SNAI2, ZEB1 and TWIST) and, consequently, decreased expression of E-cadherin, increased expression of mesenchymal markers, and acquisition of the mesenchymal phenotype. This study revealed that MCPIP1 may act as a tumor suppressor that prevents EMT by stabilizing Wnt inhibitors and decreasing the levels of active β-catenin and EMT inducers., (© 2021. The Author(s).)

Background: Growing studies have demonstrated that long noncoding RNAs (lncRNAs) play important roles in tumor progression. In this study, we aimed to explore the potential roles of lncRNA LINC00958 (LINC00958) and its biological functions in epithelial ovarian cancer (EOC)., Methods: The expression of LINC00958 in 11 cases of EOC and adjacent nontumor specimens and five cell lines was detected by qRT-PCR. CCK-8, colony formation, and flow cytometry assays were conducted to study the cell viabilities of EOC cells. Wound scratch and transwell analyses were carried out for the examination of cell invasion and migration of EOC cells. The targeting associations between LINC00958 and STAT1 were demonstrated by ChIP analyses combined with luciferase reporter assays. The related proteins of Wnt/ β -catenin signaling were determined using RT-PCR., Results: Higher levels of LINC00958 were observed in EOC tissues and cell lines. Our data also revealed that high LINC00958 expression was partly induced by STAT1. Functionally, knockdown of LINC00958 suppressed the proliferation, migration, and invasion of EOC cells. Mechanistic investigation showed that the inhibitory effect of LINC00958 knockdown on EOC cells was mediated by the Wnt/ β -catenin signaling., Conclusion: Our findings suggested that STAT1-induced overexpression of LINC00958 promoted EOC progression by modulating Wnt/ β -catenin signaling., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2021 Min Xie et al.)

Glioblastoma (GBM) is an extremely aggressive brain tumor awaiting novel, efficient, and minimally toxic treatment. Curcuminoids (CCM), polyphenols from Curcuma longa , and sodium butyrate (NaBu), a histone deacetylase inhibitor naturally occurring in the human body, await elucidation as potential anti-GBM agents. Thus, the aim of this study was to analyze CCM and NaBu both separately and as a combination treatment using three GBM cell lines. MTT was used for cytotoxicity evaluation, and the combination index was calculated for synergism prediction. Cell cycle, apoptosis, and reactive oxygen species (ROS) generation were analyzed using flow cytometry. DNA methylation was verified by MS-HRM and mRNA expression by qPCR. The permeability through the blood-brain barrier (BBB) and through the nasal cavity was evaluated using PAMPA model. The results of this study indicate that CCM and NaBu synergistically reduce the viability of GBM cells inducing apoptosis and cell cycle arrest. These effects are mediated via ROS generation and changes in gene expression, including upregulation of Wnt/β-catenin pathway antagonists, SFRP1 , and RUNX3 , and downregulation of UHRF1 , the key epigenetic regulator. Moreover, NaBu ameliorated CCM permeability through the BBB and the nasal cavity. We conclude that CCM and NaBu are promising agents with anti-GBM properties.

The contributions of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) to breast cancer are critical areas of investigation. In this study, we identified a novel lncRNA RP11-283G6.5 which was lowly expressed in breast cancer and whose low expression was correlated with poor overall survival and disease-free survival of breast cancer patients. Functional experiments revealed that ectopic expression of RP11-283G6.5 confined breast cancer cellular growth, migration, and invasion, and promoted cellular apoptosis. Conversely, RP11-283G6.5 silencing facilitated breast cancer cellular growth, migration, and invasion, and repressed cellular apoptosis. Moreover, RP11-283G6.5 was found to confine breast cancer tumour growth and metastasis in vivo . Mechanistically, RP11-283G6.5 competitively bound to ILF3, reduced the binding of ILF3to primary miR-188 (pri-miR-188), abolished the suppressive effect of ILF3 on pri-miR-188 processing, and therefore promoted pri-miR-188 processing, leading to the reduction of pri-miR-188 and the upregulation of mature miR-188-3p. The expression of RP11-283G6.5 was significantly positively correlated with that of miR-188-3p in breast cancer tissues. Through increasing miR-188-3p, RP11-283G6.5 decreased TMED3, a target of miR-188-3p. RP11-283G6.5 further suppressed Wnt/β-catenin signalling via decreasing TMED3. Rescue assays revealed that inhibition of miR-188-3p, overexpression of TMED3 or blocking Wnt/β-catenin signalling all attenuated the roles of RP11-283G6.5 in breast cancer. Collectively, these findings demonstrated that RP11-283G6.5 is a tumour suppressive lncRNA in breast cancer via modulating miR-188-3p/TMED3/Wnt/β-catenin signalling. This study indicated that RP11-283G6.5 might be a promising prognostic biomarker and therapeutic target for breast cancer.

Background and Objectives: Metabolic shifting from mitochondrial respiration to glycolysis characterizes malignant cells from its normal counterparts and is attributed to overactivation of oncogenic signaling pathways. Hence, this study intended to investigate the influence of canagliflozin (CAN) and/or γ-irradiation (γ-IR) on HepG2 cell proliferation, crosstalk between phosphatidylinositol 3-kinases (PI3K)/AKT/glycogen synthase kinase-3-β (GSK3-β)/mTOR and Wnt/β-catenin signaling pathways, and their regulation of diverse processes, such as endoplasmic reticulum (ER) stress, autophagy, and apoptosis., Materials and Methods: HepG2 cells were treated with different doses of CAN and then exposed to different doses of γ-IR to achieve optimization that was based on cytotoxicity and clonogenic assays, respectively. The effects of CAN and/or γ-IR on glycolytic metabolism, cellular bioenergetics, oxidative stress, ER stress and autophagy biomarkers, expression of PI3K/AKT/GSK3-β/mTOR and Wnt/β-Catenin signaling pathways, and apoptotic markers were monitored., Results: CAN enhanced the antitumor potential of γ-IR as displayed by a significant inhibition of clonogenic survival in HepG2 cells via inhibition of glucose uptake, lactate release, and modulation of ER stress-mediated autophagy; switched it to apoptosis; as well as disabled signaling pathways which contribute to metabolic reprogramming and tumor progression induced by γ-IR that confer radioresistance and treatment failure., Conclusion: Our study sheds light on the effective combination of CAN and γ-IR in hepatocellular carcinoma treatment and necessitates CAN treatment prior to γ-IR to overcome metabolic reprogramming-associated radioresistance and improve curative outcomes., Competing Interests: None

Down-regulated lncRNA AC061961.2 in dilated cardiomyopathy (DCM) patients was previous reported. Whether AC061961.2 has regulatory effect on DCM still need exploration. Here, we tried to investigate the effect of AC061961.2 on DCM. After DCM model rat was established through injecting Adriamycin, left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were measured by echocardiography. Histopathological changes and apoptosis were detected by hematoxylin-eosin, Masson staining, and TUNEL. After cardiomyocytes were isolated and identified by immunofluorescence, DCM cell model was established by injecting adriamycin. After transfected with overexpressed-AC061961.2 plasmids, cell apoptosis was detected by flow cytometry. The expressions of AC061961.2, β-catenin, Axin2, c-Myc, CRP78, CHOP, Caspase-3, Bcl-2, and Bax in cardiomyocytes and heart tissues were detected by RT-qPCR or western blot. LVEDD and LVESD were increased while LVEF and LVFS were decreased in DCM rats. The histopathological of heart tissues showed a typical sign of DCM. Apoptosis were increased in heart tissues of DCM rats. In DCM rats, the expressions of AC061961.2, β-catenin, Axin2, c-Myc, and Bcl-2 were decreased, the expressions of CRP78, CHOP, Caspase-3, and Bax were increased. After the overexpression of AC061961.2, levels of β-catenin, Axin2, c-Myc, and Bcl-2 were increased, while levels of CRP78, CHOP, Caspase-3, and Bax were decreased, compared with that in DCM cardiomyocytes. LncRNA AC061961.2 overexpression inhibited endoplasmic reticulum stress induced apoptosis in DCM rats and cardiomyocytes via activating Wnt/β-catenin pathway.

Small Open Reading Frames (smORFs) coding for peptides of less than 100 amino-acids are an enigmatic and pervasive gene class, found in the tens of thousands in metazoan genomes. Here we reveal a short 80 amino-acid peptide (Pegasus) which enhances Wingless/Wnt1 protein short-range diffusion and signalling. During Drosophila wing development, Wingless has sequential functions, including late induction of proneural gene expression and wing margin development. Pegasus mutants produce wing margin defects and proneural expression loss similar to those of Wingless. Pegasus is secreted, and co-localizes and co-immunoprecipitates with Wingless, suggesting their physical interaction. Finally, measurements of fixed and in-vivo Wingless gradients support that Pegasus increases Wingless diffusion in order to enhance its signalling. Our results unveil a new element in Wingless signalling and clarify the patterning role of Wingless diffusion, while corroborating the link between small open reading frame peptides, and regulation of known proteins with membrane-related functions., (© 2021. The Author(s).)

Arginyltransferase (ATE1) plays critical roles in many biological functions including cardiovascular development, angiogenesis, adipogenesis, muscle contraction, and metastasis of cancer. However, the role of ATE1 in hepatocellular carcinoma (HCC) remains unknown. In this study, we find that ATE1 plays an essential role in growth and malignancy of liver cancer. ATE1 expression is significantly reduced in human HCC samples compared with normal liver tissue. In addition, low ATE1 expression is correlated with aggressive clinicopathologic features and is an independent poor prognostic factor for overall survival and disease-free survival of patients with HCC. Lentivirus-mediated ATE1 knockdown significantly promoted liver cancer growth, migration, and disease progression in vitro and in vivo . Opposing results were observed when ATE1 was upregulated. Mechanistically, ATE1 accelerated the degradation of β-catenin and inhibited Wnt signaling by regulating turnover of Regulator of G Protein Signaling 5 (RGS5). Loss- and gain-of-function assays confirmed that RGS5 was a key effector of ATE1-mediated regulation of Wnt signaling. Further studies indicated that RGS5 might be involved in regulating the activity of GSK3-β, a crucial component of the cytoplasmic destruction complex. Treatment with a GSK inhibitor (CHIR99021) cooperated with ablation of ATE1 or RGS5 overexpression to promote Wnt/β-catenin signaling, but overexpression of ATE1 or RGS5 knockdown did not reverse the effect of GSK inhibitor. IMPLICATIONS: ATE1 inhibits liver cancer progression by suppressing Wnt/β-catenin signaling and can serve as a potentially valuable prognostic biomarker for HCC., (©2021 The Authors; Published by the American Association for Cancer Research.)

To explore the effect and underlying molecular mechanism of long non-coding RNA (lncRNA)-H19 on ovarian cancer (OC) cells, a total of 41 cases of OC and adjacent normal tissues were collected. H19 and microRNA (miR)-140 expressions in OC tissues and cells were detected using quantitative real-time polymerase chain reaction (qRT-RCR). The correlation between H19 expression and prognosis of OC patient was analyzed. siRNA (si)-H19 and si-negative control (NC) were transfected into OC cells. Cell proliferation was checked by cell counting kit-8 assay and colony formation assay, and cell migration and invasion were analyzed via Transwell assay. The targeted binding relationship between H19 and miR-140 was predicted and verified, miR-140 downstream gene was predicted and Wnt1 was screened out. The impact of in-miR-140 on the si-H19-induced decreased OC cell proliferation and migration was evaluated. H19 expression was upregulated in OC tissues and cells, and its overexpression was associated with a poor prognosis of OC. si-H19 remarkably reduced OC cell proliferation and migration. H19 upregulated Wnt1 expression through targeting miR-140 in OC cells. Altogether, miR-140 was notably downregulated in OC, and in-miR-140 partially inhibited the si-H19-induced decrease of OC cell proliferation and migration. H19 competitively bound to miR-140 to upregulate Wnt1, thereby promoting OC cell proliferation and migration., (© 2021 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Background: Alzheimer’s disease is one of the neurodegenerative disorders typified by the aggregate of amyloid-β (Aβ) and phosphorylated tau protein. Oxidative stress and neuroinflammation, because of Aβ peptides, are strongly involved in the pathophysiology of Alzheimer’s disease (AD). Linagliptin shows neuroprotective properties against AD pathological processes through alleviation of neural inflammation and AMPK activation., Methods: We assessed the benefits of linagliptin pretreatment (at 10, 20, and 50 nM concentrations), against Aβ1-42 toxicity (20 μM) in SH-SY5Y cells. The concentrations of secreted cytokines, such as TNF-α, IL-6, and IL-1β, and signaling proteins, including pCREB, Wnt1, and PKCε, were quantified by ELISA., Results: We observed that Aβ led to cellular inflammation, which was assessed by measuring inflammatory cytokines (TNF-α, IL-1β, and IL-6). Moreover, Aβ1-42 treatment impaired pCREB, PKCε, and Wnt1 signaling in human SH-SY5Y neuroblastoma cells. Addition of Linagliptin significantly reduced IL-6 levels in the lysates of cells, treated with Aβ1-42. Furthermore, linagliptin prevented the downregulation of Wnt1 in Aβ1-42-treated cells exposed., Conclusion: The current findings reveal that linagliptin alleviates Aβ1-42-induced inflammation in SH-SY5Y cells, probably through the suppression of IL-6 release, and some of its benefits are mediated through the activation of the Wnt1 signaling pathway.

Although many studies have confirmed the relationship between obesity and endometrial cancer (EC), the molecular mechanism between obesity and EC progression has not been elucidated. Overexpression of fat mass and the obesity associated protein FTO leads to weight gain, although recently it has been discovered that FTO can serve as a demethylase which erases N6-methyladenosine (m6A) modification and regulates the metabolization of mRNAs. In this study, we found high expression of FTO in metastatic EC and that this action promote both metastasis and invasion in vivo and in vitro. Mechanistically, FTO can catalyse demethylation modification in 3'UTR region of HOXB13 mRNA, thereby abolishing m6A modification recognition with the YTHDF2 protein. Decreasing HOXB13 mRNA decay and increasing HOXB13 protein expression was accompanied by WNT signalling pathway activation and the expression of downstream proteins, leading to tumour metastasis and invasion. We also found the WNT signalling pathway inhibitor ICG-001 can block HOXB13 gene-induced tumour metastasis, therefore ICG-001 may be a promising molecular intervention. This study provides insight into the relationship between obesity and the pathogenesis of endometrial cancer while highlighting future areas of research.

Angiopoietin-like 3 (ANGPTL3), which is involved in new blood vessel growth, has been reported to exhibit an abnroaml expression in many different cancers. However, the expressing pattern and functions of ANGPTL3 renal cell carcinoma (RCC) were rarely reported. In this study, we observed that ANGPTL3 expression was distinctly downregulated in both RCC specimens from TCGA datasets and cell lines. Survival assays also revealed that patients with low ANGPTL3 expression exhibited a shorter overall survival and disease-free survival than those with high ANGPTL3 expression. Cell counting kit-8 (CCK-8) assay, Colony formation assay, and flow cytometry showed that overexpression of ANGPTL3 distinctly suppressed the proliferation of RCC cells, and promoted apoptosis. Transwell assays and Wound healing assays revealed that ANGPTL3 upregulation suppressed the migration and invasion of RCC cells. Then, we explored whether ANGPTL3 dysregulation influenced the alteration of Wnt/ β -catenin signaling using TOP/FOP flash reporter assays and western blot. The results showed that overexpression of ANGPTL3 distinctly suppressed the activity of Wnt/ β -catenin signaling. Overall, our results confirmed that overexpression of ANGPTL3 was related to the malignancy and good prognosis of RCC patients, and ANGPTL3 upregulation inhibited the tumor proliferation and metastasis via the Wnt/ β -catenin pathway. ANGPTL3 may be a novel therapeutic target and a prognostic biomarker for RCC patients., Competing Interests: The authors declared that they have no conflict of interest., (Copyright © 2021 Yu-jian Zhang et al.)