Activation of the Wnt/β-catenin pathway crucially depends on the polymerization of dishevelled 2 (DVL2) into biomolecular condensates. However, given the low affinity of known DVL2 self-interaction sites and its low cellular concentration, it is unclear how polymers can form. Here, we detect oligomeric DVL2 complexes at endogenous protein levels in human cell lines, using a biochemical ultracentrifugation assay. We identify a low-complexity region (LCR4) in the C-terminus whose deletion and fusion decreased and increased the complexes, respectively. Notably, LCR4-induced complexes correlated with the formation of microscopically visible multimeric condensates. Adjacent to LCR4, we mapped a conserved domain (CD2) promoting condensates only. Molecularly, LCR4 and CD2 mediated DVL2 self-interaction via aggregating residues and phenylalanine stickers, respectively. Point mutations inactivating these interaction sites impaired Wnt pathway activation by DVL2. Our study discovers DVL2 complexes with functional importance for Wnt/β-catenin signaling. Moreover, we provide evidence that DVL2 condensates form in two steps by pre-oligomerization via high-affinity interaction sites, such as LCR4, and subsequent condensation via low-affinity interaction sites, such as CD2.
- MeSH
- beta-katenin metabolismus genetika MeSH
- HEK293 buňky MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- protein dishevelled * metabolismus genetika MeSH
- signální dráha Wnt * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Members of the casein kinase 1 (CK1) family are important regulators of multiple signaling pathways. CK1α is a well-known negative regulator of the Wnt/β-catenin pathway, which promotes the degradation of β-catenin via its phosphorylation of Ser45. In contrast, the closest paralog of CK1α, CK1α-like, is a poorly characterized kinase of unknown function. In this study, we show that the deletion of CK1α, but not CK1α-like, resulted in a strong activation of the Wnt/β-catenin pathway. Wnt-3a treatment further enhanced the activation, which suggests there are at least two modes, a CK1α-dependent and Wnt-dependent, of β-catenin regulation. Rescue experiments showed that only two out of ten naturally occurring splice CK1α/α-like variants were able to rescue the augmented Wnt/β-catenin signaling caused by CK1α deficiency in cells. Importantly, the ability to phosphorylate β-catenin on Ser45 in the in vitro kinase assay was required but not sufficient for such rescue. Our compound CK1α and GSK3α/β KO models suggest that the additional nonredundant function of CK1α in the Wnt pathway beyond Ser45-β-catenin phosphorylation includes Axin phosphorylation. Finally, we established NanoBRET assays for the three most common CK1α splice variants as well as CK1α-like. Target engagement data revealed comparable potency of known CK1α inhibitors for all CK1α variants but not for CK1α-like. In summary, our work brings important novel insights into the biology of CK1α, including evidence for the lack of redundancy with other CK1 kinases in the negative regulation of the Wnt/β-catenin pathway at the level of β-catenin and Axin.
- MeSH
- alternativní sestřih MeSH
- beta-katenin * metabolismus genetika MeSH
- fosforylace MeSH
- GSK3B metabolismus genetika MeSH
- HEK293 buňky MeSH
- kasein kinasa Ialfa * metabolismus genetika MeSH
- kinasa 3 glykogensynthasy metabolismus genetika MeSH
- lidé MeSH
- protein Wnt3A metabolismus genetika MeSH
- signální dráha Wnt * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
BACKGROUND: Glucocorticoids are commonly used in children with different chronic diseases. Growth failure represents a so far untreatable undesired side-effect. As lithium chloride (LiCl) is known to induce cell renewal in various tissues, we hypothesized that LiCl may prevent glucocorticoid-induced growth failure. METHODS: We monitored growth of fetal rat metatarsals cultured ex-vivo with dexamethasone and/or LiCl, while molecular mechanisms were explored through RNA sequencing by implementing the differential gene expression and gene set analysis. Quantification of β-catenin in human growth plate cartilage cultured with dexamethasone and/or LiCl was added for verification. RESULTS: After 14 days of culture, the length of dexamethasone-treated fetal rat metatarsals increased by 1.4 ± 0.2 mm compared to 2.4 ± 0.3 mm in control bones (p < 0.001). The combination of LiCl and dexamethasone led to bone length increase of 1.9 ± 0.3 mm (p < 0.001 vs. dexamethasone alone). By adding lithium, genes for cell cycle and Wnt/β-catenin, Hedgehog and Notch signaling, were upregulated compared to dexamethasone alone group. CONCLUSIONS: LiCl has the potential to partially rescue from dexamethasone-induced bone growth impairment in an ex vivo model. Transcriptomics identified cell renewal and proliferation as candidates for the underlying mechanisms. Our observations may open up the development of a new treatment strategy for bone growth disorders. IMPACT: LiCl is capable to prevent glucocorticoid-induced growth failure in rat metatarsals in vitro. The accompanying drug-induced transcriptomic changes suggested cell renewal and proliferation as candidate underlying mechanisms. Wnt/beta-catenin pathway could be one of those novel mechanisms.
- MeSH
- beta-katenin * metabolismus MeSH
- chlorid lithný * farmakologie MeSH
- dexamethason * farmakologie MeSH
- glukokortikoidy farmakologie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- metatarzální kosti * účinky léků MeSH
- potkani Sprague-Dawley MeSH
- proliferace buněk účinky léků MeSH
- růstová ploténka účinky léků metabolismus MeSH
- signální dráha Wnt účinky léků MeSH
- vývoj kostí účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Coronary heart disease (CHD) is one of the most commonly seen cardiovascular conditions across the globe. Junctional cadherin 5 associated (JCAD) protein is found in the intercellular junctions of endothelial cells and linked to cardiovascular diseases. Nonetheless, the influence of JCAD on cardiomyocyte injury caused by CHD is unclear. A model of H2O2-induced H9c2 cell injury was constructed, and JCAD mRNA and protein levels were assessed by qRT-PCR and Western blot. The impacts of JCAD on the proliferation or apoptosis of H9c2 cells were explored by CCK-8 assay, Western blot and TUNEL staining. The effect of JCAD on the inflammatory response and vascular endothelial function of H9c2 cells was detected using ELISA kits. The levels of Wnt/β-catenin pathway-related proteins were assessed by Western blot. H2O2 treatment led to a rise in the levels of JCAD in H9c2 cells. Over-expression of JCAD promoted H2O2-induced cellular injury, leading to notably elevated contents of inflammatory factors, along with vascular endothelial dysfunction. In contrast to over-expression of JCAD, silencing of JCAD attenuated H2O2-induced cellular injury and inhibited apoptosis, inflammatory response and vascular endothelial dysfunction. Notably, JCAD could regulate the Wnt/β-catenin pathway, while DKK-1, Wnt/β-catenin pathway antagonist, counteracted the enhancing impact of JCAD over-expression on H2O2-induced H9c2 cell injury, further confirming that JCAD acts by regulating the Wnt/β-catenin pathway. In summary, over-expression of JCAD promoted H2O2-induced H9c2 cell injury by activating the Wnt/β-catenin pathway, while silencing of JCAD attenuated the H2O2-induced cell injury.
- MeSH
- apoptóza * účinky léků MeSH
- beta-katenin metabolismus MeSH
- buněčné linie MeSH
- down regulace * účinky léků MeSH
- kadheriny metabolismus MeSH
- kardiomyocyty * metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- peroxid vodíku * farmakologie MeSH
- proliferace buněk účinky léků MeSH
- signální dráha Wnt * účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Pediatric central nervous system (CNS) tumors represent the most common cause of cancer-related death in children aged 0-14 years. They differ from their adult counterparts, showing extensive clinical and molecular heterogeneity as well as a challenging histopathological spectrum that often impairs accurate diagnosis. Here, we use DNA methylation-based CNS tumor classification in combination with copy number, RNA-seq, and ChIP-seq analysis to characterize a newly identified CNS tumor type. In addition, we report histology, patient characteristics, and survival data in this tumor type. We describe a biologically distinct pediatric CNS tumor type (n = 31 cases) that is characterized by focal high-level amplification and resultant overexpression of either PLAGL1 or PLAGL2, and an absence of recurrent genetic alterations characteristic of other pediatric CNS tumor types. Both genes act as transcription factors for a regulatory subset of imprinted genes (IGs), components of the Wnt/β-Catenin pathway, and the potential drug targets RET and CYP2W1, which are also specifically overexpressed in this tumor type. A derived PLAGL-specific gene expression signature indicates dysregulation of imprinting control and differentiation/development. These tumors occurred throughout the neuroaxis including the cerebral hemispheres, cerebellum, and brainstem, and were predominantly composed of primitive embryonal-like cells lacking robust expression of markers of glial or neuronal differentiation (e.g., GFAP, OLIG2, and synaptophysin). Tumors with PLAGL1 amplification were typically diagnosed during adolescence (median age 10.5 years), whereas those with PLAGL2 amplification were diagnosed during early childhood (median age 2 years). The 10-year overall survival was 66% for PLAGL1-amplified tumors, 25% for PLAGL2-amplified tumors, 18% for male patients, and 82% for female patients. In summary, we describe a new type of biologically distinct CNS tumor characterized by PLAGL1/2 amplification that occurs predominantly in infants and toddlers (PLAGL2) or adolescents (PLAGL1) which we consider best classified as a CNS embryonal tumor and which is associated with intermediate survival. The cell of origin and optimal treatment strategies remain to be defined.
- MeSH
- dítě MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- kojenec MeSH
- lidé MeSH
- metylace DNA MeSH
- nádorové supresorové proteiny genetika MeSH
- nádory centrálního nervového systému * genetika MeSH
- předškolní dítě MeSH
- primitivní neuroektodermové nádory * genetika MeSH
- proteiny buněčného cyklu genetika MeSH
- proteiny vázající RNA genetika MeSH
- signální dráha Wnt genetika MeSH
- transkripční faktory genetika metabolismus MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Canonical Wnt signaling is essential for a plethora of biological processes ranging from early embryogenesis to aging. Malfunctions of this crucial signaling pathway are associated with various developmental defects and diseases, including cancer. Although TCF/LEF transcription factors (TCF/LEFs) are known to be essential for this pathway, the regulation of their intracellular levels is not completely understood. Here, we show that the lysine demethylase KDM2A promotes the proteasomal destabilization of TCF/LEFs independently of its demethylase domain. We found that the KDM2A-mediated destabilization of TCF/LEFs is dependent on the KDM2A zinc finger CXXC domain. Furthermore, we identified the C-terminal region of TCF7L2 and the CXXC domain of KDM2A as the domains responsible for the interaction between the two proteins. Our study is also the first to show that endogenous TCF/LEF proteins undergo KDM2A-mediated proteasomal degradation in a neddylation-dependent manner. Here, we reveal a completely new mechanism that affects canonical Wnt signaling by regulating the levels of TCF/LEF transcription factors through their KDM2A-promoted proteasomal degradation.
- MeSH
- beta-katenin * metabolismus MeSH
- lysin * MeSH
- signální dráha Wnt MeSH
- zinkové prsty MeSH
- Publikační typ
- časopisecké články MeSH
Stem cell regulation plays a crucial role during development and homeostasis. Here, an essential source of Wnts from Gli1+ stem/progenitor cells was identified in the murine molar. Loss of Wnt production in Gli1+ apical stem/progenitor cells led to loss of Axin2 at the root apex, mis-regulation of SOX9, loss of BMP and Hh signaling, and truncation of root development. In the absence of Wnt signals, the root epithelium lost its integrity and epithelial identity. This phenotype could be partially mimicked by loss of Sox9 in the Gli1 population. Stabilization of Wnt signaling in the apical papilla led to rapid unordered differentiation of hard tissues and fragmentation of the epithelial root sheath. Wnt signaling from Gli1+ stem/progenitor cells, therefore, orchestrates root development, coordinating mesenchymal and epithelial interactions via SOX9 to regulate stem/progenitor cell expansion and differentiation. Our results demonstrate that disparate stem/progenitor cell populations are unified in their fundamental signaling interactions.
Wnt signalling is known to generate cellular asymmetry via Wnt/planar cell polarity pathway (Wnt/PCP). Wnt/PCP acts locally (i) to orient membrane polarity and asymmetric establishment of intercellular junctions via conserved set of PCP proteins most specifically represented by Vangl and Prickle, and (ii) to asymmetrically rearrange cytoskeletal structures via downstream effectors of Dishevelled (Dvl). This process is best described on stable phenotypes of epithelial cells. Here, however, we review the activity of Wnt signalling in migratory cells which experience the extensive rearrangements of cytoskeleton and consequently dynamic asymmetry, making the localised effects of Wnt signalling easier to distinguish. Firstly, we focused on migration of neuronal axons, which allows to study how the pre-existent cellular asymmetry can influence Wnt signalling outcome. Then, we reviewed the role of Wnt signalling in models of mesenchymal migration including neural crest, melanoma, and breast cancer cells. Last, we collected evidence for local Wnt signalling in amoeboid cells, especially lymphocytes. As the outcome of this review, we identify blank spots in our current understanding of this topic, propose models that synthesise the current observations and allow formulation of testable hypotheses for the future research.
- MeSH
- crista neuralis MeSH
- epitelové buňky MeSH
- obratlovci MeSH
- polarita buněk * fyziologie MeSH
- signální dráha Wnt * fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Adjuvant therapy and radiotherapy improves the survival of patients with metastatic and locally advanced gastric cancer (GC). However, the resistance to radiotherapy limits its clinical usage. Rhotekin 2 (RTKN2) functions as an oncogene and confers resistance to ultraviolet B-radiation and apoptosis- inducing agents. Here, the role of RTKN2 in radiosensitivity of GC cell lines was investigated. RTKN2 was found to be elevated in GC tissues and cells. A series of functional assays revealed that overexpression of RTKN2 induced GC cell proliferation, promoted GC cell migration and invasion, while inhibiting GC cell apoptosis. However, silence of RTKN2 promoted GC cell apoptosis, while repressing GC cell proliferation, invasion and migration. GC cells were exposed to irradiation, and data from cell survival and apoptotic assays showed that knock-down of RTKN2 enhanced radiosensitivity of GC through up-regulation of apoptosis and down-regulation of proliferation in irradiation-exposed GC cells. Moreover, the protein expression of β-catenin and c-Myc in GC cells was enhanced by RTKN2 over-expression, but reduced by RTKN2 silence. Interference of RTKN2 down-regulated nuclear β-catenin expression, while up-regulating cytoplasmic β-catenin in GC. In conclusion, RTKN2 contributed to cell growth and radioresistance in GC through activation of Wnt/β-catenin signalling.
- MeSH
- beta-katenin metabolismus MeSH
- intracelulární signální peptidy a proteiny metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory žaludku * genetika patologie radioterapie MeSH
- pohyb buněk MeSH
- proliferace buněk MeSH
- regulace genové exprese u nádorů MeSH
- signální dráha Wnt MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Proliferation and migration of retinal endothelial cells (RECs) contribute to the development of diabetic retinopathy. PLAG1 (pleomorphic adenoma gene 1) functions as a zinc-finger transcription factor to participate in the development of lipoblastomas or pleomorphic adenomas of the salivary glands through regulation of cell proliferation and migration. The role of PLAG1 in diabetic retinopathy was investigated in this study. Firstly, RECs were induced under high glucose conditions, which caused reduction in viability and induction of apoptosis in the RECs. Indeed, PLAG1 was elevated in high glucosetreated RECs. Functional assays showed that silence of PLAG1 increased viability and suppressed apoptosis in high glucose-induced RECs, accompanied with up-regulation of Bcl-2 and down-regulation of Bax and cleaved caspase-3. Moreover, migration of RECs was promoted by high glucose conditions, while repressed by knockdown of PLAG1. High glucose also triggered angiogenesis of RECs through up-regulation of vascular endothelial growth factor (VEGF). However, interference of PLAG1 reduced VEGF expression to retard the angiogenesis. Silence of PLAG1 also attenuated high glucose-induced up-regulation of Wnt3a, β-catenin and c-Myc in RECs. Moreover, silence of PLAG1 ameliorated histopathological changes in the retina of STZ-induced diabetic rats through down-regulation of β-catenin. In conclusion, knockdown of PLAG1 suppressed high glucose-induced angiogenesis and migration of RECs, and attenuated diabetic retinopathy by inactivation of Wnt/ β-catenin signalling.
- MeSH
- beta-katenin metabolismus MeSH
- diabetická retinopatie * MeSH
- DNA vazebné proteiny metabolismus MeSH
- endoteliální buňky metabolismus MeSH
- experimentální diabetes mellitus * MeSH
- glukosa toxicita MeSH
- kaspasa 3 MeSH
- krysa rodu rattus MeSH
- patologická angiogeneze MeSH
- protein X asociovaný s bcl-2 MeSH
- retina metabolismus MeSH
- signální dráha Wnt MeSH
- transkripční faktory MeSH
- vaskulární endoteliální růstový faktor A MeSH
- zinek MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
