Canonical Wnt Signaling Pathway Research Articles

Wnt signaling in cardiac development and heart diseases.

The Wnt signaling pathway is a fundamental cellular communication system with extensive implications in various organs including the heart. In cardiac homeostasis, it governs essential processes like cellular proliferation, differentiation, and apoptosis, ensuring the heart's structural and functional integrity from embryonic stages and throughout life. Both canonical and non-canonical Wnt signaling pathways play a critical role during embryonic heart development in a region- and stage-specific manner. Canonical Wnt signaling also plays a significant role in heart diseases such as myocardial infarction and heart failure. However, the role of non-canonical Wnt signaling in heart diseases has not been fully elucidated. Wnt5a is a major ligand that activates non-canonical Wnt pathway, and recent studies start to clarify the role of the Wnt5a signaling axis in cardiac health and disease. In this review, we will briefly summarize the previous findings on the role of Wnt signaling pathways in heart development and diseases, and then focus on the role of Wnt5a signaling in heart failure progression. The multifaceted roles of the Wnt signaling pathway highlight its therapeutic potential for various types of heart diseases.

PAX1 represses canonical Wnt signaling pathway and plays dual roles during endoderm differentiation

BackgroundPaired box 1 (PAX1) is a transcription factor and essential for the development of pharyngeal pouches-derived tissues, including thymus. PAX1 mutations are identified in Severe Combined Immunodeficiency (SCID) patients with Otofaciocervical Syndrome Type 2 (OTFCS2). However, despite the critical roles of PAX1 in embryonic development and diseases, detailed insights into its molecular mode of action are critically missing.MethodsThe repressing roles of PAX1 and SCID associated mutants on Wnt signaling pathway were investigated by luciferase reporter assays, qRT-PCR and in situ hybridization in HEK293FT, HCT116 cells and zebrafish embryos, respectively. Co-immunoprecipitation (co-IP) and western blotting assays were carried out to identify the molecular mechanisms underlying PAX1’s role on Wnt signaling pathway. hESC based endoderm differentiation, flow cytometry, high-throughput sequencing data analysis, and qRT-PCR assays were utilized to determine the roles of PAX1 during endoderm differentiation.ResultsHere, we show that PAX1 represses canonical Wnt signaling pathway in vertebrate cells. Mechanically, PAX1 competes with SUMO E3 ligase PIASy to bind to TCF7L2, thus perturbing TCF7L2 SUMOylation level, further reducing its transcriptional activity and protein stability. Moreover, we reveal that PAX1 plays dual roles in hESC-derived definitive and foregut/pharyngeal endoderm cells, which give rise to the thymus epithelium, by inhibiting Wnt signaling. Importantly, our data show PAX1 mutations found in SCID patients significantly compromise the suppressing ability of PAX1 on Wnt signaling.ConclusionsOur study presents a novel molecular mode of action of PAX1 in regulation of canonical Wnt signaling and endoderm differentiation, thus providing insights for the molecular basis of PAX1 associated SCID, offering better understanding of the behavior of PAX1 in embryogenesis.

Cadmium modulates intestinal Wnt/β-catenin signaling ensuing intestinal barrier disruption and systemic inflammation

The intricate architecture of the intestinal epithelium, crucial for nutrient absorption, is constantly threatened by environmental factors. The epithelium undergoes rapid turnover, which is essential for maintaining homeostasis, under the control of intestinal stem cells (ISCs). The central regulator, Wnt/β-catenin signaling plays a key role in intestinal integrity and turnover. Despite its significance, the impact of environmental factors on this pathway has been largely overlooked. This study, for the first time, investigates the influence of Cd on the intestinal Wnt signaling pathway using a mouse model. In this study, male BALB/c mice were administered an environmentally relevant Cd dose (0.98 mg/kg) through oral gavage to investigate the intestinal disruption and Wnt signaling pathway. Various studies, including histopathology, immunohistochemistry, RT-PCR, western blotting, ELISA, intestinal permeability assay, and flow cytometry, were conducted to study Cd-induced changes in the intestine. The canonical Wnt signaling pathway experienced significant downregulation as a result of sub-chronic Cd exposure, which caused extensive damage throughout the small intestine. Increased intestinal permeability and a skewed immune response were also observed. To confirm that Wnt signaling downregulation is the key driver of Cd-induced gastrointestinal toxicity, mice were co-exposed to LiCl (a recognized Wnt activator) and Cd. The results clearly showed that the harmful effects of Cd could be reversed, which is strong evidence that Cd mostly damages the intestine through the Wnt/β-catenin signalling axis. In conclusion, this research advances the current understanding of the role of Wnt/β catenin signaling in gastrointestinal toxicity caused by diverse environmental pollutants.

Clinical features, treatment, and follow-up of OPPG and high-bone-mass disorders: LRP5 is a key regulator of bone mass.

Low-density lipoprotein receptor-related protein 5 (LRP5) is involved in the canonical Wnt signaling pathway. The gain-of-function mutation leads to high bone mass (LRP5-HBM), while the loss-of-function mutation leads to osteoporosis-pseudoglioma syndrome (OPPG). In this study, the clinical manifestations, disease-causing mutations, treatment, and follow-up were summarized to improve the understanding of these two diseases. Two OPPG patients and four LRP5-HBM patients were included in this study. The clinical characteristics, biochemical and radiological examinations, pathogenic mutations, and structural analysis were summarized. Furthermore, several patients were followed up to observe the treatment effect and disease progress. Congenital blindness, persistent bone pain, low bone mineral density (BMD), and multiple brittle fractures were the main clinical manifestations of OPPG. Complex heterozygous mutations were detected in two OPPG patients. The c.1455G > T mutation in exon 7 was first reported. During the follow-up, BMD of two patients was significantly improved after bisphosphonate treatment. On the contrary, typical clinical features of LRP5-HBM included extremely high BMD without fractures, torus palatinus and normal vision. X-ray showed diffuse osteosclerosis. Two heterozygous missense mutations were detected in four patients. In addition, age-related bone loss was found in one LRP5-HBM patient after 12-year of follow-up. This study deepened the understanding of the clinical characteristics, treatment, and follow-up of OPPG and LRP5-HBM; expanded the pathogenic gene spectrum of OPPG; and confirmed that bisphosphonates were effective for OPPG. Additionally, it was found that Ala242Thr mutation could not protect LRP5-HBM patients from age-related bone loss. This phenomenon deserves further study.

Wnt signaling regulates chemokine production and cell migration of circulating human monocytes

The β-catenin dependent canonical Wnt signaling pathway plays a crucial role in maintaining normal homeostasis. However, when dysregulated, Wnt signaling is closely associated with various pathological conditions, including inflammation and different types of cancer.Here, we show a new connection between the leukocyte inflammatory response and the Wnt signaling pathway. Specifically, we demonstrate that circulating human primary monocytes express distinct Wnt signaling components and are susceptible to stimulation by the classical Wnt ligand—Wnt-3a. Although this stimulation increased the levels of β-catenin protein, the expression of the classical Wnt-target genes was not affected. Intriguingly, treating circulating human monocytes with Wnt-3a induces the secretion of cytokines and chemokines, enhancing monocyte migration. Mechanistically, the enhanced monocyte migration in response to Wnt stimuli is mediated through CCL2, a strong monocyte-chemoattractant.To further explore the physiological relevance of these findings, we conducted ex-vivo experiments using blood samples of patients with rheumatic joint diseases (RJD) – conditions where monocytes are known to be dysfunctional. Wnt-3a generated a unique cytokine expression profile, which was significantly distinct from that observed in monocytes obtained from healthy donors.Thus, our results provide the first evidence that Wnt-3a may serve as a potent stimulator of monocyte-driven immune processes. These findings contribute to our understanding of inflammatory diseases and, more importantly, shed light on the role of a core signaling pathway in the circulation.

Ablation of Wnt signaling in bone marrow stromal cells overcomes microenvironment-mediated drug resistance in acute myeloid leukemia

The survival of leukemic cells is significantly influenced by the bone marrow microenvironment, where stromal cells play a crucial role. While there has been substantial progress in understanding the mechanisms and pathways involved in this crosstalk, limited data exist regarding the impact of leukemic cells on bone marrow stromal cells and their potential role in drug resistance. In this study, we identify that leukemic cells prime bone marrow stromal cells towards osteoblast lineage and promote drug resistance. This biased differentiation of stroma is accompanied by dysregulation of the canonical Wnt signaling pathway. Inhibition of Wnt signaling in stroma reversed the drug resistance in leukemic cells, which was further validated in leukemic mice models. This study evaluates the critical role of leukemic cells in establishing a drug-resistant niche by influencing the bone marrow stromal cells. Additionally, it highlights the potential of targeting Wnt signaling in the stroma by repurposing an anthelmintic drug to overcome the microenvironment-mediated drug resistance.

SARS-CoV-2 causes dysfunction in human iPSC-derived brainmicrovascular endothelial cells potentially by modulating the Wnt signaling pathway.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which is associated with various neurological symptoms, including nausea, dizziness, headache, encephalitis, and epileptic seizures. SARS-CoV-2 is considered to affect the central nervous system (CNS) by interacting with the blood-brain barrier (BBB), which is defined by tight junctions that seal paracellular gaps between brain microvascular endothelial cells (BMECs). Although SARS-CoV-2 infection of BMECs has been reported, the detailed mechanism has not been fully elucidated. Using the original strain of SARS-CoV-2, the infection in BMECs was confirmed by a detection of intracellular RNA copy number and localization of viral particles. BMEC functions were evaluated by measuring transendothelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics, and expression levels of proinflammatory genes. BMEC signaling pathway was examined by comprehensive RNA-seq analysis. We observed that iPSC derived brain microvascular endothelial like cells (iPSC-BMELCs) were infected with SARS-CoV-2. SARS-CoV-2 infection resulted in decreased TEER. In addition, SARS-CoV-2 infection decreased expression levels of tight junction markers CLDN3 and CLDN11. SARS-CoV-2 infection also increased expression levels of proinflammatory genes, which are known to be elevated in patients with COVID-19. Furthermore, RNA-seq analysis revealed that SARS-CoV-2 dysregulated the canonical Wnt signaling pathway in iPSC-BMELCs. Modulation of the Wnt signaling by CHIR99021 partially inhibited the infection and the subsequent inflammatory responses. These findings suggest that SARS-CoV-2 infection causes BBB dysfunction via Wnt signaling. Thus, iPSC-BMELCs are a useful in vitro model for elucidating COVID-19 neuropathology and drug development.

Dihydrotanshinone I exhibits antitumor effects via β-catenin downregulation in papillary thyroid cancer cell lines

Thyroid cancer is the most common endocrine carcinoma and, among its different subtypes, the papillary subtype (PTC) is the most frequent. Generally, PTCs are well differentiated, but a minor percentage of PTCs are characterized by a worse prognosis and more aggressive behavior. Phytochemicals, naturally found in plant products, represent a heterogeneous group of bioactive compounds that can interfere with cell proliferation and the regulation of the cell cycle, taking part in multiple signaling pathways that are often disrupted in tumor initiation, proliferation, and progression. In this work, we focused on 15,16-dihydrotanshinone I (DHT), a tanshinone isolated from Salvia miltiorrhiza Bunge (Danshen). We first evaluated DHT biological effect on PTC cells regarding cell viability, colony formation ability, and migration capacity. All of these parameters were downregulated by DHT treatment. We then investigated gene expression changes after DHT treatment by performing RNA-seq. The analysis revealed that DHT significantly reduced the Wnt signaling pathway, which plays a role in various diseases, including cancer. Finally, we demonstrate that DHT treatment decreases protein levels of β-catenin, a final effector of canonical Wnt signaling pathway. Overall, our data suggest a possible use of this nutraceutical as an adjuvant in the treatment of aggressive papillary thyroid carcinoma.

The impact of canonical Wnt transcriptional repressors TLE3 and TLE4 on postsynaptic transcription at the neuromuscular junction.

Here, we investigated the role of the canonical Wnt signaling pathway transcriptional regulators at the neuromuscular junction. Upon applying a denervation paradigm, the transcription levels of Ctnnb1, Tcf7l1, Tle1, Tle2, Tle3, and Tle4 were significantly downregulated. A significant decrease in canonical Wnt signaling activity was observed using the denervation paradigm in Axin2-lacZ reporter mice. Alterations in the transcriptional profile of the myogenic lineage in response to agrin (AGRN) suggested that TLE3 and TLE4, family members of groucho transducin-like enhancer of split 3 (TLE3), transcriptional repressors known to antagonize T cell factor/lymphoid enhancer factor (TCF)-mediated target gene activation, could be important regulators of canonical Wnt signaling activity at the postsynapse. Knockouts of these genes using CRISPR/Cas9 gene editing in primary skeletal muscle stem cells, called satellite cells, led to decreased AGRN-dependent acetylcholine receptor (CHRN) clustering and reduced synaptic gene transcription upon differentiation of these cells. Overall, our findings demonstrate that TLE3 and TLE4 participate in diminishing canonical Wnt signaling activity, supporting transcription of synaptic genes and CHRN clustering at the neuromuscular junction.

Abstract 4393: Expression levels of T-cell factor-4 variants in hepato-gastrointestinal cancer cells and tissues

Abstract Background: We previously identified 14 variants of T-cell factor (TCF)-4, a key transcriptional factor in the canonical Wnt signaling pathway, from human liver cancer cell lines (Exp Cell Res 2011). The functional analysis of the variants demonstrated that the SxxSS motif played crucial roles in high tumorigenicity, resistance to hypoxia (PLoS ONE 2012), and EMT (Liver Int 2013). These findings suggest that expression levels and patterns of TCF-4 variants tightly regulate diverse phenotypes of cancer cells. Currently, we developed a novel RT-PCR system to quantitatively evaluate TCF-4 variant expressions by using the peptide nucleic acid (PNA)-directed PCR clamping method. Aim: To assess mRNA expression levels of TCF-4 variants in hepato-gastrointestinal cancer cell lines and colon cancer tissues, focusing on TCF-4J and TCF-4K with the presence (K) or absence (J) of the SxxSS motif. Methods: Thirteen cell lines used in this study were as follows: seven liver cancer; one fetus-derived immortalized hepatocyte; one gastric cancer; two pancreatic cancer; and two colon cancer cell lines. The liver cancer cell lines HAK-1A and HAK-1B are clonally sister cell lines. Sets of three forward primers and four reverse primers were used to generate templates for quantitative PCR (qPCR), and three fluorescence (FAM and Cy5)-tagged primers were applied to detect each variant. Thirty-nine resected human cancer and non-cancerous tissue pairs were used in the IRB-certified study (No. 19192). Results: All TCF-4 variants except TCF-4M and TCF-4X were specifically and reproducibly detected and quantified. Cancer cells derived from the colon and stomach highly expressed the TCF-4 variants, showing the highest expression in TCF-4J, while cancer cells from the liver and pancreas expressed relatively lower levels. There was no remarkable difference in the expression profile of TCF-4 variants between HAK-1A and HAK-1B. In colon cancer tissues, TCF-4J was significantly more expressed than TCF-4K. Conclusion: Although the variants had been identified from liver cancer cells, they were universally expressed in cells of other organs. TCF-4J, a variant responsible for aggressive cell phenotype, was prominently found in cancer cells of the digestive tract, where the canonical Wnt signal pathway is known to be constitutively active. A similar variant profile between two liver cancer cell lines with identical clonal origin suggests that the variant pattern in the cells reflects organ-specific traits rather than dynamic phenotypic changes during cancer cell dedifferentiation. Citation Format: Hironori Koga, Yasuko Imamura, Tomoya Sudo, Hiroyuki Suzuki, Toshimitsu Tanaka, Takahiko Sakaue, Atsutaka Masuda, Hideki Iwamoto, Toru Nakamura, Hirohisa Yano, Takumi Kawaguchi. Expression levels of T-cell factor-4 variants in hepato-gastrointestinal cancer cells and tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4393.

Significance of the Wnt signaling pathway in coronary artery atherosclerosis.

The progression of coronary atherosclerosis is an active and regulated process. The Wnt signaling pathway is thought to play an active role in the pathogenesis of several cardiovascular diseases; however, a better understanding of this system in atherosclerosis is yet to be unraveled. In this study, real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to quantify the expression of Wnt3a, Wnt5a, and Wnt5b in the human coronary plaque, and immunohistochemistry was used to identify sites of local expression. To determine the pathologic significance of increased Wnt, human vascular smooth muscle cells (vSMCs) were treated with Wnt3a, Wnt5a, and Wnt5b recombinant proteins and assessed for changes in cell differentiation and function. RT-PCR and Western blotting showed a significant increase in the expression of Wnt3a, Wnt5a, Wnt5b, and their receptors in diseased coronary arteries compared with that in non-diseased coronary arteries. Immunohistochemistry revealed an abundant expression of Wnt3a and Wnt5b in diseased coronary arteries, which contrasted with little or no signals in normal coronary arteries. Immunostaining of Wnt3a and Wnt5b was found largely in inflammatory cells and myointimal cells. The treatment of vSMCs with Wnt3a, Wnt5a, and Wnt5b resulted in increased vSMC differentiation, migration, calcification, oxidative stress, and impaired cholesterol handling. This study demonstrates the upregulation of three important members of canonical and non-canonical Wnt signaling pathways and their receptors in coronary atherosclerosis and shows an important role for these molecules in plaque development through increased cellular remodeling and impaired cholesterol handling.

WNT-inhibitory factor 1-mediated glycolysis protects photoreceptor cells in diabetic retinopathy

BackgroundIn diabetic retinopathy (DR), hypoxia-inducible factor (HIF-1α) induces oxidative stress by upregulating glycolysis. This process leads to neurodegeneration, particularly photoreceptor cell damage, which further contributes to retinal microvascular deterioration. Further, the regulation of Wnt-inhibitory factor 1 (WIF1), a secreted Wnt signaling antagonist, has not been fully characterized in neurodegenerative eye diseases. We aimed to explore the impact of WIF1 on photoreceptor function within the context of DR.MethodTwelve-week-old C57BL/KsJ-db/db mice were intravitreally injected with WIF1 overexpression lentivirus. After 4 weeks, optical coherence tomography (OCT), transmission electron microscopy (TEM), H&E staining, and electroretinography (ERG) were used to assess the retinal tissue and function. The potential mechanism of action of WIF1 in photoreceptor cells was explored using single-cell RNA sequencing. Under high-glucose conditions, 661 W cells were used as an in vitro DR model. WIF1-mediated signaling pathway components were assessed using quantitative real-time PCR, immunostaining, and western blotting.ResultTypical diabetic manifestations were observed in db/db mice. Notably, the expression of WIF1 was decreased at the mRNA and protein levels. These pathological manifestations and visual function improved after WIF1 overexpression in db/db mice. TEM demonstrated that WIF1 restored damaged mitochondria, the Golgi apparatus, and photoreceptor outer segments. Moreover, ERG indicated the recovery of a-wave potential amplitude. Single-cell RNA sequencing and in vitro experiments suggested that WIF1 overexpression prevented the expression of glycolytic enzymes and lactate production by inhibiting the canonical Wnt signaling pathway, HIF-1α, and Glut1, thereby reducing retinal and cellular reactive oxygen species levels and maintaining 661 W cell viability.ConclusionsWIF1 exerts an inhibitory effect on the Wnt/β-catenin-HIF-1α-Glut1 glycolytic pathway, thereby alleviating oxidative stress levels and mitigating pathological structural characteristics in retinal photoreceptor cells. This mechanism helps preserve the function of photoreceptor cells in DR and indicates that WIF1 holds promise as a potential therapeutic candidate for DR and other neurodegenerative ocular disorders.Graphical

Icariin ameliorates glycolytic dysfunction in Alzheimer's disease models by activating the Wnt/β-catenin signaling pathway.

It was reported that the Wnt/β-catenin pathway is involved in the regulation of aerobic glycolysis and that brain glycolytic dysfunction results in the development of Alzheimer's disease (AD). Icariin (ICA), an active component extracted from Epimedii Folium, has been reported to produce neuroprotective effects in multiple models of AD, but its underlying mechanism remains to be fully described. We aimed to investigate the protective effects of ICA on animal and cell models of AD and confirm whether the Wnt/β-catenin pathway has functions in the neuroprotective function of ICA. The 3 × Tg-AD mice were treated with ICA. HT22 cells, the Aβ25-35 peptide and Dickkopf-1 (DKK1) agent (a specific inhibitor of the Wnt/β-catenin pathway) were used to further explore the underlying mechanism of ICA that produces anti-AD effects. Behavioral examination, western blotting assay, staining analysis, biochemical test, and lactate dehydrogenase (LDH) assays were applied. We first demonstrated that ICA significantly improved cognitive function and autonomous behavior, reduced neuronal damage, and reversed the protein levels and activities of glycolytic key enzymes, and expression of protein molecules of the canonical Wnt signaling pathway, in 3 × Tg-AD mice back to wild-type levels. Next, we further found that ICA increased cell viability and effectively improved the dysfunctional glycolysis in HT22 cells injured by Aβ25-35 . However, when canonical Wnt signaling was inhibited by DKK1, the above effects of ICA on glycolysis were abolished. In summary, ICA exerts neuroprotective effects in 3 × Tg-AD animals and AD cellular models by enhancing the function of glycolysis through activation of the Wnt/β-catenin pathway.

WNT signalling control by KDM5C during development affects cognition.

Although KDM5C is one of the most frequently mutated genes in X-linked intellectual disability1, the exact mechanisms that lead to cognitive impairment remain unknown. Here we use human patient-derived induced pluripotent stem cells and Kdm5c knockout mice to conduct cellular, transcriptomic, chromatin and behavioural studies. KDM5C is identified as a safeguard to ensure that neurodevelopment occurs at an appropriate timescale, the disruption of which leads to intellectual disability. Specifically, there is a developmental window during which KDM5C directly controls WNT output to regulate the timely transition of primary to intermediate progenitor cells and consequently neurogenesis. Treatment with WNT signalling modulators at specific times reveal that only a transient alteration of the canonical WNT signalling pathway is sufficient to rescue the transcriptomic and chromatin landscapes in patient-derived cells and to induce these changes in wild-type cells. Notably, WNT inhibition during this developmental period also rescues behavioural changes of Kdm5c knockout mice. Conversely, a single injection of WNT3A into the brains of wild-type embryonic mice cause anxiety and memory alterations. Our work identifies KDM5C as a crucial sentinel for neurodevelopment and sheds new light on KDM5C mutation-associated intellectual disability. The results also increase our general understanding of memory and anxiety formation, with the identification of WNT functioning in a transient nature to affect long-lasting cognitive function.

The origin and evolution of Wnt signalling.

The Wnt signal transduction pathway has essential roles in the formation of the primary body axis during development, cellular differentiation and tissue homeostasis. This animal-specific pathway has been studied extensively in contexts ranging from developmental biology to medicine for more than 40 years. Despite its physiological importance, an understanding of the evolutionary origin and primary function of Wnt signalling has begun to emerge only recently. Recent studies on very basal metazoan species have shown high levels of conservation of components of both canonical and non-canonical Wnt signalling pathways. Furthermore, some pathway proteins have been described also in non-animal species, suggesting that recruitment and functional adaptation of these factors has occurred in metazoans. In this Review, we summarize the current state of research regarding the evolutionary origin of Wnt signalling, its ancestral function and the characteristics of the primal Wnt ligand, with emphasis on the importance of genomic studies in various pre-metazoan and basal metazoan species.

Transcript Profiles of Microglia/Macrophage Cells at the Borders of Chronic Active and Subpial Gray Matter Lesions in Multiple Sclerosis.

Microglia/macrophages line the border of demyelinated lesions in both cerebral white matter and the cortex in the brains of multiple sclerosis patients. Microglia/macrophages associated with chronic white matter lesions are thought to be responsible for slow lesion expansion and disability progression in progressive multiple sclerosis, whereas those lining gray matter lesions are less studied. Profiling these microglia/macrophages could help to focus therapies on genes or pathways specific to lesion expansion and disease progression. We compared the morphology and transcript profiles of microglia/macrophages associated with borders of white matter (WM line) and subpial gray matter lesions (GM line) using laser capture microscopy. We performed RNA sequencing on isolated cells followed by immunocytochemistry to determine the distribution of translational products of transcripts increased in WM line microglia. Cells in the WM line appear activated, with shorter processes and larger cell bodies, whereas those in the GM line appear more homeostatic, with smaller cell bodies and multiple thin processes. Transcript profiling revealed 176 genes in WM lines and 111 genes in GM lines as differentially expressed. Transcripts associated with immune activation and iron homeostasis were increased in WM line microglia, whereas genes belonging to the canonical Wnt signaling pathway were increased in GM line microglia. We propose that the mechanisms of demyelination and dynamics of lesion expansion are responsible for differential transcript expression in WM lines and GM lines, and posit that increased expression of the Fc epsilon receptor, spleen tyrosine kinase, and Bruton's tyrosine kinase, play a key role in regulating microglia/macrophage function at the border of chronic active white matter lesions. ANN NEUROL 2024;95:907-916.

SETD3 regulates endoderm differentiation of mouse embryonic stem cells through canonical Wnt signaling pathway.

With self-renewal and pluripotency features, embryonic stem cells (ESCs) provide an invaluable tool to investigate early cell fate decisions. Pluripotency exit and lineage commitment depend on precise regulation of gene expression that requires coordination between transcription (TF) and chromatin factors in response to various signaling pathways. SET domain-containing 3 (SETD3) is a methyltransferase that can modify histones in the nucleus and actin in the cytoplasm. Through an shRNA screen, we previously identified SETD3 as an important factor in the meso/endodermal lineage commitment of mouse ESCs (mESC). In this study, we identified SETD3-dependent transcriptomic changes during endoderm differentiation of mESCs using time-course RNA-seq analysis. We found that SETD3 is involved in the timely activation of the endoderm-related gene network. The canonical Wnt signaling pathway was one of the markedly altered signaling pathways in the absence of SETD3. The assessment of Wnt transcriptional activity revealed a significant reduction in Setd3-deleted (setd3∆) mESCs coincident with a decrease in the nuclear pool of the key TF β-catenin level, though no change was observed in its mRNA or total protein level. Furthermore, a proximity ligation assay (PLA) found an interaction between SETD3 and β-catenin. We were able to rescue the differentiation defect by stably re-expressing SETD3 or activating the canonical Wnt signaling pathway by changing mESC culture conditions. Our results suggest that alterations in the canonical Wnt pathway activity and subcellular localization of β-catenin might contribute to the endoderm differentiation defect of setd3∆ mESCs.

Canonical Wnt signaling pathway (WSP) alterations in metastatic prostate cancer.

221 Background: WSP aberrations have been implicated in the progression and treatment resistance of metastatic prostate cancer (mPCa). Sustained exposure of PCa to androgen deprivation has been shown to regulate the canonical WSP, induce beta-catenin expression and stabilization, and promote androgen independence. This study interrogates a large dataset to characterize the molecular and immune landscape of canonical WSP-altered mPCa and associations with survival outcomes. Methods: We examined PCa patients who underwent tissue-based DNA and RNA sequencing through a CLIA-certified commercial assay (Caris Life Sciences). Comprehensive genomic profiling included next-generation sequencing (NGS) of DNA (592-gene panel or whole exome) and RNA (whole transcriptome), on tissue samples from primary prostatic and/or metastatic sites. Tumor samples were categorized as having aberrant canonical Wnt signaling (WSP-act) if they harbored activating mutations in CTNNB, pathogenic RSPO2 fusions, or inactivating mutations in either APC or RNF43. Subset analysis was conducted in mPCa samples with microsatellite stability (MSS), after excluding those with RNF43 (p.G659fs*) mutations which are passenger events in MSI-high cancers. Statistical significance was determined using Fisher's Exact or X2 tests with Benjamini–Hochberg correction. Overall survival (OS) data was obtained from insurance claims and was analyzed using Kaplan-Meier estimation. Results: Of all PCa samples (4,150) (N = 4,150), 17.4% (n = 722) were classified as WSP-act; 299 (41.4%) were from the primary tumor and 423 (58.6%) were from metastatic sites. WSP-act tumors were more prevalent in brain (46%), liver (31%), lung (31%) and bone (25%) metastases compared to primary prostate cancers (10%). The MSS mPCa subgroup (N=1,638) comprised 321 WSP-act and 1,317 WSP-WT tumors. WSP-act mPCa exhibited more SPOP mutations (14.2% vs. 6.3%, p < 0.001) as well as higher mRNA expression of CST1, NKD1, and ODAM (known canonical WSP activators) compared to WSP-WT cases. M2 macrophages predominated the tumor microenvironment, with a higher median fraction in WSP-act tumors compared to WSP-WT (7.2 vs. 6.1%, p < 0.001). ROR1 gene expression was significantly elevated in WSP-act tumors from both primary and metastatic sites. There was no significant difference in OS outcomes between WSP-act and WSP-WT mPCa patients. Conclusions: WSP-act prostate cancer demonstrated a pronounced upregulation of ROR1 gene expression, underscoring its potential involvement in the crosstalk between the canonical and non-canonical Wnt signaling pathways. Additionally, the augmented levels of M2 macrophages in WSP-act tumors, combined with the reported role of ROR1 in tumor immunosuppression, suggests that ROR1 may contribute to immune evasion in WSP-act mPCa. Our findings may provide rationale for developing novel therapeutic strategies targeting Wnt-activated prostate cancers.

Anti-sclerostin antibody therapy prevents post-ischemic osteonecrosis bone collapse via interleukin-6 association

Osteonecrosis of the femoral head (ONFH) is a debilitating condition characterized by subchondral bone necrosis, which frequently culminates in joint destruction. Although total hip arthroplasty is conventionally practiced to remediate ONFH, for patients under the age of 60, the outcomes can be suboptimal. Chronic inflammation, particularly that mediated by interleukin-6 (IL-6), has been conjectured to be a potential mechanism underlying the etiology of ONFH. This study aimed at exploring the interplay between IL-6, the canonical Wnt signaling pathway, and ONFH to provide insights for potential therapeutic interventions. Human ONFH specimens depicted an elevation in β-catenin expression in the transitional layer, while IL-6 levels were pronounced in the same region. Subsequently, mouse models of ischemic osteonecrosis were treated with an anti-sclerostin antibody to assess its effects on bone metabolism and cellular processes. Histological analysis revealed that the administration of anti-sclerostin antibodies effectuated early recovery from bone necrosis, reduced empty lacunae, and suppressed IL-6 expression. The treatment evidently initiated the activation of the Wnt/β-catenin signaling pathway, presenting a potential mechanism associated with IL-6-mediated inflammation. Furthermore, the antibody upregulated osteoblast formation, downregulated osteoclast formation, and increased bone volume. Micro-CT imaging demonstrated increased bone volume, prevented epiphyseal deformity, and improved compression strength.Therefore, this study yields significant findings, indicating the potency of anti-sclerostin antibodies in effectively modulating the Wnt/β-catenin pathway, associating with IL-6 expression, and preventing post-ONFH bone collapse. Additionally, this preclinical investigation in mouse models offers an avenue for prospective research on potential therapeutic interventions against human ONFH.

SENP3 Promotes Mantle Cell Lymphoma Development through Regulating Wnt10a Expression.

SUMO-specific protease 3 (SENP3), a member of the SUMO-specific protease family, reverses the SUMOylation of SUMO-2/3 conjugates. Dysregulation of SENP3 has been proven to be involved in the development of various tumors. However, its role in mantle cell lymphoma (MCL), a highly aggressive lymphoma, remains unclear. This study was aimed to elucidate the effect of SENP3 in MCL. The expression of SENP3 in MCL cells and tissue samples was detected by RT-qPCR, Western blotting or immunohistochemistry. MCL cells with stable SENP3 knockdown were constructed using short hairpin RNAs. Cell proliferation was assessed by CCK-8 assay, and cell apoptosis was determined by flow cytometry. mRNA sequencing (mRNA-seq) was used to investigate the underlying mechanism of SENP3 knockdown on MCL development. A xenograft nude mouse model was established to evaluate the effect of SENP3 on MCL growth in vivo. SENP3 was upregulated in MCL patient samples and cells. Knockdown of SENP3 in MCL cells inhibited cell proliferation and promoted cell apoptosis. Meanwhile, the canonical Wnt signaling pathway and the expression of Wnt10a were suppressed after SENP3 knockdown. Furthermore, the growth of MCL cells in vivo was significantly inhibited after SENP3 knockdown in a xenograft nude mouse model. SENP3 participants in the development of MCL and may serve as a therapeutic target for MCL.