SLMAP3 is crucial for organogenesis through mechanisms involving primary cilia formation.

SLMAP3 is a constituent of the centrosome and is known to assemble with the striatin-interacting phosphatase and kinase (STRIPAK) complex, where it has been reported to repress Hippo signalling. The global knockout of SLMAP3 in mice results in embryonic/perinatal lethality and stunted growth without changes in the phosphorylation status of YAP. Diverse phenotypes present in the SLMAP3-/- embryos include reduced body axis, small and abnormal organs resembling defects in planar cell polarity (PCP) signalling, while also displaying the notable polycystic kidneys, a known manifestation of ciliopathies. Analysis of cell polarity in primary mouse embryonic fibroblasts (MEFs) including cell migration, orientation and mitotic spindle angle did not reveal any changes due to SLMAP3 loss in these cells, although the expression of DVL3 was significantly reduced. Furthermore, MEFs lacking FGFR1OP2 or STRN3, two other STRIPAK members, did not reveal any significant changes in any of these parameters either. Significant changes in the number of ciliated cells and primary cilium length in SLMAP3 and FGFR1OP2 deficient MEFs were evident, while a reduced primary cilium length was notable in chondrocytes of SLMAP3 deficient embryos. Our findings suggest that SLMAP3 is essential for mouse embryogenesis through novel mechanisms involving the primary cilium/PCP and protein stability independent of Hippo signalling.

Exploring DIX-DIX Homo- and Hetero-Oligomers in Wnt Signaling with AlphaFold2.

Wnt signaling is involved in embryo development and cancer. The binding between the DIX domains of Axin1/2, Dishevelled1/2/3, and Coiled-coil-DIX1 is essential for Wnt/ß-catenin signaling. Structural and biological studies have revealed that DIX domains are polymerized through head-to-tail interface interactions, which are indispensable for activating ß-catenin Wnt signaling. Although different isoforms of Dvl and Axin proteins display both redundant and specific functions in Wnt signaling, the specificity of DIX-mediated interactions remains unclear due to technical challenges. Using AlphaFold2(AF2), we predict the structures of 6 homodimers and 22 heterodimers of DIX domains without templates and compare them with the reported X-ray complex structures. PRODIGY is used to calculate the binding affinities of these DIX complexes. Our results show that the Axin2 DIX homodimer has a stronger binding affinity than the Axin1 DIX homodimer. Among Dishevelled (Dvl) proteins, the binding affinity of the Dvl1 DIX homodimer is stronger than that of Dvl2 and Dvl3. The Coiled-coil-DIX1(Ccd1) DIX homodimer shows weaker binding than the Axin1 DIX homodimer. Generally, heterodimer interactions tend to be stronger than those of homodimers. Our findings provide insights into the mechanism of the Wnt signaling pathway and highlight the potential of AF2 and PRODIGY for studying protein-protein interactions in signaling pathways.

DVL/GSK3/ISL1 pathway signaling: unraveling the mechanism of SIRT3 in neurogenesis and AD therapy.

BACKGROUND: The established association between Alzheimer's disease (AD) and compromised neural regeneration is well-documented. In addition to the mitigation of apoptosis in neural stem cells (NSCs), the induction of neurogenesis has been proposed as a promising therapeutic strategy for AD. Our previous research has demonstrated the effective inhibition of NSC injury induced by microglial activation through the repression of oxidative stress and mitochondrial dysfunction by Sirtuin 3 (SIRT3). Nonetheless, the precise role of SIRT3 in neurogenesis remains incompletely understood. METHODS: In vivo, SIRT3 overexpression adenovirus was firstly injected by brain stereotaxic localization to affect the hippocampal SIRT3 expression in APP/PS1 mice, and then behavioral experiments were performed to investigate the cognitive improvement of SIRT3 in APP/PS1 mice, as well as neurogenic changes in hippocampal region by immunohistochemistry and immunofluorescence. In vitro, under the transwell co-culture condition of microglia and neural stem cells, the mechanism of SIRT3 improving neurogenesis of neural stem cells through DVL/GSK3/ISL1 axis was investigated by immunoblotting, immunofluorescence and other experimental methods. RESULTS: Our findings indicate that the overexpression of SIRT3 in APP/PS1 mice led to enhanced cognitive function and increased neurogenesis. Additionally, SIRT3 was observed to promote the differentiation of NSCs into neurons during retinoic acid (RA)-induced NSC differentiation in vitro, suggesting a potential role in neurogenesis. Furthermore, we observed the activation of the Wnt/ß-catenin signaling pathway during this process, with Glycogen Synthase Kinase-3a (GSK3a) primarily governing NSC proliferation and GSK3ß predominantly regulating NSC differentiation. Moreover, the outcomes of our study demonstrate that SIRT3 exerts a protective effect against microglia-induced apoptosis in neural stem cells through its interaction with DVLs. CONCLUSIONS: Our results show that SIRT3 overexpressing APP/PS1 mice have improved cognition and neurogenesis, as well as improved neurogenesis of NSC in microglia and NSC transwell co-culture conditions through the DVL/GSK3/ISL1 axis.

Structural basis of Frizzled 4 in recognition of Dishevelled 2 unveils mechanism of WNT signaling activation.

WNT signaling is fundamental in development and homeostasis, but how the Frizzled receptors (FZDs) propagate signaling remains enigmatic. Here, we present the cryo-EM structure of FZD4 engaged with the DEP domain of Dishevelled 2 (DVL2), a key WNT transducer. We uncover a distinct binding mode where the DEP finger-loop inserts into the FZD4 cavity to form a hydrophobic interface. FZD4 intracellular loop 2 (ICL2) additionally anchors the complex through polar contacts. Mutagenesis validates the structural observations. The DEP interface is highly conserved in FZDs, indicating a universal mechanism by which FZDs engage with DVLs. We further reveal that DEP mimics G-protein/ß-arrestin/GRK to recognize an active conformation of receptor, expanding current GPCR engagement models. Finally, we identify a distinct FZD4 dimerization interface. Our findings delineate the molecular determinants governing FZD/DVL assembly and propagation of WNT signaling, providing long-sought answers underlying WNT signal transduction.

Structural insights into Frizzled3 through nanobody modulators.

The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating ß-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.

Class-Wide Analysis of Frizzled-Dishevelled Interactions Using BRET Biosensors Reveals Functional Differences among Receptor Paralogs.

Wingless/Int-1 (WNT) signaling is mediated by WNT binding to 10 Frizzleds (FZD1-10), which propagate the signal inside the cell by interacting with different transducers, most prominently the phosphoprotein Dishevelled (DVL). Despite recent progress, questions about WNT/FZD selectivity and paralog-dependent differences in the FZD/DVL interaction remain unanswered. Here, we present a class-wide analysis of the FZD/DVL interaction using the DEP domain of DVL as a proxy in bioluminescence resonance energy transfer (BRET) techniques. Most FZDs engage in a constitutive high-affinity interaction with DEP. Stimulation of unimolecular FZD/DEP BRET sensors with different ligands revealed that most paralogs are dynamic in the FZD/DEP interface, showing distinct profiles in terms of ligand selectivity and signal kinetics. This study underlines mechanistic differences in terms of how allosteric communication between FZDs and their main signal transducer DVL occurs. Moreover, the unimolecular sensors represent the first receptor-focused biosensors to surpass the requirements for high-throughput screening, facilitating FZD-targeted drug discovery.

WNT5B promotes the malignant phenotype of non-small cell lung cancer via the FZD3-DVL3-RAC1-PCP-JNK pathway.

The WNT5B ligand regulates the non-canonical wingless-related integration site (WNT)-planar cell polarity (PCP) pathway. However, the detailed mechanism underlying the activity of WNT5B in the WNT-PCP pathway in non-small cell lung cancer (NSCLC) is unclear. In this study, we assessed the clinicopathological significance of WNT5B expression in NSCLC specimens. WNT5B-overexpression and -knockdown NSCLC cell lines were generated in vivo and in vitro, respectively. WNT5B overexpression in NSCLC specimens correlates with advanced tumor node metastasis (TNM) stage, lymph node metastasis, and poor prognosis in patients with NSCLC. Additionally, WNT5B promotes the malignant phenotype of NSCLC cells in vivo and in vitro. Interactions were identified among WNT5B, frizzled3 (FZD3), and disheveled3 (DVL3) in NSCLC cells, leading to the activation of WNT-PCP signaling. The FZD3 receptor initiates DVL3 recruitment to the membrane for phosphorylation in a WNT5B ligand-dependent manner and activates c-Jun N-terminal kinase (JNK) signaling via the small GTPase RAC1. Furthermore, the deletion of the DEP domain of DVL3 abrogated these effects. Overall, we demonstrated a novel signal transduction pathway in which WNT5B recruits DVL3 to the membrane via its DEP domain through interaction with FZD3 to promote RAC1-PCP-JNK signaling, providing a potential target for clinical intervention in NSCLC treatment.

Wnt-Ror-Dvl signalling and the dystrophin complex organize planar-polarized membrane compartments in C. elegans muscles.

Cell polarity mechanisms allow the formation of specialized membrane domains with unique protein compositions, signalling properties, and functional characteristics. By analyzing the localization of potassium channels and proteins belonging to the dystrophin-associated protein complex, we reveal the existence of distinct planar-polarized membrane compartments at the surface of C. elegans muscle cells. We find that muscle polarity is controlled by a non-canonical Wnt signalling cascade involving the ligand EGL-20/Wnt, the receptor CAM-1/Ror, and the intracellular effector DSH-1/Dishevelled. Interestingly, classical planar cell polarity proteins are not required for this process. Using time-resolved protein degradation, we demonstrate that -while it is essentially in place by the end of embryogenesis- muscle polarity is a dynamic state, requiring continued presence of DSH-1 throughout post-embryonic life. Our results reveal the unsuspected complexity of the C. elegans muscle membrane and establish a genetically tractable model system to study cellular polarity and membrane compartmentalization in vivo.

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.

Dishevelled Segment Polarity Protein 3 (DVL3) Induced by Bacterial LPS Promotes the Proliferation and Migration of Prostate Cancer Cells through the TLR4 Pathway.

BACKGROUND: Chronic inflammation is associated with various malignant tumors. Bacterial lipopolysaccharides (LPSs) play a significant part in the event and development of prostate cancer. Dishevelled segment polarity protein 3 (DVL3) is a shared component of the Wnt/ß-catenin and Notch signaling pathways, which are involved in tumor progression, chemoresistance, and maintenance of stem cell-like properties. According to reports, prostatic cancer cell invasion and proliferation are mediated by toll-like receptor 4 (TLR4). However, the role and regulation of DVL3 in prostate cancer and its relationship with TLR4 remain unclear. METHODS: Survival curves were plotted to evaluate the relationship between DVL3 expression and prognosis in patients with prostate cancer. DVL3 was silenced in PC3 and DU145 cells using small interfering RNAs (siRNAs). Subsequently, cell counting kit-8 (CCK-8) assay, colony formation assay, transwell migration assay, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) were performed to investigate the role of DVL3 in cell proliferation and migration in vitro. The protein markers of potential pathways were analyzed via western blotting. RESULTS: DVL3 expression was linked to prognosis in patients with prostate cancer; In particular, patients with high DVL3 expression had a poor prognosis. LPS stimulation increased (p < 0.01) the expression of DVL3 in PC3 cells. DVL3 regulated tumor cell proliferation and migration by mediating the increase (p < 0.01) in TLR4 expression. Knockout of TLR4 validated that TLR4 played a crucial role in LPS-induced DVL3 expression. Silencing of DVL3 decreased (p < 0.01) the LPS-induced proliferation and migration of PC3 cells. CONCLUSIONS: Bacterial LPS-induced DVL3 promoted the multiplication and migration of prostate cancer cells through the TLR4 pathway. This study offers a valuable reference for the development and clinical application of targeted drugs for prostate cancer.

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.

Delivery of microRNA-423-5p by exosome from adipose-derived stem/stromal cells inhibits DVL3 to potentiate autologous fat graft survival through adipogenesis and inflammatory response.

Autologous fat grafting represents a reconstructive technique but is limited by unstable graft retention. Based on existing reports and bioinformatics prediction, we hypothesized that delivering exosomes from human adipose-derived stem/stromal cells (hADSC-Exo) would increase fat graft survival and further explore the mechanism. hADSC-Exo were extracted and identified. An autologous fat grafting model was established using donor and recipient mice, followed by hADSC-Exo treatment. hADSC-Exo promoted the retention of autologous fat grafts in mice, along with increased adipocyte activity, angiogenesis, and decreased inflammation in grafts. Moreover, hADSC-Exo potentiated the adipose differentiation of 3T3-L1 cells, enhanced the angiogenic and migratory capacity of human umbilical vein endothelial cells, and inhibited the inflammation and viability of RAW 264.7 cells. The therapeutic effect of hADSC-Exo on fat grafting was associated with the delivery of microRNA (miR)-423-5p. Deletion of miR-423-5p in Exo impaired the function of hADSC-Exo on fat retention. miR-423-5p bound to DVL3 to suppress DVL3 expression, and DVL3 deletion promoted adipose differentiation of 3T3-L1 cells. In conclusion, our findings further widen the theoretical basis of the clinical application of hADSC-Exo in autologous fat grafts.

Presenilin-2 knock-In mice show severe depressive behavior via DVL3 downregulation.

INTRODUCTION: Alzheimer's disease (AD) is the most common form of dementia. Depression is one of the most critical psychiatric complications of AD, and 20%-30% of patients with AD experience symptoms of depression. Phospho-glycogen synthase kinase-3 beta (GSK3ß) is known to be associated with AD and depression. Furthermore, the role of disheveled (DVL) is known to regulate GSK3ß. Moreover, presenilin-2 (PS2) and DVL have cross-talk with each other. Also, it is widely hypothesized that stress leads to hypersecretion of cortisol and is thus associated with depression. Dickkopf WNT signaling pathway inhibitor-1 (DKK-1) is a crucial factor regulating depression and both amyloid beta (Aß) and phosphorylation of tau are widely known as a biomarker of AD. METHODS: To investigate the relationship between AD and depression, and possible pathways connecting the two diseases, we examined memory function and depression-related behavior test results in PS2 knock-in AD mice (PS2 MT). Next, we confirmed that there are relationships between DVL, depression, and cognitive disease through the comparative toxicogenomics database (https://ctdbase.org) and STRING (https://string-db.org) database. RESULTS: PS2 knock-in mice showed much more severe memory impairment and depression than PS2 wild-type mice (PS2 WT). In AD-related behavioral experiments, PS2 MT mice showed more memory dysfunction compared with PS2 WT group mice. Moreover, Aß and phosphorylation of tau showed higher expression in PS2 MT mice than in PS2 WT mice. Depression-related behavioral tests showed that PS2 MT mice exhibited more depressive behaviors than PS2 WT mice. Furthermore, both higher cortisol levels and higher expression of DKK-1 were found in PS2 MT mice relative to PS2 WT mice. The results indicated that there is a relationship between DVL and the release of AD-related mediators and expression of the depression-related glucocorticoid receptor and DKK-1. In the PS2 knock-in group, DVL was significantly decreased compared with the PS2 WT group. CONCLUSION: Depression increases the risk of developing AD and other forms of dementia. Recent evidence indicates that depression symptoms could trigger changes in memory and thinking over time. However, it is recognized that there are no drugs to facilitate a full recovery for both AD and depression. However, our results suggest that AD and depression could be associated, and DVL could be a significant target for the association between AD and depression.

HCMV UL8 interaction with ß-catenin and DVL2 regulates viral reactivation in CD34+ hematopoietic progenitor cells.

IMPORTANCE: CD34+ hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34+ HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/ß-catenin pathway as an important component of viral reactivation. We further define that pUL8 and ß-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with ß-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics.

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.

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.

WNT stimulation induces dynamic conformational changes in the Frizzled-Dishevelled interaction.

Frizzleds (FZDs) are G protein-coupled receptors (GPCRs) that bind to WNT family ligands. FZDs signal through multiple effector proteins, including Dishevelled (DVL), which acts as a hub for several downstream signaling pathways. To understand how WNT binding to FZD stimulates intracellular signaling and influences downstream pathway selectivity, we investigated the dynamic changes in the FZD5-DVL2 interaction elicited by WNT-3A and WNT-5A. Ligand-induced changes in bioluminescence resonance energy transfer (BRET) between FZD5 and DVL2 or the isolated FZD-binding DEP domain of DVL2 revealed a composite response consisting of both DVL2 recruitment and conformational dynamics in the FZD5-DVL2 complex. The combination of different BRET paradigms enabled us to identify ligand-dependent conformational dynamics in the FZD5-DVL2 complex and distinguish them from ligand-induced recruitment of DVL2 or DEP to FZD5. The observed agonist-induced conformational changes at the receptor-transducer interface suggest that extracellular agonist and intracellular transducers cooperate through transmembrane allosteric interaction with FZDs in a ternary complex reminiscent of that of classical GPCRs.

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.