BACKGROUND: The WNT/planar-cell-polarity (PCP) pathway is a key regulator of cell polarity and directional cell movements. Core PCP proteins such as Van Gogh-like2 (VANGL2) are evolutionarily highly conserved; however, the mammalian PCP machinery is still poorly understood mainly due to lack of suitable models and quantitative methodology. WNT/PCP has been implicated in many human diseases with the most distinguished positive role in the metastatic process, which accounts for more than 90% of cancer related deaths, and presents therefore an attractive target for pharmacological interventions. However, cellular assays for the assessment of PCP signaling, which would allow a more detailed mechanistic analysis of PCP function and possibly also high throughput screening for chemical compounds targeting mammalian PCP signaling, are still missing. RESULTS: Here we describe a mammalian cell culture model, which correlates B lymphocyte migration of patient-derived MEC1 cells and asymmetric localization of fluorescently-tagged VANGL2. We show by live cell imaging that PCP proteins are polarized in MEC1 cells and that VANGL2 polarization is controlled by the same mechanism as in tissues i.e. it is dependent on casein kinase 1 activity. In addition, destruction of the actin cytoskeleton leads to migratory arrest and cell rounding while VANGL2-EGFP remains polarized suggesting that active PCP signaling visualized by polarized distribution of VANGL2 is a cause for and not a consequence of the asymmetric shape of a migrating cell. CONCLUSIONS: The presented imaging-based methodology allows overcoming limitations of earlier approaches to study the mammalian WNT/PCP pathway, which required in vivo models and analysis of complex tissues. Our system investigating PCP-like signaling on a single-cell level thus opens new possibilities for screening of compounds, which control asymmetric distribution of proteins in the PCP pathway.

• MeSH
• B-Lymphocytes metabolism   pathology   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell genetics   immunology   pathology   MeSH
• Intracellular Signaling Peptides and Proteins genetics   immunology   MeSH
• Humans MeSH
• Membrane Proteins genetics   immunology   MeSH
• Cell Line, Tumor MeSH
• Cell Movement genetics   immunology   MeSH
• Cell Polarity genetics   immunology   MeSH
• Wnt Signaling Pathway genetics   immunology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The planar cell polarity (PCP) pathway is a conserved pathway that regulates cell migration and polarity in various contexts. Here we show that key PCP pathway components such as Vangl2, Celsr1, Prickle1, FZD3, FZD7, Dvl2, Dvl3, and casein kinase 1 (CK1)-ε are upregulated in B lymphocytes of patients with chronic lymphocytic leukemia (CLL). Elevated levels of PCP proteins accumulate in advanced stages of the disease. Here, we show that PCP pathway is required for the migration and transendothelial invasion of CLL cells and that patients with high expression of PCP genes, FZD3, FZD7, and PRICKLE1, have a less favorable clinical prognosis. Our findings establish that the PCP pathway acts as an important regulator of CLL cell migration and invasion. PCP proteins represent an important class of molecules regulating pathogenic interaction of CLL cells with their microenvironment.

• MeSH
• B-Lymphocytes pathology   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell metabolism   pathology   MeSH
• Humans MeSH
• Membrane Proteins metabolism   MeSH
• Mice MeSH
• Cell Movement * MeSH
• Cell Polarity * MeSH
• Wnt Signaling Pathway MeSH
• Signal Transduction MeSH
• Transplantation, Heterologous MeSH
• Up-Regulation MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Autosomal-dominant mutations in the Park8 gene encoding Leucine-rich repeat kinase 2 (LRRK2) have been identified to cause up to 40% of the genetic forms of Parkinson's disease. However, the function and molecular pathways regulated by LRRK2 are largely unknown. It has been shown that LRRK2 serves as a scaffold during activation of WNT/β-catenin signaling via its interaction with the β-catenin destruction complex, DVL1-3 and LRP6. In this study, we examine whether LRRK2 also interacts with signaling components of the WNT/Planar Cell Polarity (WNT/PCP) pathway, which controls the maturation of substantia nigra dopaminergic neurons, the main cell type lost in Parkinson's disease patients. METHODS: Co-immunoprecipitation and tandem mass spectrometry was performed in a mouse substantia nigra cell line (SN4741) and human HEK293T cell line in order to identify novel LRRK2 binding partners. Inhibition of the WNT/β-catenin reporter, TOPFlash, was used as a read-out of WNT/PCP pathway activation. The capacity of LRRK2 to regulate WNT/PCP signaling in vivo was tested in Xenopus laevis' early development. RESULTS: Our proteomic analysis identified that LRRK2 interacts with proteins involved in WNT/PCP signaling such as the PDZ domain-containing protein GIPC1 and Integrin-linked kinase (ILK) in dopaminergic cells in vitro and in the mouse ventral midbrain in vivo. Moreover, co-immunoprecipitation analysis revealed that LRRK2 binds to two core components of the WNT/PCP signaling pathway, PRICKLE1 and CELSR1, as well as to FLOTILLIN-2 and CULLIN-3, which regulate WNT secretion and inhibit WNT/β-catenin signaling, respectively. We also found that PRICKLE1 and LRRK2 localize in signalosomes and act as dual regulators of WNT/PCP and β-catenin signaling. Accordingly, analysis of the function of LRRK2 in vivo, in X. laevis revelaed that LRKK2 not only inhibits WNT/β-catenin pathway, but induces a classical WNT/PCP phenotype in vivo. CONCLUSIONS: Our study shows for the first time that LRRK2 activates the WNT/PCP signaling pathway through its interaction to multiple WNT/PCP components. We suggest that LRRK2 regulates the balance between WNT/β-catenin and WNT/PCP signaling, depending on the binding partners. Since this balance is crucial for homeostasis of midbrain dopaminergic neurons, we hypothesize that its alteration may contribute to the pathophysiology of Parkinson's disease.

• MeSH
• beta Catenin metabolism   MeSH
• Dopaminergic Neurons metabolism   MeSH
• Cadherins metabolism   MeSH
• Humans MeSH
• Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 genetics   metabolism   MeSH
• Mutation genetics   MeSH
• Cell Polarity physiology   MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Proteomics methods   MeSH
• Wnt Signaling Pathway physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

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
• Neural Crest MeSH
• Epithelial Cells MeSH
• Vertebrates MeSH
• Cell Polarity * physiology   MeSH
• Wnt Signaling Pathway * physiology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

In this review, we discuss the intricate roles of the Wnt signalling network in the development and progression of mature B-cell-derived haematological malignancies, with a focus on chronic lymphocytic leukaemia (CLL) and related B-cell lymphomas. We review the current literature and highlight the differences between the β-catenin-dependent and -independent branches of Wnt signalling. Special attention is paid to the role of the non-canonical Wnt/planar cell polarity (PCP) pathway, mediated by the Wnt-5-receptor tyrosine kinase-like orphan receptor (ROR1)-Dishevelled signalling axis in CLL. This is mainly because the Wnt/PCP co-receptor ROR1 was found to be overexpressed in CLL and the Wnt/PCP pathway contributes to numerous aspects of CLL pathogenesis. We also discuss the possibilities of therapeutically targeting the Wnt signalling pathways as an approach to disrupt the crucial interaction between malignant cells and their micro-environment. We also advocate the need for research in this direction for other lymphomas, namely, diffuse large B-cell lymphoma, Hodgkin lymphoma, mantle cell lymphoma, Burkitt lymphoma and follicular lymphoma where the Wnt signalling pathway probably plays a similar role. LINKED ARTICLES: This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.

• MeSH
• Lymphoma, B-Cell diagnosis   metabolism   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell diagnosis   metabolism   MeSH
• Humans MeSH
• Wnt Signaling Pathway * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

The primary cilium, a microtubule-based organelle found in most cells, is a centre for mechano-sensing fluid movement and cellular signalling, notably through the Hedgehog pathway. We recently found that each lens fibre cell has an apically situated primary cilium that is polarised to the side of the cell facing the anterior pole of the lens. The direction of polarity is similar in neighbouring cells so that in the global view, lens fibres exhibit planar cell polarity (PCP) along the equatorial-anterior polar axis. Ciliogenesis has been associated with the establishment of PCP, although the exact relationship between PCP and the role of cilia is still controversial. To test the hypothesis that the primary cilia have a role in coordinating the precise alignment/orientation of the fibre cells, IFT88, a key component of the intraflagellar transport (IFT) complex, was removed specifically from the lens at different developmental stages using several lens-specific Cre-expressing mouse lines (MLR10- and LR-Cre). Irrespective of which Cre-line was adopted, both demonstrated that in IFT88-depleted cells, the ciliary axoneme was absent or substantially shortened, confirming the disruption of primary cilia formation. However no obvious histological defects were detected even when IFT88 was removed from the lens placode as early as E9.5. Specifically, the lens fibres aligned/oriented towards the poles to form the characteristic Y-shaped sutures as normal. Consistent with this, in primary lens epithelial explants prepared from these conditional knockout mouse lenses, the basal bodies still showed polarised localisation at the apical surface of elongating cells upon FGF-induced fibre differentiation. We further investigated the lens phenotype in knockouts of Bardet-Biedl Syndrome (BBS) proteins 4 and 8, the components of the BBSome complex which modulate ciliary function. In these BBS4 and 8 knockout lenses, again we found the pattern of the anterior sutures formed by the apical tips of elongating/migrating fibres were comparable to the control lenses. Taken together, these results indicate that primary cilia do not play an essential role in the precise cellular alignment/orientation of fibre cells. Thus, it appears that in the lens cilia are not required to establish PCP.

• MeSH
• Cilia physiology   MeSH
• Epithelial Cells ultrastructure   MeSH
• Cells, Cultured MeSH
• Mice, Knockout MeSH
• Tumor Suppressor Proteins genetics   MeSH
• Lens, Crystalline ultrastructure   MeSH
• Cell Polarity MeSH
• Microtubule-Associated Proteins genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

PURPOSE: ROR1, a receptor in the noncanonical Wnt/planar cell polarity (PCP) pathway, is upregulated in malignant B cells of chronic lymphocytic leukemia (CLL) patients. It has been shown that the Wnt/PCP pathway drives pathogenesis of CLL, but which factors activate the ROR1 and PCP pathway in CLL cells remains unclear. EXPERIMENTAL DESIGN: B lymphocytes from the peripheral blood of CLL patients were negatively separated using RosetteSep (StemCell) and gradient density centrifugation. Relative expression of WNT5A, WNT5B, and ROR1 was assessed by quantitative real-time PCR. Protein levels, protein interaction, and downstream signaling were analyzed by immunoprecipitation and Western blotting. Migration capacity of primary CLL cells was analyzed by the Transwell migration assay. RESULTS: By analyzing the expression in 137 previously untreated CLL patients, we demonstrate that WNT5A and WNT5B genes show dramatically (five orders of magnitude) varying expression in CLL cells. High WNT5A and WNT5B expression strongly associates with unmutated IGHV and shortened time to first treatment. In addition, WNT5A levels associate, independent of IGHV status, with the clinically worst CLL subgroups characterized by dysfunctional p53 and mutated SF3B1. We provide functional evidence that WNT5A-positive primary CLL cells have increased motility and attenuated chemotaxis toward CXCL12 and CCL19 that can be overcome by inhibitors of Wnt/PCP signaling. CONCLUSIONS: These observations identify Wnt-5a as the crucial regulator of ROR1 activity in CLL and suggest that the autocrine Wnt-5a signaling pathway allows CLL cells to overcome natural microenvironmental regulation.

• MeSH
• Autocrine Communication physiology   MeSH
• B-Lymphocytes metabolism   MeSH
• Chemotaxis physiology   MeSH
• Leukemia, Lymphocytic, Chronic, B-Cell metabolism   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Cell Movement physiology   MeSH
• Wnt Proteins metabolism   MeSH
• Proto-Oncogene Proteins metabolism   MeSH
• Gene Expression Regulation, Neoplastic physiology   MeSH
• Wnt Signaling Pathway physiology   MeSH
• Receptor Tyrosine Kinase-like Orphan Receptors metabolism   MeSH
• Up-Regulation physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH

Savčí chlupy jsou uspořádány podle osy přední–zadní a jsou spojeny s velice přísnou, ale jen málo pochopenou polarizací globálních buněk, tedy fenomenon známý jako PCP (planar cell polarity). Během embryogeneze probíhají změny ve tvaru buněk a jejich polarizaci, směrem anteriorním se naklánějí, morfologicky jsou polarizovány a oddíly jsou uspořádány podle osy A-P, čili rodí se folikul. S jeho iniciací koinciduje asymetrická redistribuce Vangl2, Celsr1 a Fzd6 v bazální vrstvě. Nulové mutace Vangl2 a Celsr1 jsou zapotřebí pro jejich planární lokalizaci in vivo a fyzicky se spojují do komplexu in vitro. Nakonec je podán důkaz, že homotypické intracelulární interakce of Celsr1 jsou nezbytné pro získání Vangl2 a Fzd6 pro cell-cell kontakty.

• Publication type
• Abstracts MeSH

Wnt and BMP signaling pathways are two key molecular machineries regulating development and homeostasis. The efficient coordination of Wnt and BMP is essential in many developmental processes such as establishment of antero-posterior and dorso-ventral body axis, regulation of convergent extension, or development of various organ systems. SMAD ubiquitination regulatory factor (Smurf) family of E3 ubiquitin ligases are important and evolutionary conserved regulators of TGF-β/BMP signaling pathways. Smurf2 has been previously shown to regulate Wnt/planar cell polarity (PCP) signaling pathway by ubiquitinating Prickle1, one of the key components of PCP. We explored the role of Smurf2 in Wnt pathways in further detail and identified that Smurf2 is also a ubiquitin ligase of Dishevelled (DVL), the key cytoplasmic signal transducer in the Wnt pathway. Interestingly, the Smurf2 and DVL relationship expands beyond substrate-E3 ligase. We can show that DVL activates Smurf2, which allows Smurf2 to ubiquitinate its substrates from Wnt/PCP (Prickle1) as well as TGF-β/BMP (Smad2) pathways more efficiently. Using SMAD7 as an example of Smurf2 activator we show that DVL and SMAD7 both activates Smurf2 activity. In HEK293 cells the deficiency of DVL phenocopies absence of Smurf2 and leads to the increased phosphorylation of R-Smads. Smurf2-DVL connection provides a novel and intriguing point of crosstalk for Wnt and BMP pathways.

• MeSH
• Models, Biological MeSH
• HEK293 Cells MeSH
• Bone Morphogenetic Proteins metabolism   MeSH
• Humans MeSH
• Tumor Suppressor Proteins metabolism   MeSH
• Dishevelled Proteins metabolism   MeSH
• LIM Domain Proteins metabolism   MeSH
• Proteolysis MeSH
• Wnt Signaling Pathway * MeSH
• Signal Transduction MeSH
• Transforming Growth Factor beta metabolism   MeSH
• Ubiquitination MeSH
• Ubiquitin-Protein Ligases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Autosomal-dominant polycystic kidney disease (ADPKD) is an inherited disease that results in multiple kidney cysts, and it is a common cause of end-stage renal disease. Recent studies have shown that disease progression can be slowed by simultaneous disruption of the primary cilium and polycystins. The exact genetic mechanism of this process is still unknown. The aim of the present study was to characterize the mutation profile of ciliary signalling pathways in the renal epithelial cells of ADPKD patients. In our study, we performed an analysis of 110 genes encoding the components of Sonic Hedgehog, Hippo, Notch, Wnt and planar cell polarity signalling (PCP) by targeted next-generation sequencing. We analysed 10 formalin-fixed, paraffinembedded (FFPE) tissue samples of patients with ADPKD. We identified a unique mutation profile in each of the analysed ADPKD samples, which was characterized by the presence of pathogenic variants in eight to 11 genes involved in different signalling pathways. Despite the significant genetic heterogeneity of ADPKD, we detected five genes whose genetic variants affected most ADPKD samples. The pathogenic variants in NCOR2 and LRP2 genes were present in all analysed samples of ADPKD. In addition, eight out of 10 samples showed a pathogenic variant in the MAML2 and FAT4 genes, and six out of 10 samples in the CELSR1 gene. In our study, we identified the signalling molecules that may contribute to the cystogenesis and may represent potential targets for the development of new ADPKD treatments.

• MeSH
• DNA-Binding Proteins genetics   MeSH
• Adult MeSH
• Nuclear Proteins genetics   MeSH
• Cadherins genetics   MeSH
• TRPP Cation Channels genetics   MeSH
• Nuclear Receptor Co-Repressor 2 genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• Mutation genetics   MeSH
• Tumor Suppressor Proteins genetics   MeSH
• Pilot Projects MeSH
• Cell Polarity genetics   physiology   MeSH
• Polycystic Kidney Diseases genetics   metabolism   pathology   MeSH
• Polycystic Kidney, Autosomal Dominant genetics   metabolism   pathology   MeSH
• Disease Progression MeSH
• Low Density Lipoprotein Receptor-Related Protein-2 genetics   MeSH
• Signal Transduction genetics   physiology   MeSH
• Transcription Factors genetics   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH