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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with unknown etiology, characterized by motor neuron degeneration, and there is no highly effective treatment. The canonical WNT/β-catenin signaling pathway has a critical role in the physiological and pathophysiological processes of the central nervous system. In this study, we investigated the regulatory mechanism of the WNT/β-catenin signaling pathway from the perspective of ligand-receptor binding and its relationship with the degeneration of ALS motor neurons. We used hSOD1-G93A mutant ALS transgenic mice and hSOD1-G93A mutant NSC34 cells combined with morphological and molecular biology techniques to determine the role of the WNT/β-catenin pathway in ALS. Our findings demonstrated that WNT5A regulates the WNT/β-catenin signaling pathway by binding to the FZD4 receptor in the pathogenesis of ALS and affects the proliferation and apoptosis of ALS motor neurons. Therefore, these findings may lead to the development of novel therapies to support the survival of ALS motor neurons.

The identification of characteristic differences between cancer stem cells and their normal counterparts remains a key challenge for cancer treatment. Here, we investigated the role of immunoglobulin superfamily member 8 (Igsf8, also known as EWI-2, PGRL, and CD316) on normal and malignant hematopoietic stem cells, mainly using the conditional knockout model. Deletion of Igsf8 did not affect steady state hematopoiesis, but it led to a significant improvement of survival in mouse myeloid leukemia models. Deletion of Igsf8 significantly depletes leukemia stem cells (LSCs) through enhanced apoptosis and β-catenin degradation. At a molecular level, we found that activation of β-catenin in LSCs depends on Igsf8, which promotes the association of FZD4 with its co-receptor LRP6 in the presence of Igsf8. Similarly, IGSF8 inhibition blocks the colony-forming ability of LSCs and improves the survival of recipients in xenograft models of myeloid leukemia. Collectively, these data indicate strong genetic evidence identifying Igsf8 as a key regulator of myeloid leukemia and the possibility that targeting IGSF8 may serve as a new therapeutic approach against myeloid leukemia.

The organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes (1). However, the mechanisms that generate cellular asymmetries within the oocyte, and then transduce that polarity to organismal scale body axes are poorly understood outside of select model organisms. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled is a cytoplasmic Wnt pathway effector required for axis development in diverse species (2–4), but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dishevelled localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes, but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-reassembly mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome association and Frizzled receptors pre-localized within the oocyte cortex. Our results reveal a cell cycle-linked mechanism by which maternal cellular polarity is transduced to the embryo through spatially-regulated Dishevelled dynamics.

Background Wnt genes code for ligands that activate signaling pathways during development in Metazoa. Through the canonical Wnt (cWnt) signaling pathway, these genes regulate important processes in bilaterian development, such as establishing the anteroposterior axis and posterior growth. In Arthropoda, Wnt ligands also regulate segment polarity, and outgrowth and patterning of developing appendages. Arthropods are part of a lineage called Panarthropoda that includes Onychophora and Tardigrada. Previous studies revealed potential roles of Wnt genes in regulating posterior growth, segment polarity, and growth and patterning of legs in Onychophora. Unlike most other panarthropods, tardigrades lack posterior growth, but retain segmentation and appendages. Here, we investigated Wnt genes in tardigrades to gain insight into potential roles that these genes play during development of the highly compact and miniaturized tardigrade body plan. Results We analyzed published genomes for two representatives of Tardigrada, Hypsibius exemplaris and Ramazzottius varieornatus. We identified single orthologs of Wnt4, Wnt5, Wnt9, Wnt11, and WntA, as well as two Wnt16 paralogs in both tardigrade genomes. We only found a Wnt2 ortholog in H. exemplaris. We could not identify orthologs of Wnt1, Wnt6, Wnt7, Wnt8, or Wnt10. We identified most other components of cWnt signaling in both tardigrade genomes. However, we were unable to identify an ortholog of arrow/Lrp5/6, a gene that codes for a Frizzled co-receptor of Wnt ligands. Additionally, we found that some other animals that have lost several Wnt genes and are secondarily miniaturized, like tardigrades, are also missing an ortholog of arrow/Lrp5/6. We analyzed the embryonic expression patterns of Wnt genes in H. exemplaris during developmental stages that span the establishment of the AP axis through segmentation and leg development. We detected expression of all Wnt genes in H. exemplaris besides one of the Wnt16 paralogs. During embryo elongation, expression of several Wnt genes was restricted to the posterior pole or a region between the anterior and posterior poles. Wnt genes were expressed in distinct patterns during segmentation and development of legs in H. exemplaris, rather than in broadly overlapping patterns. Conclusions Our results indicate that Wnt signaling has been highly modified in Tardigrada. While most components of cWnt signaling are conserved in tardigrades, we conclude that tardigrades have lost Wnt1, Wnt6, Wnt7, Wnt8, and Wnt10, along with arrow/Lrp5/6. Our expression data may indicate a conserved role of Wnt genes in specifying posterior identities during establishment of the AP axis. However, the loss of several Wnt genes and the distinct expression patterns of Wnt genes during segmentation and leg development may indicate that combinatorial interactions among Wnt genes are less important during tardigrade development compared to many other animals. Based on our results, and comparisons to previous studies, we speculate that the loss of several Wnt genes in Tardigrada may be related to a reduced number of cells and simplified development that accompanied miniaturization and anatomical simplification in this lineage.

Although WNT signaling is frequently dysregulated in solid tumors, drugging this pathway has been challenging due to off-tumor effects. Current clinical pan-WNT inhibitors are nonspecific and lead to adverse effects, highlighting the urgent need for more specific WNT pathway–targeting strategies. We identified elevated expression of the WNT receptor Frizzled class receptor 7 (FZD7) in multiple solid cancers in The Cancer Genome Atlas, particularly in the mesenchymal and proliferative subtypes of ovarian serous cystadenocarcinoma, which correlate with poorer median patient survival. Moreover, we observed increased FZD7 protein expression in ovarian tumors compared with normal ovarian tissue, indicating that FZD7 may be a tumor-specific antigen. We therefore developed a novel antibody–drug conjugate, septuximab vedotin (F7-ADC), which is composed of a chimeric human–mouse antibody to human FZD7 conjugated to the microtubule-inhibiting drug monomethyl auristatin E (MMAE). F7-ADC selectively binds human FZD7, potently kills ovarian cancer cells in vitro, and induces regression of ovarian tumor xenografts in murine models. To evaluate F7-ADC toxicity in vivo, we generated mice harboring a modified Fzd7 gene where the resulting Fzd7 protein is reactive with the human-targeting F7-ADC. F7-ADC treatment of these mice did not induce acute toxicities, indicating a potentially favorable safety profile in patients. Overall, our data suggest that the antibody–drug conjugate approach may be a powerful strategy to combat FZD7-expressing ovarian cancers in the clinic.

Introduction Accumulating evidence highlights the critical roles of circular RNAs (circRNAs) in the malignant progression of cancers. In this study, we investigated the expression pattern of a newly identified circRNA (hsa_circ_0017109) in non–small cell lung cancer (NSCLC), and examined its downstream molecular targets. Methods Quantitative real-time PCR (qRT-PCR) and Western blotting (WB) were conducted to quantify gene and protein expression. In vitro functional assays such as colony formation assay, cell counting kit-8 (CCK-8) and flow cytometry were used to study cell proliferation and apoptosis. RNA pull-down assay, luciferase reporter assay and RNA immunoprecipitation were performed to validate molecular interaction. Mouse xenograft model of NSCLC cells was used to assess the role of circ_0017109 in tumorigenesis. Results Circ_0017109 was upregulated in NSCLC tumor samples and cells. Silencing circ_0017109 impaired cell proliferation and promoted apoptosis in NSCLC cells, and circ_0017109 knockdown suppressed in vivo tumorigenesis of NSCLC cells in mouse xenograft model. MiR-671-5p was identified as a target of circ_0017109, and circ_0017109 negatively impacted on miR-671-5p expression. MiR-671-5p downregulated FZD4 and dampened the activity of Wnt/β-catenin signaling pathway. Circ_0017109 modulated FZD4 expression by suppressing miR-671-5p activity. Conclusions Elevated circ_0017109 expression promotes tumor progression of NSCLC by modulating miR-671-5p/FZD4/β-catenin axis.

Dishevelled is a cytoplasmic hub that transduces Wnt signals to cytoplasmic effectors, which can be broadly characterised as canonical (β-catenin dependent) and noncanonical, to specify cell fates and behaviours during development. To transduce canonical Wnt signals, Dishevelled binds to the intracellular face of Frizzled through its DEP domain and polymerises through its DIX domain to assemble dynamic signalosomes. Dishevelled also contains a PDZ domain, whose function remains controversial. Here, we use genome editing to delete the PDZ domain-encoding region from Drosophila dishevelled. Canonical Wingless signalling is entirely normal in these deletion mutants; however, they show defects in multiple contexts controlled by noncanonical Wnt signalling, such as planar polarity. We use nuclear magnetic resonance spectroscopy to identify bona fide PDZ-binding motifs at the C termini of different polarity proteins. Although deletions of these motifs proved aphenotypic in adults, we detected changes in the proximodistal distribution of the polarity protein Flamingo (also known as Starry night) in pupal wings that suggest a modulatory role of these motifs in polarity signalling. We also provide new genetic evidence that planar polarity relies on the DEP-dependent recruitment of Dishevelled to the plasma membrane by Frizzled.

Circular RNA (circRNA) exerts a crucial role in the progression of many cancers, including hepatocellular carcinoma (HCC). However, the function of circ_0001955 in HCC progression has been poorly studied. Elucidating the role and molecular mechanism of circ_0001955 in HCC progression. Quantitative real-time PCR was employed to detect the expression of circ_0001955 and miR-646. Cell counting kit 8 assay, Ethynyl-2-deoxyuridine assay, flow cytometry, transwell assay, and tube formation assay were conducted to measure cell proliferation, metastasis, angiogenesis and apoptosis. Dual-luciferase reporter assay and biotin-labeled RNA pull-down assay were performed to analyze the interactions among circ_0001955, miR-646 and frizzled class receptor 4 (FZD4). Moreover, animal experiments were performed to examine the influence of circ_0001955 on HCC tumor growth in vivo. Circ_0001955 was a highly expressed circRNA in HCC tissues and cells. Circ_0001955 knockdown inhibited the proliferation, metastasis, angiogenesis, and enhanced the apoptosis of HCC cells. Meanwhile, miR-646 could be sponged by circ_0001955, and its inhibitor could reverse the negative regulation of circ_0001955 knockdown on HCC progression. Further, FZD4 was a target of miR-646, and its overexpression could invert the inhibition effect of miR-646 mimic on HCC progression. Besides, our results also indicated that circ_0001955 promoted FZD4 expression by sponging miR-646. Animal experiment results showed that circ_0001955 silencing restrained HCC tumor growth in vivo. Our findings suggested that circ_0001955 might play a positive role in HCC progression via regulating the miR-646/FZD4 axis, indicating that circ_0001955 might be a potential therapeutic target for HCC.

Circular RNAs (circRNA) are a key regulator of cancer progression, including colorectal cancer (CRC). Nevertheless, the role of circRASSF2 in CRC remains unclear. Quantitative real-time PCR was used to measure the expression of circRASSF2 and miR-195-5p. Cell counting kit 8 assay, colony formation assay, flow cytometry and transwell assay were used to determine the proliferation, apoptosis, migration and invasion of cells, respectively. The levels of proliferation, metastasis and Wnt/β-catenin signaling pathway-related proteins, as well as Frizzled 4 (FZD4) protein, were determined using western blot analysis. Furthermore, a dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay were used to illumine the mechanism of circRASSF2. Animal experiments were used to determine the role of circRASSF2 in the tumor growth of CRC in vivo. Our study reported that circRASSF2 was upregulated in CRC tissues and cells, and its high expression was related to the poor prognosis of CRC patients. CircRASSF2 knockdown could inhibit proliferation, migration, invasion, and enhance apoptosis in CRC cells, and its overexpression had the opposite effect. Besides, our data revealed that circRASSF2 could sponge miR-195-5p, and miR-195-5p could target FZD4. The rescue experiments indicated that both miR-195-5p inhibitor and FZD4 overexpression could reverse the negative regulation of circRASSF2 silencing on CRC progression. Moreover, circRASSF2 could positively regulate the activity of Wnt/β-catenin signaling pathway by the miR-195-5p/FZD4 axis. In addition, circRASSF2 knockdown restrained the tumor growth of CRC in vivo. Our findings suggested that circRASSF2 might function as a tumor promoter to accelerate the progression of CRC via regulating the miR-195-5p/FZD4/Wnt/β-catenin pathway.

Defining traits of platinum-tolerant cancer cells could expose new treatment vulnerabilities. Here, new markers associated with platinum-tolerant cells and tumors were identified using in vitro and in vivo ovarian cancer models treated repetitively with carboplatin and validated in human specimens. Platinum-tolerant cells and tumors were enriched in ALDH+ cells, formed more spheroids, and expressed increased levels of stemness-related transcription factors compared with parental cells. Additionally, platinum-tolerant cells and tumors exhibited expression of the Wnt receptor Frizzled-7 (FZD7). Knockdown of FZD7 improved sensitivity to platinum, decreased spheroid formation, and delayed tumor initiation. The molecular signature distinguishing FZD7+ from FZD7− cells included epithelial-to-mesenchymal (EMT), stemness, and oxidative phosphorylation-enriched gene sets. Overexpression of FZD7 activated the oncogenic factor Tp63, driving upregulation of glutathione metabolism pathways, including glutathione peroxidase 4 (GPX4), which protected cells from chemotherapy-induced oxidative stress. FZD7+ platinum-tolerant ovarian cancer cells were more sensitive and underwent ferroptosis after treatment with GPX4 inhibitors. FZD7, Tp63, and glutathione metabolism gene sets were strongly correlated in the ovarian cancer Tumor Cancer Genome Atlas (TCGA) database and in residual human ovarian cancer specimens after chemotherapy. These results support the existence of a platinum-tolerant cell population with partial cancer stem cell features, characterized by FZD7 expression and dependent on the FZD7–β-catenin–Tp63–GPX4 pathway for survival. The findings reveal a novel therapeutic vulnerability of platinum-tolerant cancer cells and provide new insight into a potential “persister cancer cell” phenotype.Significance:Frizzled-7 marks platinum-tolerant cancer cells harboring stemness features and altered glutathione metabolism that depend on GPX4 for survival and are highly susceptible to ferroptosis.

Microphthalamia-associated transcription factor (MITF) is a critical mediator in melanocyte differentiation and exerts oncogenic functions in melanoma progression. However, the role of MITF in non-small cell lung cancer (NSCLC) is still unknown. We found that MITF is dominantly expressed in the low-invasive CL1-0 lung adenocarcinoma cells and paired adjacent normal lung tissues. MITF expression is significantly associated with better overall survival and disease-free survival in NSCLC and serves as an independent prognostic marker. Silencing MITF promotes tumor cell migration, invasion and colony formation in lung adenocarcinoma cells. In xenograft mouse model, MITF knockdown enhances metastasis and tumorigenesis, but decreases angiogenesis in the Matrigel plug assay. Whole transcriptome profiling of the landscape of MITF regulation in lung adenocarcinoma indicates that MITF is involved in cell development, cell cycle, inflammation and WNT signaling pathways. Chromatin immunoprecipitation assays revealed that MITF targets the promoters of FZD7, PTGR1 and ANXA1. Moreover, silencing FZD7 reduces the invasiveness that is promoted by silencing MITF. Strikingly, MITF has significantly inverse correlations with the expression of its downstream genes in lung adenocarcinoma. In summary, we demonstrate the suppressive role of MITF in lung cancer progression, which is opposite to the canonical oncogenic function of MITF in melanoma.

Precise Norrin and β-catenin (Norrin/β-catenin; encoded by NDP and CTNNB1, respectively) signaling is critical for proper angiogenesis. Dysregulation of this signaling leads to various diseases, of which retinal exudative vitreoretinopathy is the most prevalent. Here, we used a global knockout mouse model to show that limb development membrane protein 1 like (LMBR1L), a transmembrane protein of unknown function in angiogenesis, is essential for retinal vascular development. In vitro experiments revealed that LMBR1L depletion results in aberrant activation of the Norrin/β-catenin signaling pathway via decreased ubiquitylation of FZD4 and increased Norrin co-receptor LRP5 and p-GSK3β-Ser9 expression levels, which cause accumulation of β-catenin. Moreover, inhibition of LMBR1L in human retinal microvascular endothelial cells (HRECs) caused increased proliferation ability and defective cell migration, which might have occurred as a result of upregulated expression levels of the apical junction components. Treatment with p-GSK3β-Ser9 inhibitor AR-A014418 restored the phenotypes in LMBR1L-null HRECs, which further demonstrated the important regulatory role of LMBR1L in the Norrin/β-catenin signaling pathway. Taken together, our data reveal an essential role for LMBR1L in angiogenesis. This article has an associated First Person interview with the first author of the paper.

Significance In addition to responding to directional cues, axons join one another as they extend to form highly organized projections. We show here that growth cones communicate with each other, and this communication is mediated by planar cell polarity signaling components, which are known to mediate cell–cell interactions in tissue polarization. This interaction is, in part, mediated by an intercellular interaction between Frizzled3 and Vangl2. Leucine-rich repeat kinase 2 (LRRK2), encoded by a Parkinson’s disease gene, regulates Frizzled3 phosphorylation and the intercellular interaction between Frizzled3 and Vangl2. Both loss-of-function and gain-of-function LRRK2 mutants show axon guidance defects, including those of the dopaminergic neurons, suggesting that this Parkinson’s disease gene plays important roles in growth cone–growth cone interactions during axon development., Axon–axon interactions are essential for axon guidance during nervous system wiring. However, it is unknown whether and how the growth cones communicate with each other while sensing and responding to guidance cues. We found that the Parkinson’s disease gene, leucine-rich repeat kinase 2 (LRRK2), has an unexpected role in growth cone–growth cone communication. The LRRK2 protein acts as a scaffold and induces Frizzled3 hyperphosphorylation indirectly by recruiting other kinases and also directly phosphorylates Frizzled3 on threonine 598 (T598). In LRRK1 or LRRK2 single knockout, LRRK1/2 double knockout, and LRRK2 G2019S knockin, the postcrossing spinal cord commissural axons are disorganized and showed anterior–posterior guidance errors after midline crossing. Growth cones from either LRRK2 knockout or G2019S knockin mice showed altered interactions, suggesting impaired communication. Intercellular interaction between Frizzled3 and Vangl2 is essential for planar cell polarity signaling. We show here that this interaction is regulated by phosphorylation of Frizzled3 at T598 and can be regulated by LRRK2 in a kinase activity-dependent way. In the LRRK1/2 double knockout or LRRK2 G2019S knockin, the dopaminergic axon bundle in the midbrain was significantly widened and appeared disorganized, showing aberrant posterior-directed growth. Our findings demonstrate that LRRK2 regulates growth cone–growth cone communication in axon guidance and that both loss-of-function mutation and a gain-of-function mutation (G2019S) cause axon guidance defects in development.

Background Accumulating evidence have highlighted the importance of long noncoding RNAs (lncRNAs) in multiple cancers development and progression. Cancer susceptibility candidate 9 (CASC9) is a novel long non-coding RNA and plays important regulatory role in diverse biological processes of cancers. However, the clinical significance and molecular mechanism of CASC9 in bladder cancer is still unknown. Methods Comprehensive lncRNAs profiling analysis were conducted to identify lncRNAs profile alterations and uncover valuable lncRNA candidates for bladder cancer. The expression level of CASC9 was determined in a total of 106 patients with bladder cancer. Loss-of-function experiments were performed to identify the functions of CASC9 in tumor growth and metastasis of bladder cancer in vitro and in vivo. Bioinformatics analysis and further experiments were performed to explore the molecular mechanisms underlying the functions of CASC9. Results This study found that CASC9 expression was markedly upregulated in bladder cancer and related to histological grade, TNM stage and prognosis. Knockdown of CASC9 inhibited tumor growth and metastasis of bladder cancer in vitro and in vivo. Mechanistically, we found that CASC9 functions as a miRNA sponge to positively regulate FZD6 expression and subsequently activates Wnt/β-catenin signaling pathway, thus playing an oncogenic role in bladder cancer pathogenesis. Conclusion In summary, lncRNA CASC9 plays a critical regulatory role in bladder cancer. The CASC9/miR-497-5p/ FZD6 axis provides insights for regulatory mechanism of bladder cancer, and new strategies for clinical practice.

This study aimed to investigate if wingless-related integration site (Wnt) signaling participates in the high osteogenic potential of titanium with nanotopography (Ti-Nano). We showed that among the several components of the Wnt signaling pathway, Frizzled 6 (Fzd6) was the transcript most intensely modulated by nanotopography compared with the untreated Ti surface (Ti-Machined). Then, we investigated whether and how Fzd6 participates in the regulation of osteoblast differentiation caused by nanotopography. The Fzd6 silencing with CRISPR-Cas9 transfection in MC3T3-E1 cells induced a more pronounced inhibition of osteoblast differentiation of cells cultured on nanotopography than those cultured on Ti-Machined. The analysis of the expression of calcium-calmodulin-dependent protein kinase II and β-catenin demonstrated that Fzd6 disruption inhibited the osteoblast differentiation induced by Ti-Nano by preventing the activation of Wnt/β-catenin but not that of Wnt/Ca2+ signaling, which is usually triggered by the receptor Fzd6. These findings elucidate the biological function of Fzd6 as a receptor that triggers Wnt/β-catenin signaling and the cellular mechanisms modulated by nanotopography during osteoblast differentiation.

B cell dysfunction due to obesity can be associated with alterations in the levels of micro-RNAs (miRNAs). However, the role of miRNAs in these processes remains elusive. Here, we show that miR-802 is increased in the pancreatic islets of obese mouse models and demonstrate that inducible transgenic overexpression of miR-802 in mice causes impaired insulin transcription and secretion. We identify Foxo1 as a transcription factor of miR-802 promoting its transcription, and NeuroD1 and Fzd5 as targets of miR-802-dependent silencing. Repression of NeuroD1 in β cell and primary islets impairs insulin transcription and reduction of Fzd5 in β cell, which, in turn, impairs Ca2+ signaling, thereby repressing calcium influx and decreasing insulin secretion. We functionally create a novel network between obesity and β cell dysfunction via miR-802 regulation. Elucidation of the impact of obesity on microRNA expression can broaden our understanding of pathophysiological development of diabetes., Obesity predisposes to type 2 diabetes, but the mechanisms of obesity-associated β cell dysfunction are incompletely understood. Here the authors report that obesity increases the levels of miR-802, which impairs insulin transcription and secretion by targeting NeuroD1 and Fzd5.

Wnt molecules play crucial roles in development and adult homeostasis through their receptors Frizzled proteins (Fzds). Fzds mediate canonical -catenin pathway and various noncanonical -catenin-independent pathways. Aberrant Fzd signaling is involved in many diseases including cancer. Wnt/-catenin is a well-established oncogenic pathway involved in almost every aspect of tumor development. However, Fzd-mediated noncanonical Wnt pathways function as both tumor promoters and tumor suppressors depending on cellular context. Fzd-targeted therapies have proven to be effective on cultured tumor cells, tumor cell xenografts, mouse tumor models, and patient-derived xenografts (PDX). Moreover, Fzd-targeted therapies synergize with chemotherapy in preclinical models. However, the occurrence of fragility fractures in patients treated with Fzd-targeted agents such as OMP-54F28 and OMP-18R5 limits the development of this combination. Along with new insights on signaling, roles, and modulation mechanisms of Fzds in human tumors, more Fzd-related therapeutic targets will be developed.

Frizzled-2 plays an important role in maintaining normal hepatic cell functionality. This study aimed to investigate the role of inhibition of Frizzled-2 in protecting rat liver BRL-3A cells from Hypoxia/Reoxygenation (H/R). In vitro H/R hepatic cell model was established by culturing BRL-3A cells under H/R condition. Frizzled-2 siRNA was transfected into BRL-3A cells to inhibit Frizzled-2 signaling. Wnt5a and Frizzled-2 were significantly increased in BRL-3A cells upon H/R treatment. H/R treatment induced cell cytotoxicity, the early apoptosis rate and the intracellular Ca2+ level in BRL-3A cells while silencing frizzled-2 gene decreased the H/R induced cell cytotoxicity, apoptosis and intracellular Ca2+ level. In vivo mice study further showed the up-regulation of Frizzled-2/Wnt 5 pathway and cleaved Caspase-3 expression in liver tissues under ischemia and reperfusion injury (IRI). In summary, inhibition of Frizzled-2 by its siRNA may protects BRL-3A cells by attenuating the H/R induced cell cytotoxicity and apoptosis.

The planar cell polarity (PCP) signaling pathway is a potent developmental regulator of directional cell behaviors such as migration, asymmetric division and morphological polarization that are critical for shaping the body axis and the complex three-dimensional architecture of tissues and organs. PCP is considered a noncanonical Wnt pathway due to the involvement of Wnt ligands and Frizzled family receptors in the absence of the beta-catenin driven gene expression observed in the canonical Wnt cascade. At the heart of the PCP mechanism are protein complexes capable of generating molecular asymmetries within cells along a tissue-wide axis that are translated into polarized actin and microtubule cytoskeletal dynamics. PCP has emerged as an important regulator of developmental, homeostatic and disease processes in the respiratory system. It acts along other signaling pathways to create the elaborately branched structure of the lung by controlling the directional protrusive movements of cells during branching morphogenesis. PCP operates in the airway epithelium to establish and maintain the orientation of respiratory cilia along the airway axis for anatomically directed mucociliary clearance. It also regulates the establishment of the pulmonary vasculature. In adult tissues, PCP dysfunction has been linked to a variety of chronic lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease, and idiopathic pulmonary arterial hypertension, stemming chiefly from the breakdown of proper tissue structure and function and aberrant cell migration during regenerative wound healing. A better understanding of these (impaired) PCP mechanisms is needed to fully harness the therapeutic opportunities of targeting PCP in chronic lung diseases.

The activation of the Wnt/β-catenin signaling pathway is critical for skeletal development but surprisingly little is known about the requirements for the specific frizzled (Fzd) receptors that recognize Wnt ligands. To define the contributions of individual Fzd proteins to osteoblast function, we profiled the expression of all 10 mammalian receptors during calvarial osteoblast differentiation. Expression of Fzd4 was highly upregulated during in vitro differentiation and therefore targeted for further study. Mice lacking Fzd4 in mature osteoblasts had normal cortical bone structure but reduced cortical tissue mineral density and also exhibited an impairment in the femoral trabecular bone acquisition that was secondary to a defect in the mineralization process. Consistent with this observation, matrix mineralization, markers of osteoblastic differentiation, and the ability of Wnt3a to stimulate the accumulation of β-catenin were reduced in cultures of calvarial osteoblasts deficient for Fzd4. Interestingly, Fzd4-deficient osteoblasts exhibited an increase in the expression of Fzd8 both in vitro and in vivo, which suggests that the two receptors may exhibit overlapping functions. Indeed, ablating a single Fzd8 allele in osteoblast-specific Fzd4 mutants produced a more severe effect on bone acquisition. Taken together, our data indicate that Fzd4 is required for normal bone development and mineralization despite compensation from Fzd8.

Despite the experimental evidence pointing to a significant role of the Wnt family of proteins in physiological and pathological rodent spinal cord functioning, its potential relevance in the healthy and traumatically injured human spinal cord as well as its therapeutic potential in spinal cord injury (SCI) are still poorly understood. To get further insight into these interesting issues, we first demonstrated by quantitative Real-Time PCR and simple immunohistochemistry that detectable mRNA expression of most Wnt components, as well as protein expression of all known Wnt receptors, can be found in the healthy human spinal cord, supporting its potential involvement in human spinal cord physiology. Moreover, evaluation of Frizzled (Fz) 1 expression by double immunohistochemistry showed that its spatio-temporal and cellular expression pattern in the traumatically injured human spinal cord is equivalent to that observed in a clinically relevant model of rat SCI and suggests its potential involvement in SCI progression/outcome. Accordingly, we found that long-term lentiviral-mediated overexpression of the Fz1 ligand Wnt1 after rat SCI improves motor functional recovery, increases myelin preservation and neuronal survival, and reduces early astroglial reactivity and NG2+ cell accumulation, highlighting the therapeutic potential of Wnt1 in this neuropathological situation.

We recently published that type 2 diabetes promotes cell centrosome amplification via upregulation of Rho-associated protein kinase 1 (ROCK1) and 14-3-3 protein-σ (14-3-3σ). This study further investigates the molecular mechanisms underlying diabetes-associated centrosome amplification. We found that treatment of cells with high glucose, insulin, and palmitic acid levels increased the intracellular and extracellular protein levels of Wingless-type MMTV integration site family member 6 (Wnt6) as well as the cellular level of β-catenin. The treatment also activated β-catenin and promoted its nuclear translocation. Treatment of cells with siRNA species for Wnt6, Frizzled-4 (FZD4), or β-catenin as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture medium could all attenuate the treatment-triggered centrosome amplification. Moreover, we showed that secreted Wnt6-FZD4-β-catenin was the signaling pathway that was upstream of ROCK1 and 14-3-3σ. We found that advanced glycation end products (AGEs) were also able to increase the cellular and extracellular levels of Wnt6, the cellular protein level of β-catenin, and centrosome amplification. Treatment of the cells with siRNA species for Wnt6 or FZD4 as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture could all inhibit the AGEs-elicited centrosome amplification. In colon tissues from a diabetic mouse model, the protein levels of Wnt6 and 14-3-3σ were increased. In conclusion, our results showed that the pathophysiological factors in type 2 diabetes, including AGEs, were able to induce centrosome amplification. It is suggested that secreted Wnt6 binds to FZD4 to activate the canonical Wnt6 signaling pathway, which is upstream of ROCK1 and 14-3-3σ, and that this is the cell signaling pathway underlying diabetes-associated centrosome amplification.

The blood-brain barrier (BBB) protects the central nervous system (CNS) from harmful blood-borne factors. Although BBB dysfunction is a hallmark of several neurological disorders, therapies to restore BBB function are lacking. An attractive strategy is to repurpose developmental BBB regulators, such as Wnt7a, into BBB-protective agents. However, safe therapeutic use of Wnt ligands is complicated by their pleiotropic Frizzled signaling activities. Taking advantage of the Wnt7a/b-specific Gpr124/Reck co-receptor complex, we genetically engineered Wnt7a ligands into BBB-specific Wnt activators. In a “hit-and-run” adeno-associated virus–assisted CNS gene delivery setting, these new Gpr124/Reck-specific agonists protected BBB function, thereby mitigating glioblastoma expansion and ischemic stroke infarction. This work reveals that the signaling specificity of Wnt ligands is adjustable and defines a modality to treat CNS disorders by normalizing the BBB.

The mammalian tongue is a highly specialized muscular organ. The Wnt5a ligand regulates muscle development by mediating the activation of several noncanonical Wnt signaling pathways in a receptor context-dependent fashion. However, there is poor information on the expression and behavior of Wnt5a proteins during muscle development of the embryonic tongue.The spatiotemporal distribution profiles of the Wnt5a ligand and its receptors, receptor tyrosine kinase-like orphan receptor 2 (Ror2), Frizzled2 (Fzd2), and Frizzled5 (Fzd5), in the developing tongue muscles of prenatal mice from embryonic day 12.5-18.5 were analyzed using immunofluorescence (IF) double staining of a target protein and desmin, a marker protein of myogenic cells. Immunolabeling images were subjected to digital detection analysis using the WinROOF 2018 version 4.19.0 image processing software when needed.IF signals of the Wnt5a ligand protein and its receptors Ror2 and Fzd2 were detected in developing myoblasts and myotubes of the embryonic tongue, but they were undetectable in mature myofibers equipped with sarcomere structures. Fzd2 expression was specific for desmin-positive developing muscle cells, whereas those of Ror2 and the Wnt5a ligand were widespread and nonselective for desmin-positive cells and that of Fzd5 was predominant in desmin-negative cells of the epithelium and subepithelial mesenchyme.Developing muscle cells but not mature myofibers of the mouse embryonic tongue express the Wnt5a ligand and its receptors Ror2 and Fzd2, which may mediate Wnt5a signaling in the development processes of tongue muscle fibers.

Emerging evidence indicates that circRNAs are broadly expressed in osteosarcoma (OS) cells and play a crucial role in OS progression. Recently, cancer-specific circRNA circPRKAR1B has been identified by high-throughput sequencing and is recorded in publicly available databases. Nevertheless, the detailed functions and underlying mechanisms of circPRKAR1B in OS remains poorly understood. By functional experiments, we found that circPRKAR1B enhanced OS cell proliferation, migration, and promotes OS epithelial–mesenchymal transition (EMT). Mechanistic investigations suggested that circPRKAR1B promotes OS progression through sponging miR-361-3p to modulate the expression of FZD4. Subsequently, we identified that EIF4A3 promoted cirPRKAR1B formation through binding to the downstream target of circPRKAR1B on PRKAR1B mRNA. Further rescue study revealed that overexpression of the Wnt signalling could impair the onco-suppressor activities of the silencing of circPRKAR1B. Interestingly, further experiments indicated that circPRKAR1B is involved in the sensitivity of chemoresistance in OS. On the whole, our results demonstrated that circPRKAR1B exerted oncogenic roles in OS and suggested the circPRKAR1B/miR-361-3p/FZD4 axis plays an important role in OS progression and might be a potential therapeutic target.

Background Osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is closely associated with bone diseases. Circular RNAs are reported to be involved in BMSC differentiation. CircSmg5 (circ_0001145) has been identified to be downregulated in an osteoporosis mouse model. In this study, we aimed to explore the function and regulatory mechanism of circSmg5 in BMSC osteogenic differentiation. Methods The Alizarin Red staining and alkaline phosphatase staining assays were performed to explore the osteogenic differentiation of BMSCs. The interaction between circ_0001145, miR-194-5p, and frizzled class receptor 6 (Fzd6) was analyzed by luciferase reporter assay. The nuclear translocation of β-catenin was assessed using immunofluorescence staining. Results CircSmg5 is in stable circular structure. CircSmg5 expression was elevated in the process of BMSC osteogenic differentiation. CircSmg5 overexpression promoted the osteogenic differentiation of BMSCs. CircSmg5 bound with miR-194-5p, whose expression was decreased in the osteogenic differentiation of BMSCs. MiR-194-5p directly targeted the 3'UTR of Fzd6. The mRNA and protein levels of Fzd6 were positively modulated by circSmg5 and negatively regulated by miR-194-5p in BMSCs. Conclusion CircSmg5 was demonstrated to promote the BMSC osteogenic differentiation by targeting the miR-194-5p/Fzd6 axis to activate the Wnt/β-catenin signaling.

Oligodendrocyte (OL) maturation arrest in human white matter injury contributes significantly to the failure of endogenous remyelination in multiple sclerosis (MS) and newborn brain injuries such as hypoxic ischemic encephalopathy (HIE) that cause cerebral palsy. In this study, we identify an oligodendroglial-intrinsic factor that controls OL maturation specifically in the setting of injury. We find a requirement for the ring finger protein Rnf43 not in normal development but in neonatal hypoxic injury and remyelination in the adult mammalian CNS. Rnf43, but not the related Znrf3, is potently activated by Wnt signaling in OL progenitor cells (OPCs) and marks activated OPCs in human MS and HIE. Rnf43 is required in an injury-specific context, and it promotes OPC differentiation through negative regulation of Wnt signal strength in OPCs at the level of Fzd1 receptor presentation on the cell surface. Inhibition of Fzd1 using UM206 promotes remyelination following exvivo and invivo demyelinating injury.

Previous studies have demonstrated miRNAs derived from plants and parasites can modulate mammalian gene expression and cell phenotype in a cross-kingdom manner, leading to occurrence of diseases or strengthening resistance of host to diseases such as cancer. In this study, we identified a schistosome miRNA (named Sja-miR-71a) through screening of 57

Background Destruction of blood–brain barrier (BBB) ​​is one of the main mechanisms of secondary brain injury following intracerebral hemorrhage (ICH). Frizzled-7 is a key protein expressed on the surface of endothelial cells that controls vascular permeability through the Wnt-canonical pathway involving WNT1-inducible signaling pathway protein 1 (WISPI). This study aimed to investigate the role of Frizzled-7 signaling in BBB preservation after ICH in mice. Methods Adult CD1 mice were subjected to sham surgery or collagenase-induced ICH. Frizzled-7 activation or knockdown was performed by administration of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) by intracerebroventricular injection at 48 h before ICH induction. WISP1 activation or WISP1 knockdown was performed to evaluate the underlying signaling pathway. Post-ICH assessments included neurobehavior, brain edema, BBB permeability, hemoglobin level, western blot and immunofluorescence. Results The brain expressions of Frizzled-7 and WISP1 significantly increased post-ICH. Frizzled-7 was expressed in endothelial cells, astrocytes, and neurons after ICH. Activation of Frizzled-7 significantly improved neurological function, reduced brain water content and attenuated BBB permeability to large molecular weight substances after ICH. Whereas, knockdown of Frizzled-7 worsened neurological function and brain edema after ICH. Activation of Frizzled-7 significantly increased the expressions of Dvl, β-Catenin, WISP1, VE-Cadherin, Claudin-5, ZO-1 and reduced the expression of phospho-β-Catenin. WISP1 knockdown abolished the effects of Frizzled-7 activation on the expressions of VE-Cadherin, Claudin-5 and ZO-1 at 24 h after ICH. Conclusions Frizzled-7 activation potentially attenuated BBB permeability and improved neurological deficits after ICH through Dvl​​/β-Catenin/WISP1 pathway. Frizzled-7 may be a potential target for the development of ICH therapeutic drugs.

Progressive cardiac fibrosis accelerates the development of heart failure. Here, we aimed to explore serum Wnt5a and Wnt11 levels in hypertension patients, the roles of Wnt5a and Wnt11 in cardiac fibrosis and potential mechanisms under pressure overload. The pressure overload mouse model was built by transverse aortic constriction (TAC). Cardiac fibrosis was analyzed by Masson’s staining. Serum Wnt5a or Wnt11 was elevated and associated with diastolic dysfunction in hypertension patients. TAC enhanced the expression and secretion of Wnt5a or Wnt11 from cardiomyocytes (CMs), cardiac fibroblasts (CFs), and cardiac microvascular endothelial cells (CMECs). Knockdown of Wnt5a and Wnt11 greatly improved cardiac fibrosis and function at 4 weeks after TAC. In vitro, shWnt5a or shWnt11 lentivirus transfection inhibited pro-fibrotic effects in CFs under mechanical stretch (MS). Similarly, conditional medium from stretched-CMs transfected with shWnt5a or shWnt11 lentivirus significantly suppressed the pro-fibrotic effects induced by conditional medium from stretched-CMs. These data suggested that CMs- or CFs-derived Wnt5a or Wnt11 showed a pro-fibrotic effect under pressure overload. In vitro, exogenous Wnt5a or Wnt11 activated ERK and p38 (fibrotic-related signaling) pathway, promoted the phosphorylation of EGFR, and increased the expression of Frizzled 5 (FZD5) in CFs. Inhibition or knockdown of EGFR greatly attenuated the increased FZD5, p-p38, and p-ERK levels, and the pro-fibrotic effect induced by Wnt5a or Wnt11 in CFs. Si-FZD5 transfection suppressed the increased p-EGFR level, and the fibrotic-related effects in CFs treated with Wnt5a or Wnt11. In conclusion, pressure overload enhances the secretion of Wnt5a or Wnt11 from CMs and CFs which promotes cardiac fibrosis by activation the crosstalk of FZD5 and EGFR. Thus, Wnt5a or Wnt11 may be a novel therapeutic target for the prevention of cardiac fibrosis under pressure overload.

The collective polarization of cellular structures and behaviors across a tissue plane is a near universal feature of epithelia known as planar cell polarity (PCP). This property is controlled by the core PCP pathway, which consists of highly conserved membrane-associated protein complexes that localize asymmetrically at cell junctions. Here, we introduce three new mouse models for investigating the localization and dynamics of transmembrane PCP proteins: Celsr1, Fz6 and Vangl2. Using the skin epidermis as a model, we characterize and verify the expression, localization and function of endogenously tagged Celsr1-3xGFP, Fz6-3xGFP and tdTomato-Vangl2 fusion proteins. Live imaging of Fz6-3xGFP in basal epidermal progenitors reveals that the polarity of the tissue is not fixed through time. Rather, asymmetry dynamically shifts during cell rearrangements and divisions, while global, average polarity of the tissue is preserved. We show using super-resolution STED imaging that Fz6-3xGFP and tdTomato-Vangl2 can be resolved, enabling us to observe their complex localization along junctions. We further explore PCP fusion protein localization in the trachea and neural tube, and discover new patterns of PCP expression and localization throughout the mouse embryo.

MicroRNAs are small, non-coding RNAs that regulate the expression of target genes. Previous research has demonstrated that microRNA-200a regulates cell apoptosis, tumor progression, and autoimmune disease. Preliminary studies found that microRNA-200a was differently expressed in the skin of Cashmere goats of various coat colors. However, the role of microRNA-200a in skin pigmentation remained poorly understood. In the current study, we investigated the effect of microRNA-200a on pigmentation in Cashmere goats. The expression of target genes was detected by real-time quantitative PCR, western blot, and immunohistochemistry staining both in vivo and in vitro. Luciferase reporter assays were used to demonstrate the relationship between microRNA-200a and its target genes Wnt family member 5A and frizzled class receptor 4 (WNT5A and FZD4) in HEK293T cells. BALB/c mice were injected with antagomiR-200a to detect melanin content and the expression of microRNA-200a and its target genes. The results demonstrated that the expression of microRNA-200a was significantly higher in brown tissue. Luciferase reporter assays confirmed that microRNA-200a targeted WNT5A and FZD4. The expression of WNT5A and FZD4 in the skin of brown Cashmere goats was significantly lower than that in white Cashmere goats by the detection of mRNA and protein levels. Overexpression/inhibition of microRNA-200a in keratinocytes decreased/increased the mRNA and protein expression of WNT5A and FZD4, respectively. In addition, the expression of WNT5A and FZD4 increased in the skin of BALB/c mice injected with antagomiR-200a, but the melanin content decreased. In summary, this study indicated that microRNA-200a regulates skin pigmentation by targeting WNT5A and FZD4 in Cashmere goats.

Wnt signaling is critical for proper development of the embryo and for tissue homeostasis in the adult. Activation of this signaling cascade is initiated by binding of the secreted Wnts to their receptors. With the mammalian genome encoding multiple Wnts and Wnt receptors, a longstanding question in the field has been how Wnt-receptor specificities are achieved. Emerging from these studies is a picture of exquisite control over Wnt protein production, secretion, distribution, and receptor interactions, culminating in activation of downstream signaling cascades that control a myriad of biological processes. Here we discuss mechanisms by which Wnt protein activities are tuned and illustrate how the multiple layers of regulation can be leveraged for therapeutic interventions in disease.

Planar cell polarity (PCP) regulates the orientation of external structures. A core group of proteins that includes Frizzled forms the heart of the PCP regulatory system. Other PCP mechanisms that are independent of the core group likely exist, but their underlying mechanisms are elusive. Here, we show that tissue flow is a mechanism governing core group-independent PCP on the Drosophila notum. Loss of core group function only slightly affects bristle orientation in the adult central notum. This near-normal PCP results from tissue flow-mediated rescue of random bristle orientation during the pupal stage. Manipulation studies suggest that tissue flow can orient bristles in the opposite direction to the flow. This process is independent of the core group and implies that the apical extracellular matrix functions like a “comb” to align bristles. Our results reveal the significance of cooperation between tissue dynamics and extracellular substances in PCP establishment.

Frizzled

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by joint inflammation, synovial hyperplasia, cartilage degeneration, bone erosion, and pannus. Immunoglobulin D (IgD) plays an important role in autoimmune diseases although the content of it in vivo is low. Increased concentrations of anti-IgD autoantibodies have been detected in many RA patients. IgD-Fc-Ig fusion protein is constructed by connecting human IgD Fc domain and IgG1 Fc domain, which specifically block the IgD/ IgDR pathway and regulate the function of cells expressing IgDR to treat RA. The expression levels of Wnt5A and Frizzled 5 are higher in RA synovial tissue specimens. The complex of Wnt5A-Fzd5-LRP5/6-CTHRC1 promotes the expression of hypoxia inducible factor-1α by activating nuclear factor kappa-B (NF-κB), leading to high expression of VEGF and participating in angiogenesis. VEGF is the strongest angiogenic factor found so far. Here, we aimed to explore whether IgD participates in synovitis by binding to IgDR and regulating the activation of Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway in fibroblast synovial cells (FLSs), whether IgD-Fc-Ig fusion protein inhibits VEGF production in FLS of CIA and explore mechanism. We found that IgDR is expressed on MH7A and FLS. IgD promotes VEGF expression by activating Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway in MH7A and FLS. After activation of Fzd5 with Wnt5A, IgD-Fc-Ig reduced VEGF-A level in the culture supernatant of MH7A stimulation by IgD. The expressions of CTHRC1, Fzd5, p-P65 and VEGF in MH7A and FLSs were down-regulated after IgD-Fc-Ig treatment. IgD-Fc-Ig suppressed the combination of CTHRC1 and Fzd5 as well. By using the animal model, we demonstrated that IgD-Fc-Ig suppress ankle CTHRC1 and Fzd5 production resulted in inhibition of index of joint inflammation of CIA rats, which were consistent with vitro results. Conclusively, IgD-Fc-Ig inhibits IgD and Wnt5A-induced angiogenesis and joint inflammation by suppressing the combination of CTHRC1 and Fzd5. Our results show that IgD-Fc-Ig exerts its suppressive effect on IgD and Wnt5A by Wnt5A-Fzd5-CTHRC1-NF-κB signaling pathway.