Dishevelled2 activates WGEF via its interaction with a unique internal peptide motif of the GEF.

The Wnt-planar cell polarity (Wnt-PCP) pathway is crucial in establishing cell polarity during development and tissue homoeostasis. This pathway is found to be dysregulated in many pathological conditions, including cancer and autoimmune disorders. The central event in Wnt-PCP pathway is the activation of Weak-similarity guanine nucleotide exchange factor (WGEF) by the adapter protein Dishevelled (Dvl). The PDZ domain of Dishevelled2 (Dvl2PDZ) binds and activates WGEF by releasing it from its autoinhibitory state. However, the actual Dvl2PDZ binding site of WGEF and the consequent activation mechanism of the GEF have remained elusive. Using biochemical and molecular dynamics studies, we show that a unique "internal-PDZ binding motif" (IPM) of WGEF mediates the WGEF-Dvl2PDZ interaction to activate the GEF. The residues at P2, P0, P-2 and P-3 positions of IPM play an important role in stabilizing the WGEFpep-Dvl2PDZ interaction. Furthermore, MD simulations of modelled Dvl2PDZ-WGEFIPM peptide complexes suggest that WGEF-Dvl2PDZ interaction may differ from the reported Dvl2PDZ-IPM interactions. Additionally, the apo structure of human Dvl2PDZ shows conformational dynamics different from its IPM peptide bound state, suggesting an induced fit mechanism for the Dvl2PDZ-peptide interaction. The current study provides a model for Dvl2 induced activation of WGEF.

A vertebrate Vangl2 translational variant required for planar cell polarity.

First described in the milkweed bug Oncopeltus fasciatus, planar cell polarity (PCP) is a developmental process essential for embryogenesis and development of polarized structures in Metazoans. This signaling pathway involves a set of evolutionarily conserved genes encoding transmembrane (Vangl, Frizzled, Celsr) and cytoplasmic (Prickle, Dishevelled) molecules. Vangl2 is of major importance in embryonic development as illustrated by its pivotal role during neural tube closure in human, mouse, Xenopus, and zebrafish embryos. Here, we report on the molecular and functional characterization of a Vangl2 isoform, Vangl2-Long, containing an N-terminal extension of about 50 aa, which arises from an alternative near-cognate AUA translation initiation site, lying upstream of the conventional start codon. While missing in Vangl1 paralogs and in all invertebrates, including Drosophila, this N-terminal extension is conserved in all vertebrate Vangl2 sequences. We show that Vangl2-Long belongs to a multimeric complex with Vangl1 and Vangl2. Using morpholino oligonucleotides to specifically knockdown Vangl2-Long in Xenopus, we found that this isoform is functional and required for embryo extension and neural tube closure. Furthermore, both Vangl2 and Vangl2-Long must be correctly expressed for the polarized distribution of the PCP molecules Pk2 and Dvl1 and for centriole rotational polarity in ciliated epidermal cells. Altogether, our study suggests that Vangl2-Long significantly contributes to the pool of Vangl2 molecules present at the plasma membrane to maintain PCP in vertebrate tissues.

Ginsenoside Rg3 suppresses vasculogenic mimicry by impairing DVL3-maintained stemness via PAAD cell-derived exosomal miR-204 in pancreatic adenocarcinoma.

BACKGROUND: Vasculogenic mimicry (VM) is an angiogenesis-independent process that potentially contributes to the poor clinical outcome of anti-angiogenesis therapy in multiple malignant cancers, including pancreatic adenocarcinoma (PAAD). Several studies have shown that ginsenoside Rg3, a bioactive component of ginseng, holds considerable potential for cancer treatment. Our previous work has proved that Rg3 can inhibit VM formation in PAAD. However, its underlying mechanism remains unclear. PURPOSE: To explore the underlying mechanism by which Rg3 affects VM formation in PAAD. METHODS: We first investigated the effects of Rg3 on the cellular phenotypes of two PAAD cell lines (SW-1990 and PCI-35), and the expression of EMT- and stemness-related proteins. SW-1990 cells were adopted to construct xenograft models, and the anti-tumor effects of Rg3 in vivo were validated. Subsequently, we isolated the exosomes from the two PAAD cell lines with Rg3 treatment or not, and explored whether Rg3 regulated VM via PAAD cell-derived exosomes. MiRNA sequencing, clinical analysis, and rescue experiments were performed to investigate whether and which miRNA was involved. Subsequently, the target gene of miRNA was predicted using the miRDB website (https://mirdb.org/), and rescue experiments were further conducted to validate those in vitro and in vivo. RESULTS: Rg3 indeed exhibited excellent anti-tumor effects both in vitro and in vivo, with inhibitory effects on EMT and stemness of PAAD cells. More interestingly, Rg3-treated PAAD cell-derived exosomes suppressed the tube-forming ability of HUVEC and PAAD cells, with a decrease in stemness-related protein expression, indicating that Rg3 inhibited both angiogenesis and VM processes. Subsequently, we found that Rg3 induced the up-regulation of miR-204 in PAAD cell-derived exosomes, and miR-204 alone inhibited tube and sphere formation abilities of PAAD cells like exosomes. Specifically, miR-204 down-regulated DVL3 expression, which was involved in regulating cancer cell stemness, and ultimately affected VM. The in vivo experiments further indicated that Rg3-treated SW-1990 cell-derived exosome-inhibited tumor growth, VM formation, and stemness-related protein expression can be abrogated by DVL3 overexpression. CONCLUSION: Ginsenoside Rg3 increased the PAAD cell-derived exosomal miR-204 levels, which subsequently inhibited its target genes DVL3 expression in the receptor PAAD cells, and the down-regulated DVL3 broke stemness maintenance, ultimately suppressing VM formation of PAAD. Our findings revealed a novel mechanism by which Rg3 exerted its anti-tumor activity in PAAD via inhibiting VM, and provided a promising strategy to make up for the deficiency of anti-angiogenesis therapy in cancer.

A Van Gogh/Vangl tyrosine phosphorylation switch regulates its interaction with core Planar Cell Polarity factors Prickle and Dishevelled.

Epithelial tissues can be polarized along two axes: in addition to apical-basal polarity they are often also polarized within the plane of the epithelium, known as planar cell polarity (PCP). PCP depends upon the conserved Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl in mammals). Here, taking advantage of the complementary features of Drosophila wing and mouse skin PCP establishment, we dissect how Vang/Vangl phosphorylation on a specific conserved tyrosine residue affects its interaction with two cytoplasmic core PCP factors, Dishevelled (Dsh/Dvl1-3 in mammals) and Prickle (Pk/Pk1-3). We demonstrate that Pk and Dsh/Dvl bind to Vang/Vangl in an overlapping region centered around this tyrosine. Strikingly, Vang/Vangl phosphorylation promotes its binding to Prickle, a key effector of the Vang/Vangl complex, and inhibits its interaction with Dishevelled. Thus phosphorylation of this tyrosine appears to promote the formation of the mature Vang/Vangl-Pk complex during PCP establishment and conversely it inhibits the Vang interaction with the antagonistic effector Dishevelled. Intriguingly, the phosphorylation state of this tyrosine might thus serve as a switch between transient interactions with Dishevelled and stable formation of Vang-Pk complexes during PCP establishment.

Disheveled3 enhanced EMT and cancer stem-like cells properties via Wnt/ß-catenin/c-Myc/SOX2 pathway in colorectal cancer.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) and cancer stem-like cells (CSLCs) play crucial role in tumor metastasis and drug-resistance. Disheveled3 (DVL3) is involved in malignant behaviors of cancer. However, the role and potential mechanism of DVL3 remain elusive in EMT and CSLCs of colorectal cancer (CRC). METHODS: UALCAN and PrognoScan databases were employed to evaluate DVL3 expression in CRC tissues and its correlation with CRC prognosis, respectively. Transwell, sphere formation and CCK8 assay were used to assess metastasis, stemness and drug sensitivity of CRC cells, respectively. Western blotting and dual luciferase assay were performed to analyze the protein expression and Wnt/ß-catenin activation, respectively. Lentiviral transfection was used to construct the stable cell lines. Animal studies were performed to analyze the effect of silencing DVL3 on tumorigenicity and metastasis of CRC cells in vivo. RESULTS: DVL3 was overexpressed in CRC tissues and several CRC cell lines. DVL3 expression was also higher in CRC tissues with lymph node metastasis than tumor tissues without metastasis, and correlated with poor prognosis of CRC patients. DVL3 positively regulated the abilities of migration, invasion and EMT-like molecular changes in CRC cells. Moreover, DVL3 promoted CSLCs properties and multidrug resistance. We further identified that Wnt/ß-catenin was crucial for DVL3-mediated EMT, stemness and SOX2 expression, while silencing SOX2 inhibited DVL3-mediated EMT and stemness. Furthermore, c-Myc, a direct target gene of Wnt/ß-catenin, was required for SOX2 expression and strengthened EMT and stemness via SOX2 in CRC cells. Finally, knockdown of DVL3 suppressed tumorigenicity and lung metastasis of CRC cells in nude mice. CONCLUSION: DVL3 promoted EMT and CSLCs properties of CRC via Wnt/ß-catenin/c-Myc/SOX2 axis, providing a new strategy for successful CRC treatment.

Dishevelled segment polarity protein 2 promotes gastric cancer progression through Wnt/ß-catenin pathway.

Dishevelled family proteins (DVL1-3), key scaffold proteins, act on canonical and non-canonical Wnt/ß-catenin signaling pathway. DVL has been implicated in various tumor progression. However, its role and underlying mechanisms in gastric cancer (GC) remain unclear. The aim of this study was to investigate the role of DVL in GC development using cell lines and 209 GC specimens. We analyzed three orthologs of DVL in GC tissues and paired adjacent non-tumor tissues, and only DVL2 is highly expressed in GC tissues. We also analyzed clinicopathological data on DVL2 expression in gastric cancer specimens. In immunohistochemistry, DVL2 expression was up-regulated in GC tissues compared with paired adjacent non-tumor tissues (153/209, 73.2%). DVL2 expression level was significantly correlated with many clinicopathological parameters such as T stage (P < 0.001) and N stage (P < 0.001). Survival analysis showed that the overall survival (OS) of patients with high expression of DVL2 was significantly shorter than those with low expression. Multivariate Cox regression analysis revealed that DVL2 expression was an important and independent prognostic factor for gastric cancer patients (P = 0.011, HR=1.78, 95%CI (1.14-2.79). Depletion of endogenous DVL2 using short hairpin RNA (shRNA) inhibited GC cell proliferation, migration, and invasion. The abnormal activation of Wnt/ß-catenin signaling pathway is mainly achieved through the abnormal expression of DVL2. DVL2 is highly expressed in gastric cancer tissues, which may be a new independent risk factor for the prognosis of gastric cancer patients. In gastric cancer, DVL2 overexpression plays a crucial role in the occurrence and development of gastric cancer, so it may become a new, effective and complementary therapeutic target for gastric cancer.

Mechanistic studies in Drosophila and chicken give new insights into functions of DVL1 in dominant Robinow syndrome.

The study of rare genetic diseases provides valuable insights into human gene function. The autosomal dominant or autosomal recessive forms of Robinow syndrome are genetically heterogeneous, and the common theme is that all the mutations lie in genes in Wnt signaling pathways. Cases diagnosed with Robinow syndrome do survive to adulthood with distinct skeletal phenotypes, including limb shortening and craniofacial abnormalities. Here, we focus on mutations in dishevelled 1 (DVL1), an intracellular adaptor protein that is required for both canonical (ß-catenin-dependent) or non-canonical (requiring small GTPases and JNK) Wnt signaling. We expressed human wild-type DVL1 or DVL1 variants alongside the endogenous genome of chicken and Drosophila. This design is strategically suited to test for functional differences between mutant and wild-type human proteins in relevant developmental contexts. The expression of variant forms of DVL1 produced a major disorganization of cartilage and Drosophila wing morphology compared to expression of wild-type DVL1. Moreover, the variants caused a loss of canonical and gain of non-canonical Wnt signaling in several assays. Our data point to future therapies that might correct the levels of Wnt signaling, thus improving skeletal growth.

The Wnt pathway protein Dvl1 targets somatostatin receptor 2 for lysosome-dependent degradation.

The Somatostatin receptor 2 (Sstr2) is a heterotrimeric G protein-coupled receptor that is highly expressed in neuroendocrine tumors and is a common pharmacological target for intervention. Unfortunately, not all neuroendocrine tumors express Sstr2, and Sstr2 expression can be downregulated with prolonged agonist use. Sstr2 is rapidly internalized following agonist stimulation and, in the short term, is quantitatively recycled back to the plasma membrane. However, mechanisms controlling steady state expression of Sstr2 in the absence of agonist are less well described. Here, we show that Sstr2 interacts with the Wnt pathway protein Dvl1 in a ligand-independent manner to target Sstr2 for lysosomal degradation. Interaction of Sstr2 with Dvl1 does not affect receptor internalization, recycling, or signaling to adenylyl cyclase but does suppress agonist-stimulated ERK1/2 activation. Importantly, Dvl1-dependent degradation of Sstr2 can be stimulated by overexpression of Wnts and treatment of cells with Wnt pathway inhibitors can boost Sstr2 expression in neuroendocrine tumor cells. Taken together, this study identifies for the first time a mechanism that targets Sstr2 for lysosomal degradation that is independent of Sstr2 agonist and can be potentiated by Wnt ligand. Intervention in this signaling mechanism has the potential to elevate Sstr2 expression in neuroendocrine tumors and enhance Sstr2-directed therapies.

Hyperoxia exposure upregulates Dvl-1 and activates Wnt/ß-catenin signaling pathway in newborn rat lung.

BACKGROUND: Bronchopulmonary dysplasia is a serious and lifelong pulmonary disease in premature neonates that influences around one-quarter of premature newborns. The wingless-related integration site /ß-catenin signaling pathway, which is abnormally activated in the lungs with pulmonary fibrosis, affects cell differentiation and lung development. METHODS: Newborn rats were subjected to hyperoxia exposure. Histopathological changes to the lungs were evaluated through immunohistochemistry, and the activation of disheveled and Wnt /ß-catenin signaling pathway components was assessed by Western blotting and real-time PCR. The abilities of proliferation, apoptosis and migration were detected by Cell Counting Kit-8, flow cytometry and scratch wound assay, respectively. RESULTS: Contrasting with normoxic lungs, hyperoxia-exposed lungs demonstrated larger alveoli, fewer alveoli and thicker alveolar septa. Superoxide dismutase activity was significantly decreased (7th day: P < 0.05; 14th day: P < 0.01) and malondialdehyde significantly increased (7th day: P < 0.05; 14th day: P < 0.01) after hyperoxia exposure. Protein and mRNA expression levels of ß-catenin, Dvl-1, CTNNBL1 and cyclin D1 were significantly upregulated by hyperoxia exposure on 7th day (P < 0.01) and 14th day (P < 0.01). In hyperoxic conditions, Dvl-l downregulation and Dvl-l downregulation + MSAB treatment significantly increased the proliferation rates, decreased the apoptosis rates and improved the ability of cell migration. In hyperoxic conditions, Dvl-l downregulation could decrease the mRNA expression levels of GSK3ß, ß-catenin, CTNNBL1 and cyclin D1 and decrease the protein relative expression levels of GSK3ß, p-GSK3ß, ß-catenin, CTNNBL1 and cyclin D1. CONCLUSIONS: We confirmed the positive role of Dvl-1 and the Wnt/ß-catenin signaling pathway in promoting BPD in hyperoxia conditions and provided a promising therapeutic target.

Dishevelled 2 regulates cancer cell proliferation and T cell mediated immunity in HER2-positive breast cancer.

BACKGROUND: Dishevelled paralogs (DVL1, 2, 3) are key mediators of Wnt pathway playing a role in constitutive oncogenic signaling influencing the tumor microenvironment. While previous studies showed correlation of ß-catenin with T cell gene expression, little is known about the role of DVL2 in modulating tumor immunity. This study aimed to uncover the novel interaction between DVL2 and HER2-positive (HER2+) breast cancer (BC) in regulating tumor immunity and disease progression. METHODS: DVL2 loss of function studies were performed with or without a clinically approved HER2 inhibitor, Neratinib in two different HER2+ BC cell lines. We analyzed RNA (RT-qPCR) and protein (western blot) expression of classic Wnt markers and performed cell proliferation and cell cycle analyses by live cell imaging and flow cytometry, respectively. A pilot study in 24 HER2+ BC patients was performed to dissect the role of DVL2 in tumor immunity. Retrospective chart review on patient records and banked tissue histology were performed. Data were analyzed in SPSS (version 25) and GraphPad Prism (version 7) at a significance p < 0.05. RESULTS: DVL2 regulates the transcription of immune modulatory genes involved in antigen presentation and T cell maintenance. DVL2 loss of function down regulated mRNA expression of Wnt target genes involved in cell proliferation, migration, invasion in HER2+ BC cell lines (±Neratinib). Similarly, live cell proliferation and cell cycle analyses reveal that DVL2 knockdown (±Neratinib) resulted in reduced proliferation, higher growth arrest (G1), limited mitosis (G2/M) compared to non-targeted control in one of the two cell lines used. Analyses on patient tissues who received neoadjuvant chemotherapy (n = 14) further demonstrate that higher DVL2 expression at baseline biopsy pose a significant negative correlation with % CD8α levels (r = - 0.67, p < 0.05) while have a positive correlation with NLR (r = 0.58, p < 0.05), where high NLR denotes worse cancer prognosis. These results from our pilot study reveal interesting roles of DVL2 proteins in regulating tumor immune microenvironment and clinical predictors of survival in HER2+ BC. CONCLUSION: Our study demonstrates potential immune regulatory role of DVL2 proteins in HER2+ BC. More in-depth mechanistic studies of DVL paralogs and their influence on anti-tumor immunity may provide insight into DVLs as potential therapeutic targets benefiting BC patients.

Dvl proteins regulate SMAD1, AHR, mTOR, BRD7 protein expression while differentially regulating canonical and non-canonical Wnt signaling pathways in CML cell lines.

Dishevelled (Dvl) is a scaffold protein that transmits Wnt signals to downstream effector molecules via both canonical and non-canonical Wnt signaling pathways. Deregulated activation of Dvl proteins has been reported in various solid tumors. However, it is not clear which pathway and proteins are responsible for observed aberrant activities and their relevance in disease prognosis. In addition, there is relatively limited knowledge on the role Dvl proteins may have in hematologic malignancy etiopathogenesis. In this study, we demonstrated that Dvl genes are not expressed in normal bone marrow but are expressed at different levels in the bone marrow of patients with chronic myeloid leukemia. We showed SMAD1, AHR, mTOR, BRD7 protein expressions are significantly affected by Dvl silencing and overexpression in CML cell lines. Wnt/ß-catenin and Wnt/PCP signaling pathway components are effectively repressed after Dvl silencing in K562 cells, while regulator of Wnt/Ca2+ signaling showed increase in both CML cell lines. Targeting Dvl proteins increases imatinib susceptibility of the K562 and MEG-01 cell lines. In light of our data, Dvl could be a potential therapeutic target in the treatment of CML.

Heterozygous variants in the DVL2 interaction region of DACT1 cause CAKUT and features of Townes-Brocks syndrome 2.

Most patients with congenital anomalies of the kidney and urinary tract (CAKUT) remain genetically unexplained. In search of novel genes associated with CAKUT in humans, we applied whole-exome sequencing in a patient with kidney, anorectal, spinal, and brain anomalies, and identified a rare heterozygous missense variant in the DACT1 (dishevelled binding antagonist of beta catenin 1) gene encoding a cytoplasmic WNT signaling mediator. Our patient's features overlapped Townes-Brocks syndrome 2 (TBS2) previously described in a family carrying a DACT1 nonsense variant as well as those of Dact1-deficient mice. Therefore, we assessed the role of DACT1 in CAKUT pathogenesis. Taken together, very rare (minor allele frequency ≤ 0.0005) non-silent DACT1 variants were detected in eight of 209 (3.8%) CAKUT families, significantly more frequently than in controls (1.7%). All seven different DACT1 missense variants, predominantly likely pathogenic and exclusively maternally inherited, were located in the interaction region with DVL2 (dishevelled segment polarity protein 2), and biochemical characterization revealed reduced binding of mutant DACT1 to DVL2. Patients carrying DACT1 variants presented with kidney agenesis, duplex or (multi)cystic (hypo)dysplastic kidneys with hydronephrosis and TBS2 features. During murine development, Dact1 was expressed in organs affected by anomalies in patients with DACT1 variants, including the kidney, anal canal, vertebrae, and brain. In a branching morphogenesis assay, tubule formation was impaired in CRISPR/Cas9-induced Dact1-/- murine inner medullary collecting duct cells. In summary, we provide evidence that heterozygous hypomorphic DACT1 variants cause CAKUT and other features of TBS2, including anomalies of the skeleton, brain, distal digestive and genital tract.

Letting go: Dishevelled phase separation recruits Axin to stabilize ß-catenin.

Dishevelled exerts a molecular force that guides cell fate, but how it does so remains enigmatic. In this issue, Kang et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202205069) show Dvl2 undergoes liquid-liquid phase separation to stabilize ß-catenin by pulling Axin into its biomolecular condensate at the plasma membrane.

Dishevelled phase separation promotes Wnt signalosome assembly and destruction complex disassembly.

The amplitude of Wnt/ß-catenin signaling is precisely controlled by the assembly of the cell surface-localized Wnt receptor signalosome and the cytosolic ß-catenin destruction complex. How these two distinct complexes are coordinately controlled remains largely unknown. Here, we demonstrated that the signalosome scaffold protein Dishevelled 2 (Dvl2) undergoes liquid-liquid phase separation (LLPS). Dvl2 LLPS is mediated by an intrinsically disordered region and facilitated by components of the signalosome, such as the receptor Fzd5. Assembly of the signalosome is initiated by rapid recruitment of Dvl2 to the membrane, followed by slow and dynamic recruitment of Axin1. Axin LLPS mediates assembly of the ß-catenin destruction complex, and Dvl2 attenuates LLPS of Axin. Compared with the destruction complex, Axin partitions into the signalosome at a lower concentration and exhibits a higher mobility. Together, our results revealed that Dvl2 LLPS is crucial for controlling the assembly of the Wnt receptor signalosome and disruption of the phase-separated ß-catenin destruction complex.

PI(4,5)P2-stimulated positive feedback drives the recruitment of Dishevelled to Frizzled in Wnt-ß-catenin signaling.

In the Wnt-ß-catenin pathway, Wnt binding to Frizzled (Fzd) and LRP5 or LRP6 (LRP5/6) co-receptors inhibits the degradation of the transcriptional coactivator ß-catenin by recruiting the cytosolic effector Dishevelled (Dvl). Polymerization of Dvl at the plasma membrane recruits the ß-catenin destruction complex, enabling the phosphorylation of LRP5/6, a key step in inhibiting ß-catenin degradation. Using purified Fzd proteins reconstituted in lipid nanodiscs, we investigated the factors that promote the recruitment of Dvl to the plasma membrane. We found that the affinity of Fzd for Dvl was not affected by Wnt ligands, in contrast to other members of the GPCR superfamily for which the binding of extracellular ligands affects the affinity for downstream transducers. Instead, Fzd-Dvl binding was enhanced by increased concentration of the lipid PI(4,5)P2, which is generated by Dvl-associated lipid kinases in response to Wnt and which is required for LRP5/6 phosphorylation. Moreover, binding to Fzd did not promote Dvl DEP domain dimerization, which has been proposed to be required for signaling downstream of Fzd. Our findings suggest a positive feedback loop in which Wnt-stimulated local PI(4,5)P2 production enhances Dvl recruitment and further PI(4,5)P2 production to support Dvl polymerization, LRP5/6 phosphorylation, and ß-catenin stabilization.

Nuclear Dishevelled: An enigmatic role in governing cell fate and Wnt signaling.

The Dishevelled gene was first identified in Drosophila mutants with disoriented hair and bristle polarity and subsequent work has now demonstrated its importance in critical and diverse aspects of biology. Since those early discoveries, Dishevelled has been shown to coordinate a plethora of developmental and cellular processes that range from controlling cell polarity during gastrulation to partnering with chromatin modifying enzymes to regulate histone methylation at genomic loci. While the role of DVL in development is well-respected and the cytosolic function of DVL has been studied more extensively, its nuclear role continues to remain murky. In this review we highlight some of the seminal discoveries that have contributed to the field, but the primary focus is to discuss recent advances with respect to the nuclear role of Dishevelled. This nuclear function of Dishevelled is a dimension which is proving to be increasingly important yet remains enigmatic.

Exosomal Dvl3 promoted the aggressive phenotypic transformation of RA-FLS via wnt pathway.

OBJECTIVE: This study was performed to explore the function and mechanism of Dvl3 in RA-FLS by exosome intervention. METHODS: The expression pattern of Dvl3 was examined by IHC, WB, and qPCR. Modified exosomes obtained from culturing supernatant of RA-FLS infected with Dvl3 over expression (OE) lentivirus were administrated to the target RA-FLS. The ability of survival, migration, and the production of inflammatory factor influenced by exosomal Dvl3 were detected by CKK8 kits, Tunel, migration test, qPCR, and enzyme-linked immunosorbent assay (ELISA) respectively; Flow cytometry analysis was conducted to explorer the inflammatory moderate role of exosomes on CD4+ T cells. The possible downstream pathways of Dvl3 were screened by qPCR and WB and verified by double luciferase reporter experiment. RESULTS: The expression level of Dvl3 was significantly increased in RA and CIA. Exosomes from the OE group could significantly promote cell proliferation activity, migration/invasion ability. The augment of TNF-α, IL-1ß, IL-17, and IL-21 was observed in exosomal Dvl3-OE group. Th1 and Th17 cells polarisation and cytokines related were both enhanced by Exosomal Dvl3. Over expression of Dvl3 was accompanied by the significant increase of ß-catenin and RhoA activities. CONCLUSION: This study discovered the high expression of Dvl3 of exosomes derived from RA patients which may possessed the ability to promote phenotypic transformation of RA-FLS through Wnt pathway.

CYP2E1 plays a suppressive role in hepatocellular carcinoma by regulating Wnt/Dvl2/ß-catenin signaling.

OBJECTIVE: Knowledge of the role of CYP2E1 in hepatocarcinogenesis is largely based on epidemiological and animal studies, with a primary focus on the role of CYP2E1 in metabolic activation of procarcinogens. Few studies have directly assessed the effects of CYP2E1 on HCC malignant phenotypes. METHODS: The expression of CYP2E1 in HCC tissues was determined by qRT-PCR, western blotting and immunohistochemistry. Overexpression of CYP2E1 in HCC cell was achieved by lentivirus transfection. The function of CYP2E1 were detected by CCK-8, wound healing, transwell assays, xenograft models and pulmonary metastasis model. TOP/FOPFlash reporter assay, western blotting, functional rescue experiments, Co-immunoprecipitation and reactive oxygen species detection were conducted to reveal the underlying mechanism of the tumor suppressive role of CYP2E1. RESULTS: CYP2E1 expression is down-regulated in HCC tissues, and this downregulation was associated with large tumor diameter, vascular invasion, poor differentiation, and shortened patient survival time. Ectopic expression of CYP2E1 inhibits the proliferation, invasion and migration and epithelial-to-mesenchymal transition of HCC cells in vitro, and inhibits tumor formation and lung metastasis in nude mice. Mechanistic investigations show that CYP2E1 overexpression significantly inhibited Wnt/ß-catenin signaling activity and decreased Dvl2 expression in HCC cells. An increase in Dvl2 expression restored the malignant phenotype of HCC cells. Notably, CYP2E1 promoted the ubiquitin-mediated degradation of Dvl2 by strengthening the interaction between Dvl2 and the E3 ubiquitin ligase KLHL12 in CYP2E1-stable HCC cells. CYP2E1-induced ROS accumulation was a critical upstream event in the Wnt/ß-Catenin pathway in CYP2E1-overexpressing HCC cells. CONCLUSIONS: These results provide novel insight into the role of CYP2E1 in HCC and the tumor suppressor role of CYP2E1 can be attributed to its ability to manipulate Wnt/Dvl2/ß-catenin pathway via inducing ROS accumulation, which provides a potential target for the prevention and treatment of HCC.

Cell cortex regulation by the planar cell polarity protein Prickle1.

The planar cell polarity pathway regulates cell polarity, adhesion, and rearrangement. Its cytoplasmic core components Prickle (Pk) and Dishevelled (Dvl) often localize as dense puncta at cell membranes to form antagonizing complexes and establish cell asymmetry. In vertebrates, Pk and Dvl have been implicated in actomyosin cortex regulation, but the mechanism of how these proteins control cell mechanics is unclear. Here we demonstrate that in Xenopus prechordal mesoderm cells, diffusely distributed, cytoplasmic Pk1 up-regulates the F-actin content of the cortex. This counteracts cortex down-regulation by Dvl2. Both factors act upstream of casein kinase II to increase or decrease cortical tension. Thus, cortex modulation by Pk1 and Dvl2 is translated into mechanical force and affects cell migration and rearrangement during radial intercalation in the prechordal mesoderm. Pk1 also forms puncta and plaques, which are associated with localized depletion of cortical F-actin, suggesting opposite roles for diffuse and punctate Pk1.

E2F1-activated LINC01224 drives esophageal squamous cell carcinoma cell malignant behaviors via targeting miR-6884-5p/DVL3 axis and activating Wnt/ß-catenin signaling pathway.

Current evidence has unveiled that long non-coding RNAs (lncRNAs) are pivotal regulators in the development of cancers. This study aimed to investigate the potential mechanisms of LINC01224 in esophageal squamous cell carcinoma (ESCC) cells. RT-qPCR analysis was done to test LINC01224 expression in ESCC cells. Functional assays were conducted to assess the influences of LINC01224 on ESCC cell functions. Mechanism assays were carried out to detect the regulatory mechanisms of LINC01224 at post-transcriptional and transcriptional levels. Briefly, LINC01224 expression was remarkably high in ESCC cells. LINC01224 silence restricted the proliferative, migratory, and invasive capabilities of ESCC cells. Moreover, LINC01224 could combine with miR-6884-5p by acting as a ceRNA. Further, DVL3 was proved to be targeted by miR-6884-5p. Importantly, LINC01224 could switch on Wnt/ß-catenin signaling pathway by via enhancing DVL3 expression. Additionally, E2F1 could serve as a transcription factor to stimulate LINC01224 transcription. In summary, our study elucidated that E2F1-activated LINC01224 regulated miR-6884-5p/DVL3 to actuate the Wnt/ß-catenin signaling pathway, which facilitates multiple phenotype of ESCC cells, including proliferation, migration, and invasion. Our findings might offer potential therapeutic targets for ESCC treatment.