Non-canonical Wnt signalling modulates the endothelial shear stress flow sensor in vascular remodelling.
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ABSTRACT:
SUBMITTER: Franco CA
PROVIDER: S-EPMC4798962 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Reactive oxygen species regulate canonical Wnt signaling. However, the role of the redox regulatory protein p66(Shc) in the canonical Wnt pathway is not known. We investigated whether p66(Shc) is essential for canonical Wnt signaling in the endothelium and determined whether the canonical Wnt pathway induces vascular endothelial dysfunction via p66(Shc)-mediated oxidative stress.The canonical Wnt ligand Wnt3a induced phosphorylation (activation) of p66(Shc) in endothelial cells. Wnt3a-stimulated dephosphorylation of ?-catenin, and ?-catenin-dependent transcription, was inhibited by knockdown of p66(Shc). Exogenous H2O2-induced ?-catenin dephosphorylation was also mediated by p66(Shc). Moreover, p66(Shc) overexpression dephosphorylated ?-catenin and increased ?-catenin-dependent transcription, independent of Wnt3a ligand. P66(Shc)-induced ?-catenin dephosphorylation was inhibited by antioxidants N-acetyl cysteine and catalase. Wnt3a upregulated endothelial NADPH oxidase-4, and ?-catenin dephosphorylation was suppressed by knocking down NADPH oxidase-4 and by antioxidants. Wnt3a increased H2O2 levels in endothelial cells and impaired endothelium-dependent vasorelaxation in mouse aortas, both of which were rescued by p66(Shc) knockdown. P66(Shc) knockdown also inhibited adhesion of monocytes to Wnt3a-stimulated endothelial cells. Furthermore, constitutively active ?-catenin expression in the endothelium increased vascular reactive oxygen species and impaired endothelium-dependent vasorelaxation. In vivo, high-fat diet feeding-induced endothelial dysfunction in mice was associated with increased endothelial Wnt3a, dephosphorylated ?-catenin, and phosphorylated p66(Shc). High-fat diet-induced dephosphorylation of endothelial ?-catenin was diminished in mice in which p66(Shc) was knocked down.p66(Shc) plays a vital part in canonical Wnt signaling in the endothelium and mediates Wnt3a-stimulated endothelial oxidative stress and dysfunction.
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Project description:The Wnt signalling pathways are of great importance in embryonic development and oncogenesis. Canonical and non-canonical Wnt signalling pathways are known, with the canonical (or β-catenin dependent) pathway being perhaps the best studied of these. While structural knowledge of proteins and interactions involved in canonical Wnt signalling has accumulated over the past 20 years, the pace of discovery has increased in recent years, with the structures of several key proteins and assemblies in the pathway being released. In this review, we provide a brief overview of canonical Wnt signalling, followed by a comprehensive overview of currently available X-ray, NMR and cryoEM data elaborating the structures of proteins and interactions involved in canonical Wnt signalling. While the volume of structures available is considerable, numerous gaps in knowledge remain, particularly a comprehensive understanding of the assembly of large multiprotein complexes mediating key aspects of pathway, as well as understanding the structure and activation of membrane receptors in the pathway. Nonetheless, the presently available data affords considerable opportunities for structure-based drug design efforts targeting canonical Wnt signalling.
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Project description:We previously reported pro-survival effects of Wnt3a and Wnt5a proteins in vascular smooth muscle cells (VSMCs). Wnt5a achieved this through induction of Wnt1-inducible signalling pathway protein-1 (WISP-1) consequent to ?-catenin/CREB-dependent, TCF-independent, signalling. However, we found that as atherosclerosis advances, although Wnt5a protein was increased, WISP-1 was reduced. We hypothesized this disconnect could be due to aging. In this study, we elucidate the mechanism underlying Wnt3a pro-survival signalling and demonstrate the differential effect of age on Wnt3a- and Wnt5a-mediated survival. We show Wnt3a protein was expressed in human atherosclerotic coronary arteries and co-located with macrophages and VSMCs. Meanwhile, Wnt3a stimulation of primary mouse VSMCs increased ?-catenin nuclear translocation and TCF, but not CREB, activation. Wnt3a increased mRNA expression of the pro-survival factor WISP-2 in a TCF-dependent manner. Functionally, ?-catenin/TCF inhibition or WISP-2 neutralization significantly impaired Wnt3a-mediated VSMC survival. WISP-2 was upregulated in human atherosclerosis and partly co-localized with Wnt3a. The pro-survival action of Wnt3a was effective in VSMCs from young (2 month) and old (18-20 month) mice, whereas Wnt5a-mediated rescue was impaired with age. Further investigation revealed that although Wnt5a induced ?-catenin nuclear translocation in VSMCs from both ages, CREB phosphorylation and WISP-1 upregulation did not occur in old VSMCs. Unlike Wnt5a, pro-survival Wnt3a signalling involves ?-catenin/TCF and WISP-2. While Wnt3a-mediated survival was unchanged with age, Wnt5a-mediated survival was lost due to impaired CREB activation and WISP-1 regulation. Greater understanding of the effect of age on Wnt signalling may identify targets to promote VSMC survival in elderly patients with atherosclerosis.
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Project description:Shear stress induces directed endothelial cell (EC) migration in blood vessels leading to vessel diameter increase and induction of vascular maturation. Other factors, such as EC elongation and interaction between ECs and non-vascular areas are also important. Computational models have previously been used to study collective cell migration. These models can be used to predict EC migration and its effect on vascular remodelling during embryogenesis. We combined live time-lapse imaging of the remodelling vasculature of the quail embryo yolk sac with flow quantification using a combination of micro-Particle Image Velocimetry and computational fluid dynamics. We then used the flow and remodelling data to inform a model of EC migration during remodelling. To obtain the relation between shear stress and velocity in vitro for EC cells, we developed a flow chamber to assess how confluent sheets of ECs migrate in response to shear stress. Using these data as an input, we developed a multiphase, self-propelled particles (SPP) model where individual agents are driven to migrate based on the level of shear stress while maintaining appropriate spatial relationship to nearby agents. These agents elongate, interact with each other, and with avascular agents at each time-step of the model. We compared predicted vascular shape to real vascular shape after 4 hours from our time-lapse movies and performed sensitivity analysis on the various model parameters. Our model shows that shear stress has the largest effect on the remodelling process. Importantly, however, elongation played an especially important part in remodelling. This model provides a powerful tool to study the input of different biological processes on remodelling.
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Project description:Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD-LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.
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Project description:Wnt signalling plays essential roles during embryonic development and is known to be mis-regulated in human disease. There are many molecular mechanisms that ensure tight regulation of Wnt activity. One such regulator is the heparan-sulfate-specific 6-O-endosulfatase Sulf1. Sulf1 acts extracellularly to modify the structure of heparan sulfate chains to affect the bio-availability of Wnt ligands. Sulf1 could, therefore, influence the formation of Wnt signalling complexes to modulate the activation of both canonical and non-canonical pathways. In this study, we use well-established assays in Xenopus to investigate the ability of Sulf1 to modify canonical and non-canonical Wnt signalling. In addition, we model the ability of Sulf1 to influence morphogen gradients using fluorescently tagged Wnt ligands in ectodermal explants. We show that Sulf1 overexpression has ligand-specific effects on Wnt signalling: it affects membrane accumulation and extracellular levels of tagged Wnt8a and Wnt11b ligands differently, and inhibits the activity of canonical Wnt8a but enhances the activity of non-canonical Wnt11b.
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Project description:AimsFluid shear stress (SS) is known to regulate endothelial cell (EC) function. Most of the studies, however, focused on the effects of cell-free fluid-generated wall SS on ECs. The objective of this study was to investigate how changes in blood flow altered EC signalling and endothelial function directly through wall SS and indirectly through SS effects on red blood cells (RBCs).Methods and resultsExperiments were conducted in individually perfused rat venules. We experimentally induced changes in SS that were quantified by measured flow velocity and fluid viscosity. The concomitant changes in EC [Ca2+]i and nitric oxide (NO) were measured with fluorescent markers, and EC barrier function was assessed by fluorescent microsphere accumulation at EC junctions using confocal imaging. EC eNOS activation was evaluated by immunostaining. In response to changes in SS, increases in EC [Ca2+]i and gap formation occurred only in blood or RBC solution perfused vessels, whereas SS-dependent NO production and eNOS-Ser1177 phosphorylation occurred in both plasma and blood perfused vessels. A bioluminescent assay detected SS-dependent ATP release from RBCs. Pharmacological inhibition and genetic modification of pannexin-1 channels on RBCs abolished SS-dependent ATP release and SS-induced increases in EC [Ca2+]i and gap formation.ConclusionsSS-induced EC NO production occurs in both cell free fluid and blood perfused vessels, whereas SS-induced increases in EC [Ca2+]i and EC gap formation require the presence of RBCs, attributing to SS-induced pannexin-1 channel dependent release of ATP from RBCs. Thus, changes in blood flow alter vascular EC function through both wall SS and SS exerted on RBCs, and RBC released ATP contributes to SS-induced changes in EC barrier function.
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Project description:Many organs with a high cell turnover (for example, skin, intestine and blood) are composed of short-lived cells that require continuous replenishment by somatic stem cells. Ageing results in the inability of these tissues to maintain homeostasis and it is believed that somatic stem-cell ageing is one underlying cause of tissue attrition with age or age-related diseases. Ageing of haematopoietic stem cells (HSCs) is associated with impaired haematopoiesis in the elderly. Despite a large amount of data describing the decline of HSC function on ageing, the molecular mechanisms of this process remain largely unknown, which precludes rational approaches to attenuate stem-cell ageing. Here we report an unexpected shift from canonical to non-canonical Wnt signalling in mice due to elevated expression of Wnt5a in aged HSCs, which causes stem-cell ageing. Wnt5a treatment of young HSCs induces ageing-associated stem-cell apolarity, reduction of regenerative capacity and an ageing-like myeloid-lymphoid differentiation skewing via activation of the small Rho GTPase Cdc42. Conversely, Wnt5a haploinsufficiency attenuates HSC ageing, whereas stem-cell-intrinsic reduction of Wnt5a expression results in functionally rejuvenated aged HSCs. Our data demonstrate a critical role for stem-cell-intrinsic non-canonical Wnt5a signalling in HSC ageing.
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