Liposomal packaging generates Wnt protein with in vivo biological activity.
ABSTRACT: Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.
Project description:Wnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/?-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A-WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding ?-barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.
Project description:Wnts are secreted lipid-modified glycoproteins that carry out various signaling functions during development and in adult tissue. Wnt signaling is mediated by frizzled receptors (Fzds) at the cell surface and can be modulated by the secreted frizzled-related proteins (SFRPs) and other molecular antagonists. Abnormal Wnt signaling has been implicated in several diseases. However, due to the complexity of the Wnt signal and the lack of knowledge pertaining to the binding properties of different Wnt ligands, no therapeutic agents that target this pathway exist. Using a novel enzyme-linked immunosorbent assay (ELISA)-based technique, we were able to determine the first measurements of binding affinity for specific Wnt interactions. This study shows that purified Wnt3a, Wnt7a, and Wnt5a have different binding specificities for Fzds and SFRPs.
Project description:Wnt plays important role during development and in various diseases. Because Wnts are lipidated and highly hydrophobic, they can only be purified in the presence of detergents, limiting their use in various in vitro and in vivo assays. We purified N-terminally tagged recombinant Wnt3a secreted from cells and accidentally discovered that Wnt3a co-purified with a glycoprotein afamin derived from the bovine serum included in the media. Wnt3a forms a 1:1 complex with afamin, which remains soluble in aqueous buffer after isolation, and can induce signaling in various cellular systems including the intestical stem cell growth assay. By co-expressing with afamin, biologically active afamin-Wnt complex can be easily obtained in large quantity. As afamin can also solubilize Wnt5a, Wnt3, and many more Wnt subtypes, afamin complexation will open a way to put various Wnt ligands and their signaling mechanisms under a thorough biochemical scrutiny that had been difficult for years.
Project description:Age-related fatty degeneration of the bone marrow contributes to delayed fracture-healing and osteoporosis-related fractures in the elderly. The mechanisms underlying this fatty change are unknown, but they may relate to the level of Wnt signaling within the aged marrow cavity.Transgenic mice were used in conjunction with a syngeneic bone-graft model to follow the fates of cells involved in the engraftment. Immunohistochemistry along with quantitative assays were used to evaluate Wnt signaling and adipogenic and osteogenic gene expression in bone grafts from young and aged mice. Liposomal Wnt3a protein (L-Wnt3a) was tested for its ability to restore osteogenic potential to aged bone grafts in critical-size defect models created in mice and in rabbits. Radiography, microquantitative computed tomography (micro-CT) reconstruction, histology, and histomorphometric measurements were used to quantify bone-healing resulting from L-Wnt3a or a control substance (liposomal phosphate-buffered saline solution [L-PBS]).Expression profiling of cells in a bone graft demonstrated a shift away from an osteogenic gene profile and toward an adipogenic one with age. This age-related adipogenic shift was accompanied by a significant reduction (p < 0.05) in Wnt signaling and a loss in osteogenic potential. In both large and small animal models, osteogenic competence was restored to aged bone grafts by a brief incubation with the stem-cell factor Wnt3a. In addition, liposomal Wnt3a significantly reduced cell death in the bone graft, resulting in significantly more osseous regenerate in comparison with controls.Liposomal Wnt3a enhances cell survival and reestablishes the osteogenic capacity of bone grafts from aged animals.We developed an effective, clinically applicable, regenerative medicine-based strategy for revitalizing bone grafts from aged patients.
Project description:Wnt proteins play important roles in regulating cell differentiation, proliferation and polarity. Wnts have been proposed to play roles in tissue repair and fibrosis, yet the gene expression profile of fibroblasts exposed to Wnts has not been examined. We use Affymetrix genome-wide expression profiling to show that a 6-h treatment of fibroblasts of Wnt3a results in the induction of mRNAs encoding known Wnt targets such as the fibrogenic pro-adhesive molecule connective tissue growth factor (CTGF, CCN2). Wnt3a also induces mRNAs encoding potent pro-fibrotic proteins such as TGFbeta and endothelin-1 (ET-1). Moreover, Wnt3a promotes genes associated with cell adhesion and migration, vasculature development, cell proliferation and Wnt signaling. Conversely, Wnt3a suppresses gene associated with skeletal development, matrix degradation and cell death. Results were confirmed using real-time polymerase chain reaction of cells exposed to Wnt3a and Wnt10b. These results suggest that Wnts induce genes promoting fibroblast differentiation towards angiogenesis and matrix remodeling, at the expense of skeletal development.
Project description:Colorectal cancer (CRC) is the fourth leading cause of cancer death worldwide, and constitutive activation of the Wnt signaling pathway is universal in most CRC cases. Wnt ligands (Wnts) are secreted glycoproteins and fundamentally essential for the transduction of Wnt signaling pathway. However, the 19 members of Wnts in humans imply a daunting complexity of Wnt signaling and biological effects, and our understanding of their roles in CRC tumorigenesis is still quite rudimentary. This review will give an overview of the structural characteristics and maturation process of Wnts. The expression pattern of all human Wnts in CRC tissues, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16, and their relationship with the tumorigenesis and the progression of CRC will be specifically summarized separately. Despite certain challenges, Wnt-based therapeutics for CRC emerge continuously and some are now in clinical trials. In conclusion, a deep understanding of Wnts is very helpful for a better management of this disease.
Project description:The Wnt signaling pathway plays critical roles in development. However, to date, the role of Wnts in learning and memory in adults is still not well understood. Here, we aimed to investigate the roles and mechanisms of Wnts in hippocampal-dependent contextual fear conditioning (CFC) memory formation in adult mice. CFC training induced the secretion and expression of Wnt3a and the activation of its downstream Wnt/Ca(2+) and Wnt/?-catenin signaling pathways in the dorsal hippocampus (DH). Intrahippocampal infusion of Wnt3a antibody impaired CFC acquisition and consolidation, but not expression. Using the Wnt antagonist sFRP1 or the canonical Wnt inhibitor Dkk1, we found that Wnt/Ca(2+) and Wnt/?-catenin signaling pathways were involved in acquisition and consolidation, respectively. Moreover, we found Wnt3a signaling is not only necessary but also sufficient for CFC memory. Intrahippocampal infusion of exogenous Wnt3a could enhance acquisition and consolidation of CFC. Overexpression of constitutively active ?-catenin in the DH could rescue the deficit in CFC memory consolidation, but not acquisition induced by Wnt3a antibody injection, which suggests ?-catenin signaling pathway acts downstream of Wnt3a to mediate CFC memory consolidation. Our study may help further the understanding of the precise regulation of Wnt3a in differential memory phases depending on divergent signaling pathways.
Project description:Wnts regulate important intracellular signaling events, and dysregulation of the Wnt pathway has been linked to human disease. Here, we uncover numerous Wnt canonical effectors in human platelets where Wnts, their receptors, and downstream signaling components have not been previously described. We demonstrate that the Wnt3a ligand inhibits platelet adhesion, activation, dense granule secretion, and aggregation. Wnt3a also altered platelet shape change and inhibited the activation of the small GTPase RhoA. In addition, we found the Wnt-beta-catenin signaling pathway to be functional in platelets. Finally, disruption of the Wnt Frizzled 6 receptor in the mouse resulted in a hyperactivatory platelet phenotype and a reduced sensitivity to Wnt3a. Taken together our studies reveal a novel functional role for Wnt signaling in regulating anucleate platelet function and may provide a tractable target for future antiplatelet therapy.
Project description:Wnts comprise a family of 20 lipid-modified glycoproteins in mammals and play critical roles during embryological development and organogenesis of several organ systems, including the heart. They are required for mesoderm formation and have been implicated in promoting cardiomyogenic differentiation of mammalian embryonic stem cells, but the underlying mechanisms regulating Wnt signaling during cardiomyogenesis remain poorly understood. In this report, we show that in a pluripotent mouse embryonal carcinoma stem cell line, SFRP2 inhibits cardiomyogenic differentiation by regulating Wnt3a transcription. SFRP2 inhibited early stages of cardiomyogenesis, preventing mesoderm specification and maintaining the cells in the undifferentiated state. Using a gain- and loss-of-function approach, we demonstrate that although addition of recombinant SFRP2 decreased Wnt3a transcription and cardiomyogenic differentiation, silencing of Sfrp2 led to enhanced Wnt3a transcription, mesoderm formation, and increased cardiomyogenesis. We show that the inhibitory effects of SFRP2 on Wnt transcription are secondary to interruption of a positive feedback effect of Wnt3a on its own transcription. Wnt3a increased its own transcription via the canonical pathway and TCF4 family of transcription factors, and the inhibitory effects of SFRP2 on Wnt3a transcription were associated with disruption of downstream canonical Wnt signaling. The inhibitory effects of Sfrp2 on Wnt3a expression identify Sfrp2 as a "checkpoint gene," which exerts its control on cardiomyogenesis through regulation of Wnt3a transcription.
Project description:WNT proteins are secreted symmetry breaking signals that interact with cell surface receptors of the FZD family to regulate a multitude of developmental processes. Studying selectivity between WNTs and FZDs has been hampered by the paucity of purified WNT proteins and by their apparent non-selective interactions with the FZD receptors. Here, we describe an engineered protein, called F7L6, comprised of antibody-derived single-chain variable fragments, that selectively binds to human FZD7 and the co-receptor LRP6. F7L6 potently activates WNT/?-catenin signaling in a manner similar to Wnt3a. In contrast to Wnt3a, F7L6 engages only FZD7 and none of the other FZD proteins. Treatment of human pluripotent stem (hPS) cells with F7L6 initiates transcriptional programs similar to those observed during primitive streak formation and subsequent gastrulation in the mammalian embryo. This demonstrates that selective engagement and activation of FZD7 signaling is sufficient to promote mesendodermal differentiation of hPS cells.