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Roel Nusse - One of the best experts on this subject based on the ideXlab platform.
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single molecule imaging of Wnt3a Protein diffusion on living cell membranes
Biophysical Journal, 2017Co-Authors: Anna Lippert, Roel Nusse, Jill A. Helms, Agnieszka A Janeczek, Alexandre Furstenberg, Aleks Ponjavic, W E Moerner, Nick Evans, Steven F LeeAbstract:Wnt Proteins are secreted, hydrophobic, lipidated Proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the Protein with its receptor, Frizzled, but may also regulate the transport of Wnt Protein and its localization at the cell membrane. Here, by employing single-molecule fluorescence techniques, we show that Wnt Proteins associate with and diffuse on the plasma membranes of living cells in the absence of any receptor binding. We find that labeled Wnt3A transiently and dynamically associates with the membranes of Drosophila Schneider 2 cells, diffuses with Brownian kinetics on flattened membranes and on cellular protrusions, and does not transfer between cells in close contact. In S2 receptor-plus (S2R+) cells, which express Frizzled receptors, membrane diffusion rate is reduced and membrane residency time is increased. These results provide direct evidence of Wnt3A interaction with living cell membranes, and represent, to our knowledge, a new system for investigating the dynamics of Wnt transport.
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an integral program for tissue renewal and regeneration Wnt signaling and stem cell control
Science, 2014Co-Authors: Hans Clevers, Roel NusseAbstract:BACKGROUND In adult mammalian organisms, multiple tissues—including the skin, blood, stomach, and intestines—are entrapped in a state of permanent regeneration; older cells are constantly shed, and the tissue is continuously being regenerated from resident stem cells. This phenomenon of “tissue renewal” was appreciated by Leblond in 1956, but the underlying mechanism has been unclear. It is now evident that a class of extracellular developmental signaling Proteins, known as Wnt signals, animate the continued renewal of several mammalian tissues by fuelling stem cell activity. If the Wnt pathway is inhibited, tissue renewal is crippled. This signaling pathway is an ancient evolutionary program dating from when Wnt signals arose in the simplest multicellular organisms, in which Wnts acted as primordial symmetry-breaking signals crucial for the generation of patterned tissues during embryogenesis. In vertebrates, these signals also function in pattern maintenance: They sustain tissue renewal, enabling tissues to be continuously replenished and maintained over a lifetime. Multiple adult organs are in a state of continual regeneration. In tissues such as the skin, intestines, brain, and mammary glands, Wnt signaling Proteins sustain this constant regeneration by inducing stem cells (green cells in the illustration) to grow. This leads to the robust supply of new cells (green) in order to replenish and maintain the tissue. [Image credits are available in the full article online.] ADVANCES In contrast to traditional “long-range” developmental signals, Wnts seem to act as short-range intercellular signals—acting mostly between adjacent cells. Lending credence to this notion, a membrane-tethered Wnt Protein variant can fulfill most functions of a normal Wnt Protein in Drosophila . Likely explaining the short-range nature of these signals, Wnt Proteins are attached to a lipid and therefore are hydrophobic; they cannot freely traverse the extracellular space by themselves. This provides insight into how tissue renewal is regulated. It implies that Wnt signals emanating from the stem cell microenvironment (the “niche”) may influence adjacent stem cells without affecting a broad field of cells located farther away. The concept of an external niche, however, may have to be refined because it is clear that stem cells can sometimes act as their own niche and have unexpected developmental self-organizing capacities. Last, the widespread importance of Wnt signaling in driving tissue renewal has been revealed by the identification of Axin2 and Lgr5 , genes expressed in cells that are responding to Wnt signals. Genetically labeling Axin2 + or Lgr5 + cells in a variety of tissues has revealed that such cells fuel tissue renewal in the intestines, mammary gland, skin, and brain, among other organs. OUTLOOK The amazing continuous self-regeneration of various mammalian tissues over years and decades continues to be an enigmatic terra incognita in biology. For instance, visualization of stem cells in real-time in vivo (through intravital microscopy) has shown that when some stem cells are ablated, they are replaced by more differentiated cells that are recalled to the stem cell niche, whereupon they regain stem cell identity to effect tissue repair. Therefore, lineage barriers between stem cell and differentiated fates are not always stringent and can be traversed during times of tissue damage. Reactivated Wnt signals may be instrumental in this process, and perhaps such signals could be exploited in order to enkindle tissue regeneration after injury or disease. From a pragmatic perspective, Wnt signals have already found practical use in manipulating stem cells, enabling propagation of stem cells in vitro as self-renewing cell populations and as organoids.
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Wnt5a can both activate and repress Wnt β catenin signaling during mouse embryonic development
Developmental Biology, 2012Co-Authors: Renee Van Amerongen, Christophe Fuerer, Makiko Mizutani, Roel NusseAbstract:Abstract Embryonic development is controlled by a small set of signal transduction pathways, with vastly different phenotypic outcomes depending on the time and place of their recruitment. How the same molecular machinery can elicit such specific and distinct responses, remains one of the outstanding questions in developmental biology. Part of the answer may lie in the high inherent genetic complexity of these signaling cascades, as observed for the Wnt-pathway. The mammalian genome encodes multiple Wnt Proteins and receptors, each of which show dynamic and tightly controlled expression patterns in the embryo. Yet how these components interact in the context of the whole organism remains unknown. Here we report the generation of a novel, inducible transgenic mouse model that allows spatiotemporal control over the expression of Wnt5a, a Protein implicated in many developmental processes and multiple Wnt-signaling responses. We show that ectopic Wnt5a expression from E10.5 onwards results in a variety of developmental defects, including loss of hair follicles and reduced bone formation in the skull. Moreover, we find that Wnt5a can have dual signaling activities during mouse embryonic development. Specifically, Wnt5a is capable of both inducing and repressing β-catenin/TCF signaling in vivo, depending on the time and site of expression and the receptors expressed by receiving cells. These experiments show for the first time that a single mammalian Wnt Protein can have multiple signaling activities in vivo, thereby furthering our understanding of how signaling specificity is achieved in a complex developmental context.
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Wnt Proteins Are Self-Renewal Factors for Mammary Stem Cells and Promote Their Long-Term Expansion in Culture
Cell Stem Cell, 2010Co-Authors: Yi Arial Zeng, Roel NusseAbstract:Adult stem cells have the ability to self-renew and to generate specialized cells. Self-renewal is dependent on extrinsic niche factors but few of those signals have been identified. In addition, stem cells tend to differentiate in the absence of the proper signals and are therefore difficult to maintain in cell culture. The mammary gland provides an excellent system to study self-renewal signals, because the organ develops postnatally, arises from stem cells, and is readily generated from transplanted cells. We show here that adult mammary glands contain a Wnt-responsive cell population that is enriched for stem cells. In addition, stem cells mutant for the negative-feedback regulator Axin2 and therefore sensitized to Wnt signals have a competitive advantage in mammary gland reconstitution assays. In cell culture experiments, exposure to purified Wnt Protein clonally expands mammary stem cells for many generations and maintains their ability to generate functional glands in transplantation assays. We conclude that Wnt Proteins serve as rate-limiting self-renewal signals acting directly on mammary stem cells.
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Lipid-independent Secretion of a Drosophila Wnt Protein
Journal of Biological Chemistry, 2008Co-Authors: Wendy Ching, Howard C. Hang, Roel NusseAbstract:Abstract Wnt Proteins comprise a large class of secreted signaling molecules with key roles during embryonic development and throughout adult life. Recently, much effort has been focused on understanding the factors that regulate Wnt signal production. For example, Porcupine and Wntless/Evi/Sprinter have been identified as being required in Wnt-producing cells for the processing and secretion of many Wnt Proteins. Interestingly, in this study we find that WntD, a recently characterized Drosophila Wnt family member, does not require Porcupine or Wntless/Evi/Sprinter for its secretion or signaling activity. Because Porcupine is involved in post-translational lipid modification of Wnt Proteins, we used a novel labeling method and mass spectrometry to ask whether WntD undergoes lipid modification and found that it does not. Although lipid modification is also hypothesized to be required for Wnt secretion, we find that WntD is secreted very efficiently. WntD secretion does, however, maintain a requirement for the secretory pathway component Rab1. Our results show that not all Wnt family members require lipid modification, Porcupine, or Wntless/Evi/Sprinter for secretion and suggest that different modes of secretion may exist for different Wnt Proteins.
Tommy Andersson - One of the best experts on this subject based on the ideXlab platform.
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Wnt5a induces release of exosomes containing pro angiogenic and immunosuppressive factors from malignant melanoma cells
Molecular Cancer, 2014Co-Authors: Elin J Ekstrom, Tommy Andersson, Caroline Bergenfelz, Verena Von Bulow, Filiz Serifler, Eric Carlemalm, Goran Jonsson, Karin LeanderssonAbstract:Wnt Proteins are important for developmental processes and certain diseases. Wnt5A is a non-canonical Wnt Protein that previously has been shown to play a role in the progression of malignant melanoma. High expression of Wnt5A in melanoma tumors correlates to formation of distant metastasis and poor prognosis. This has partly been described by the findings that Wnt5A expression in melanoma cell lines increases migration and invasion.
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Wnt5a stimulates migration and invasion in OSCC
Oral Oncology, 2013Co-Authors: Zdenka Prgomet, Pia Lindberg, Tommy AnderssonAbstract:Purpose Wnt5a is the most important Wnt Protein activating the non-canonical Wnt pathway. It regulates proliferation, differentiation, migration, adhesion and polarity of the cell. Clinical studies have shown that Wnt5a could act as a prognostic marker in various cancers. It seems that Wnt5a acts both as a tumor suppressor (breast, thyroid, colon and livercancer) and as a tumor promotor (malignant melanoma, pancreatic and gastric cancer). In oral squamous cell carcinoma (OSCC) a number of Wnt and Frizzled genes are expressed, mostly Wnt5a and Frizzled-5 but the role of Wnt5a has not yet been thoroughly elucidated. The aim of this study is to evaluate the role of Wnt5a in oral OSCC and its influence on the cell proliferation, migration and invasion. Material and methods The Protein content of Wnt5a in two OSCC cell lines (SCC25 and SCC9) was determined by Western blotting. The influence of rWnt5a and two Wnt5a-derived hexapeptides: Foxy5 (an agonist that mimics Wnt5a activity) and Box5 (an antagonist that inhibits Wnt5a) on the migration and proliferation in OSCC cell lines was studied by a scratch assay and BrdU. Invasion influenced by rWnt5a was studied using Boyden chambers. Results Both cell lines express Wnt5a. Dose–response curves of rWnt5a in SCC9 and SCC25 indicate that Wnt5a increases migration with statistically significant effective concentration at 0.4 μg/ml but has no effect on the proliferation of the two cell lines. To confirm this we used Foxy5 and Box5. Foxy5 increases migration with statistically significant effective concentration at 150 μM while Box5 inhibits Wnt5a effect on the migration. Foxy5 and Box5 have no influence on proliferation. Invasion was increased by rWnt5a. Conclusions The results demonstrate that Wnt5a increases migration and invasion but has no influence on proliferation in OSCC cell lines.
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Wnt 5a mrna translation is suppressed by the elav like Protein hur in human breast epithelial cells
Nucleic Acids Research, 2006Co-Authors: Karin Leandersson, Kristian Riesbeck, Tommy AnderssonAbstract:Wnt-5a is a non-transforming Wnt Protein. Since Wnt-5a mRNA and Protein levels differ within and between tumours, the potential of Wnt-5a as a prognostic factor has been debated. We have previously shown that the lack of Wnt-5a Protein is a predictor of shorter disease-free survival in human breast cancer. Recently, however, we also showed that the breast tumours lacking Wnt-5a Protein had a high or normal level of Wnt-5a mRNA that might explain the discrepancies in previous studies. We here report that Wnt-5a is regulated at the post-transcriptional level. The regulation was mediated by the Embryonic Lethal Abnormal Vision (ELAV)like Protein HuR, which inhibited translation of Wnt-5a when bound to highly conserved AU-rich sequences in the 3'-untranslated region (3'-UTR) of the Wnt-5a mRNA molecule, as shown by both HA-tagged Wnt-5a- and Luciferase-Wnt-5a-3'-UTR reporter assays. The HuR-dependent inhibition of Wnt-5a was supported by the fact that active HuR is located in the cytoplasm in invasive human breast tumours and that hypoxia-induced activation of HuR inhibits translation of both Luciferase-Wnt5a-3'-UTR and endogenous Wnt-5a Protein. We propose that the lack of Wnt-5a Protein expression in invasive human breast tumours is caused by a HuR-mediated suppression of Wnt-5a mRNA translation.. (Less)
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Wnt 5a Protein expression in primary dukes b colon cancers identifies a subgroup of patients with good prognosis
Cancer Research, 2005Co-Authors: Janna Dejmek, Annika Dejmek, Annette Safholm, Anita Sjolander, Tommy AnderssonAbstract:Oncogenic Wnt/β-catenin signaling occurs in a majority of colorectal cancers. In contrast, very little is known about the role of the nontransforming Wnt Protein family member Wnt-5a in those tumors. In the most common of the three colon cancer stages, Dukes B or lymph node–negative, the outcome is the hardest to predict. We searched for a predictive marker in this group and observed loss of or reduced Wnt-5a expression in 50% of Dukes B tumors. Such Wnt-5a negativity was a strong predictor of adverse outcome, with a relative risk of death of 3.007 (95% confidence interval, 1.336-6.769; P = 0.008) after 5 years in Wnt-5a-negative patients. Furthermore, the median survival time after diagnosis was 109.1 months for patients with Wnt-5a-positive primary tumors but only 58 months for those with Wnt-5a-negative primary tumors. To find a possible biological explanation for these results, we studied the invasive and poorly differentiated human colon cancer cell line, SW480, which does not express Wnt-5a Protein and the Wnt-5a-expressing and moderately differentiated Caco2 colon cancer cell line. We found that the addition of recombinant/purified Wnt-5a significantly reduced the migratory capacity of SW480 cells. By comparison, equivalent treatment did not significantly alter migration in the Wnt-5a-expressing Caco2 colon cancer cell line. These findings indicate that the expression of Wnt-5a in primary Dukes B colon cancer tissue constitutes a good prognostic marker for longer survival, which can be explained by the ability of Wnt-5a to impair tumor cell migration and thus reduce invasiveness and metastasis.
Karin Leandersson - One of the best experts on this subject based on the ideXlab platform.
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Wnt5a induces release of exosomes containing pro angiogenic and immunosuppressive factors from malignant melanoma cells
Molecular Cancer, 2014Co-Authors: Elin J Ekstrom, Tommy Andersson, Caroline Bergenfelz, Verena Von Bulow, Filiz Serifler, Eric Carlemalm, Goran Jonsson, Karin LeanderssonAbstract:Wnt Proteins are important for developmental processes and certain diseases. Wnt5A is a non-canonical Wnt Protein that previously has been shown to play a role in the progression of malignant melanoma. High expression of Wnt5A in melanoma tumors correlates to formation of distant metastasis and poor prognosis. This has partly been described by the findings that Wnt5A expression in melanoma cell lines increases migration and invasion.
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Wnt5a induces a tolerogenic phenotype of macrophages in sepsis and breast cancer patients
Journal of Immunology, 2012Co-Authors: Caroline Bergenfelz, Elin J Ekstrom, Catharina Medrek, Karin Jirstrom, Helena Janols, Marlene Wullt, Anders Bredberg, Karin LeanderssonAbstract:A well-orchestrated inflammatory reaction involves the induction of effector functions and, at a later stage, an active downregulation of this potentially harmful process. In this study we show that under proinflammatory conditions the noncanonical Wnt Protein, Wnt5a, induces immunosuppressive macrophages. The suppressive phenotype induced by Wnt5a is associated with induction of IL-10 and inhibition of the classical TLR4-NF-κB signaling. Interestingly, this phenotype closely resembles that observed in reprogrammed monocytes in sepsis patients. The Wnt5a-induced feedback inhibition is active both during in vitro LPS stimulation of macrophages and in patients with sepsis caused by LPS-containing, Gram-negative bacteria. Furthermore, using breast cancer patient tissue microarrays, we find a strong correlation between the expression of Wnt5a in malignant epithelial cells and the frequency of CD163+ anti-inflammatory tumor-associated macrophages. In conclusion, our data point out Wnt5a as a potential target for an efficient therapeutic modality in severe human diseases as diverse as sepsis and malignancy.
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Wnt 5a mrna translation is suppressed by the elav like Protein hur in human breast epithelial cells
Nucleic Acids Research, 2006Co-Authors: Karin Leandersson, Kristian Riesbeck, Tommy AnderssonAbstract:Wnt-5a is a non-transforming Wnt Protein. Since Wnt-5a mRNA and Protein levels differ within and between tumours, the potential of Wnt-5a as a prognostic factor has been debated. We have previously shown that the lack of Wnt-5a Protein is a predictor of shorter disease-free survival in human breast cancer. Recently, however, we also showed that the breast tumours lacking Wnt-5a Protein had a high or normal level of Wnt-5a mRNA that might explain the discrepancies in previous studies. We here report that Wnt-5a is regulated at the post-transcriptional level. The regulation was mediated by the Embryonic Lethal Abnormal Vision (ELAV)like Protein HuR, which inhibited translation of Wnt-5a when bound to highly conserved AU-rich sequences in the 3'-untranslated region (3'-UTR) of the Wnt-5a mRNA molecule, as shown by both HA-tagged Wnt-5a- and Luciferase-Wnt-5a-3'-UTR reporter assays. The HuR-dependent inhibition of Wnt-5a was supported by the fact that active HuR is located in the cytoplasm in invasive human breast tumours and that hypoxia-induced activation of HuR inhibits translation of both Luciferase-Wnt5a-3'-UTR and endogenous Wnt-5a Protein. We propose that the lack of Wnt-5a Protein expression in invasive human breast tumours is caused by a HuR-mediated suppression of Wnt-5a mRNA translation.. (Less)
Jill A. Helms - One of the best experts on this subject based on the ideXlab platform.
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Bone formation around unstable implants is enhanced by a Wnt Protein therapeutic in a preclinical in vivo model.
Clinical Oral Implants Research, 2020Co-Authors: Benjamin R. Coyac, John B. Brunski, Brian Leahy, Giuseppe Salvi, Xing Yin, Jill A. HelmsAbstract:OBJECTIVES Our objective was to test the hypothesis that local delivery of a Wnt Protein therapeutic would support osseointegration of an unstable implant placed into an oversized osteotomy and subjected to functional loading. MATERIALS AND METHODS Using a split-mouth design in an ovariectomized (OVX) rat model, 50 titanium implants were placed in oversized osteotomies. Implants were subjected to functional loading. One-half of the implants were treated with a liposomal formulation of Wnt3A Protein (L-Wnt3A); the other half received an identical liposomal formulation containing phosphate-buffered saline (PBS). Finite element modeling estimated peri-implant strains caused by functional loading. Histological, molecular, cellular, and quantitative micro-computed tomographic (µCT) imaging analyses were performed on samples from post-implant days (PID) 3, 7, and 14. Lateral implant stability was quantified at PID 7 and 14. RESULTS Finite element analyses predicted levels of peri-implant strains incompatible with new bone formation. Micro-CT imaging, histological, and quantitative immunohistochemical (IHC) analyses confirmed that PBS-treated implants underwent fibrous encapsulation. In those cases where the peri-implant environment was treated with L-Wnt3A, µCT imaging, histological, and quantitative IHC analyses demonstrated a significant increase in expression of proliferative (PCNA) and osteogenic (Runx2, Osterix) markers. One week after L-Wnt3A treatment, new bone formation was evident, and two weeks later, L-Wnt3A-treated gaps had a stiffer interface compared to PBS-treated gaps. CONCLUSION In a rat model, unstable implants undergo fibrous encapsulation. If the same unstable implants are treated with L-Wnt3A at the time of placement, then it results in significantly more peri-implant bone and greater interfacial stiffness.
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a Wnt Protein therapeutic improves the bone forming capacity of autografts from aged animals
Scientific Reports, 2018Co-Authors: Tao Chen, Sylvain Mouraret, Luis A Cordova, Bo Liu, Qiang Sun, Benjamin Salmon, Jill A. HelmsAbstract:Autografts tend to be unreliable in older patients. Some of these age-related skeletal changes appear to be attributable to a decline in endogenous Wnt signaling. We used a functional in vivo transplantation assay to demonstrate that the bone-forming capacity of an autograft can be traced back to a Wnt-responsive cell population associated with the mineralized bone matrix fraction of a bone graft. Micro-CT imaging, flow cytometry and quantitative analyses demonstrate that this mineralized fraction declines with age, along with a waning in endogenous Wnt signaling; together these factors contribute to the age-related deterioration in autograft efficacy. Using a lipid formulation to stabilize the hydrophobic Wnt3A Protein, we demonstrate that osteogenic capacity can be restored by incubating the bone graft ex vivo with Wnt3A. Compared to control bone grafts, Wnt-treated bone grafts give rise to three times more bone. These preclinical results establish a pivotal role for Wnt signaling in the age-related decline of autologous bone grafting efficacy, and demonstrate a means to restore that efficacy via local, transient amplification of endogenous Wnt signaling.
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single molecule imaging of Wnt3a Protein diffusion on living cell membranes
Biophysical Journal, 2017Co-Authors: Anna Lippert, Roel Nusse, Jill A. Helms, Agnieszka A Janeczek, Alexandre Furstenberg, Aleks Ponjavic, W E Moerner, Nick Evans, Steven F LeeAbstract:Wnt Proteins are secreted, hydrophobic, lipidated Proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the Protein with its receptor, Frizzled, but may also regulate the transport of Wnt Protein and its localization at the cell membrane. Here, by employing single-molecule fluorescence techniques, we show that Wnt Proteins associate with and diffuse on the plasma membranes of living cells in the absence of any receptor binding. We find that labeled Wnt3A transiently and dynamically associates with the membranes of Drosophila Schneider 2 cells, diffuses with Brownian kinetics on flattened membranes and on cellular protrusions, and does not transfer between cells in close contact. In S2 receptor-plus (S2R+) cells, which express Frizzled receptors, membrane diffusion rate is reduced and membrane residency time is increased. These results provide direct evidence of Wnt3A interaction with living cell membranes, and represent, to our knowledge, a new system for investigating the dynamics of Wnt transport.
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Rescuing failed oral implants via Wnt activation
Journal of Clinical Periodontology, 2016Co-Authors: Jingtao Li, Sylvain Mouraret, John B. Brunski, Girija Dhamdhere, Tao Chen, Jill A. HelmsAbstract:AIM: Implant osseointegration is not always guaranteed and once fibrous encapsulation occurs clinicians have few options other than implant removal. Our goal was to test whether a Wnt Protein therapeutic could rescue such failed implants. MATERIAL AND METHODS: Titanium implants were placed in over-sized murine oral osteotomies. A lack of primary stability was verified by mechanical testing. Interfacial strains were estimated by finite element modelling and histology coupled with histomorphometry confirmed the lack of peri-implant bone. After fibrous encapsulation was established peri-implant injections of a liposomal formulation of Wnt3A Protein (L-Wnt3A) or liposomal PBS (L-PBS) were then initiated. Quantitative assays were employed to analyse the effects of L-Wnt3A treatment. RESULTS: Implants in gap-type interfaces exhibited high interfacial strains and no primary stability. After verification of implant failure, L-Wnt3A or L-PBS injections were initiated. L-Wnt3A induced a rapid, significant increase in Wnt responsiveness in the peri-implant environment, cell proliferation and osteogenic Protein expression. The amount of peri-implant bone and bone in contact with the implant were significantly higher in L-Wnt3A cases. CONCLUSIONS: These data demonstrate L-Wnt3A can induce peri-implant bone formation even in cases where fibrous encapsulation predominates.
Junichi Takagi - One of the best experts on this subject based on the ideXlab platform.
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Active and water-soluble form of lipidated Wnt Protein is maintained by a serum glycoProtein afamin/α-albumin
eLife, 2016Co-Authors: Emiko Mihara, Hidenori Hirai, Hideki Yamamoto, Keiko Tamura-kawakami, Mami Matano, Akira Kikuchi, Toshiro Sato, Junichi TakagiAbstract:The Wnt signaling pathway helps animal cells to communicate with each other to coordinate the formation of tissues and organs. The pathway relies on a Protein called Wnt that is released from cells and binds to a receptor Protein called Frizzled on the surface of other cells to trigger changes in gene activation. Defects in the Wnt signaling pathway contribute to cancer and other diseases. Great progress has been made in understanding Wnt signaling, but certain types of experiments have been hindered because it has been difficult to isolate pure Wnt Proteins. This is partly because Wnt Proteins are attached to a fatty molecule that is important for their activity but also makes these Proteins “hydrophobic,” or repelled by water. Hydrophobic Proteins have a strong tendency to clump or aggregate when they are isolated from cells, which reduces the biological activity of Proteins. Adding detergents to the aggregates can break them apart, but can also hinder the Proteins’ activities and cannot be used in all experiments. Previous research has shown that mammalian cells grown in the presence of blood serum can produce Wnt Proteins that do not aggregate. Blood serum is a complex mixture of different molecules obtained from blood and is commonly added to cells grown in the laboratory. However, adding serum can have also undesirable effects and it is not understood why serum stops Wnt Proteins forming aggregates. Using biochemical methods, Mihara et al. have now identified the component in blood serum that prevents Wnt Proteins from aggregating. The experiments showed that a Protein in the blood serum called afamin binds tightly to Wnt Proteins. Furthermore, the complex between afamin and Wnt was biologically active, and could bind to the Frizzled receptor and trigger an appropriate response in cells. Mihara et al. then generated cells that produced both afamin and Wnt and used them to purify large amounts of biologically active Wnt/afamin complexes. This method avoids the potentially undesirable effects of using detergents or serum, and will therefore likely be useful for future experiments and therapeutic applications. Further work is also needed to understand why afamin binds to Wnt Proteins and whether this is important for Wnt signaling.
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active and water soluble form of lipidated Wnt Protein is maintained by a serum glycoProtein afamin α albumin
eLife, 2016Co-Authors: Emiko Mihara, Hidenori Hirai, Hideki Yamamoto, Mami Matano, Akira Kikuchi, Toshiro Sato, Keiko Tamurakawakami, Junichi TakagiAbstract:The Wnt signaling pathway helps animal cells to communicate with each other to coordinate the formation of tissues and organs. The pathway relies on a Protein called Wnt that is released from cells and binds to a receptor Protein called Frizzled on the surface of other cells to trigger changes in gene activation. Defects in the Wnt signaling pathway contribute to cancer and other diseases. Great progress has been made in understanding Wnt signaling, but certain types of experiments have been hindered because it has been difficult to isolate pure Wnt Proteins. This is partly because Wnt Proteins are attached to a fatty molecule that is important for their activity but also makes these Proteins “hydrophobic,” or repelled by water. Hydrophobic Proteins have a strong tendency to clump or aggregate when they are isolated from cells, which reduces the biological activity of Proteins. Adding detergents to the aggregates can break them apart, but can also hinder the Proteins’ activities and cannot be used in all experiments. Previous research has shown that mammalian cells grown in the presence of blood serum can produce Wnt Proteins that do not aggregate. Blood serum is a complex mixture of different molecules obtained from blood and is commonly added to cells grown in the laboratory. However, adding serum can have also undesirable effects and it is not understood why serum stops Wnt Proteins forming aggregates. Using biochemical methods, Mihara et al. have now identified the component in blood serum that prevents Wnt Proteins from aggregating. The experiments showed that a Protein in the blood serum called afamin binds tightly to Wnt Proteins. Furthermore, the complex between afamin and Wnt was biologically active, and could bind to the Frizzled receptor and trigger an appropriate response in cells. Mihara et al. then generated cells that produced both afamin and Wnt and used them to purify large amounts of biologically active Wnt/afamin complexes. This method avoids the potentially undesirable effects of using detergents or serum, and will therefore likely be useful for future experiments and therapeutic applications. Further work is also needed to understand why afamin binds to Wnt Proteins and whether this is important for Wnt signaling.
