Dysregulation of Wingless and Int-1 (Wnt) signaling has been strongly associated with development and progression of osteoarthritis (OA). Here, we set out to investigate the independent effects of... Show moreDysregulation of Wingless and Int-1 (Wnt) signaling has been strongly associated with development and progression of osteoarthritis (OA). Here, we set out to investigate the independent effects of either mechanical stress (MS) or inflammation on Wnt signaling in human neocartilage pellets, and to relate this Wnt signaling to OA pathophysiology. OA synovium-conditioned media (OAS-CM) was collected after incubating synovium from human end-stage OA joints for 24 h in medium. Cytokine levels in the OAS-CM were determined with a multiplex immunoassay (Luminex). Human neocartilage pellets were exposed to 20% MS, 2% OAS-CM or 1 ng/mL Interleukin-1 & beta; (IL-1 & beta;). Effects on expression levels of Wnt signaling members were determined by reverse transcription-quantitative polymerase chain reaction. Additionally, the expression of these members in articular cartilage from human OA joints was analyzed in association with joint space narrowing (JSN) and osteophyte scores. Protein levels of IL-1 & beta;, IL-6, IL-8, IL-10, tumor necrosis factor & alpha;, and granulocyte-macrophage colony-stimulating factor positively correlated with each other. MS increased noncanonical WNT5A and FOS expression. In contrast, these genes were downregulated upon stimulation with OAS-CM or IL-1 & beta;. Furthermore, Wnt inhibitors DKK1 and FRZB decreased in response to OAS-CM or IL-1 & beta; exposure. Finally, expression of WNT5A in OA articular cartilage was associated with increased JSN scores, but not osteophyte scores. Our results demonstrate that MS and inflammatory stimuli have opposite effects on canonical and noncanonical Wnt signaling in human neocartilage. Considering the extent to which MS and inflammation contribute to OA in individual patients, we hypothesize that targeting specific Wnt pathways offers a more effective, individualized approach. Show less
ObjectivesTo investigate how ANP32A, previously linked to the antioxidant response, regulates Wnt signaling as unraveled by transcriptome analysis of Anp32a-deficient mouse articular cartilage, and... Show moreObjectivesTo investigate how ANP32A, previously linked to the antioxidant response, regulates Wnt signaling as unraveled by transcriptome analysis of Anp32a-deficient mouse articular cartilage, and its implications for osteoarthritis (OA) and diseases beyond the joint.MethodsAnp32a knockdown chondrogenic ATDC5 cells were cultured in micromasses. Wnt target genes, differentiation markers and matrix deposition were quantified. Wnt target genes were determined in articular cartilage from Anp32a-deficient mice and primary human articular chondrocytes upon ANP32A silencing, using qPCR, luciferase assays and immunohistochemistry. Co-immunoprecipitation, immunofluorescence and chromatin-immunoprecipitation quantitative PCR probed the molecular mechanism via which ANP32A regulates Wnt signaling. Anp32a-deficient mice were subjected to the destabilization of the medial meniscus (DMM) OA model and treated with a Wnt inhibitor and an antioxidant. Severity of OA was assessed by cartilage damage and osteophyte formation. Human Protein Atlas data analysis identified additional organs where ANP32A may regulate Wnt signaling. Wnt target genes were determined in heart and hippocampus from Anp32a-deficient mice, and cardiac hypertrophy and fibrosis quantified.ResultsAnp32a loss triggered Wnt signaling hyper-activation in articular cartilage. Mechanistically, ANP32A inhibited target gene expression via histone acetylation masking. Wnt antagonist treatment reduced OA severity in Anp32a-deficient mice by preventing osteophyte formation but not cartilage degradation, contrasting with antioxidant treatment. Dual therapy ameliorated more OA features than individual treatments. Anp32a-deficient mice also showed Wnt hyper-activation in the heart, potentially explaining the cardiac hypertrophy phenotype found.ConclusionsANP32A is a novel translationally relevant repressor of Wnt signaling impacting osteoarthritis and cardiac disease. Show less
The immune system of mammals is responsible for protecting our body against pathogensand foreign substances (antigens), and it consists of two discrete lines of defense. The firstline called innate... Show moreThe immune system of mammals is responsible for protecting our body against pathogensand foreign substances (antigens), and it consists of two discrete lines of defense. The firstline called innate immunity and provide a quick and nonspecific defense. The innate immunityincludes different cells types, such as mast cells, macrophages, neutrophils, eosinophils,dendritic cells and natural killer (NK) cells. The second line of defense called adaptive immunityresponds in an antigen-specific manner, and comprised of B and T lymphocyte cells.This thesis focuses on one of the signals which are known to play an important role duringHSC repopulation and T cell development that is Wnt signaling pathway. Dependingon the tissue/cell types (microenvironment) and specific class of Wnt proteins binding tothe corresponding receptors on the developing lymphocytes, two discrete downstreampathways will be activated namely canonical or non-canonical Wnt pathway. The main aimof this thesis is to dissect the roles of these two distinct pathways during hematopoiesisand lymphocyte development in murine as a physiologically relevant animal model. Show less
Jansen, A.M.L.; Crobach, S.; Geurts-Giele, W.R.R.; Akker, B.E.W.M. van den; Garcia, M.V.; Ruano, D.; ... ; Morreau, H. 2017
In healthy individuals, a balance exists between bone formation and resorption. Disruption of this balance can lead to higher or lower bone mass, and disease such as osteoporosis. Treatment for... Show moreIn healthy individuals, a balance exists between bone formation and resorption. Disruption of this balance can lead to higher or lower bone mass, and disease such as osteoporosis. Treatment for osteoporosis generally inhibits bone resorption, but does not rebuild bone to a healthy strength. More knowledge on bone metabolism and new therapeutic strategies is therefore needed. Sclerostin is a protein that inhibits bone formation and is produced by osteocytes in mineralized bone matrix. Due to this specific localization and function it is viewed as one of the most interesting targets for new osteoporosis therapies. The first part of this thesis is focused on the effect of sclerostin in patients. People with inactivating mutations in sclerostin have a strongly increased bone mass. They show the effect of decreased production, and investigating these patients therefore increases knowledge on sclerostin__s mechanism of action. The regulation of sclerostin has been investigated in the second part of this thesis. In addition, antisense oligonucleotides have been used in exon skipping to decrease sclerostin expression. Finally, a new method for quantification of in vitro mineralization of bone cells was developed. This method is easy and scalable and can therefore contribute to future research into new osteoporosis therapies. Show less
Kroon, J.; in't Veld, L.S.; Buijs, J.T.; Cheung, H.; Horst, G. van der; Pluijm, G. van der 2014
Wnt genes encode highly conserved glycoproteins that play a variety of roles at different stages of development. Their functions include the regulation of cell proliferation, cell fate... Show moreWnt genes encode highly conserved glycoproteins that play a variety of roles at different stages of development. Their functions include the regulation of cell proliferation, cell fate specification, cell polarity, apoptosis, stem cell self-renewal, cell migration and tissue homeostasis. In the nervous system, Wnts act in neuronal migration, axon path finding, dendritic morphogenesis and synapse differentiation. Wnts serve as both attractive and repulsive cues during axon guidance, mediated through distinct mechanisms. The attractive responses to axonal growth are guided, at least in part, by the Frizzled receptors. Repulsive cues, on the other hand, can be mediated by the tyrosine kinase receptor Ryk. The aim of this thesis is the dissection of the basic biological and likely evolutionary conserved, functions of Wnt signaling through two different receptor families, the Ryks and Rors. We have employed genetic, biochemical, and electrophysiological approaches in order to understand the functions of these receptors and the pathways that they mediate. The studies in this thesis present novel insights into the biochemical mechanisms and the biological relevance of Wnt/Ror and Wnt/Ryk signaling for the development of a complex nervous system. Our findings can provide a starting point for the design of future therapeutic approaches for modulating the Wnt-Ryk and or Wnt-Ror pathways to treat post-injury nervous system lesions and aid neuronal regeneration. Show less
Cellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer... Show moreCellular responses to DNA damage are highly variable and strongly depend on the cellular and organismic context. Studying the DNA damage response is crucial for a better understanding of cancer formation and ageing as well as genotoxic stress-induced cancer therapy. To do justice to the multifaceted cellular changes, elicited by DNA damage, use of high-throughput techniques and integration with bioinformatics tools is of great value. This thesis summarizes recent advances in the field of systems biology studies of the DNA damage response and furthermore shows integrated approaches of the study of DNA damage response signaling networks in embryonic stem and cancer cells. By integration of transcriptional changes and the phosphorylation and metabolic response of cisplatin-treated embryonic stem cells, with RNAi-based knockdown screens we identify novel DNA damage response signaling networks, linking process such as Wnt signaling, translation arrest or altered metabolic pathways to the cellular response to DNA damage. Furthermore, genes, whose knockdown sensitizes embryonic stem cells to DNA damage-induced killing, are tested in cancer cells of varying genetic backgrounds identifying a small subset of genes, which represent potential drug targets for sensitization of cancer cells. Altogether, our systems approach for studying the DNA damage response identifies novel DNA damage-induced signaling networks and molecules, which modulate survival in the presence of DNA damage, potentially providing new targets for therapeutic intervention or biomarker discovery. Show less
By identifying and studying novel regulators, the studies described in this thesis give substantive insights into the molecular mechanisms and different levels of control of TGF-_/BMP, IL-1_ and... Show moreBy identifying and studying novel regulators, the studies described in this thesis give substantive insights into the molecular mechanisms and different levels of control of TGF-_/BMP, IL-1_ and Wnt signaling pathways. Crucially, our work for the first time demonstrated the monoubiquitination of an I-SMAD by an E2-E3 hybrid, and added an important unraveled mechanism of how monoubiquitination could affect TGF-_/BMP signaling. We found that UBE2O participates in IL-1R/TLR4-mediated NF-_B activation via a different mechanism than in BMP signaling. Future studies of ubiquitin enzymes will benefit from our model in chapter 3, which shows that UBE2O disrupts the interaction between TRAF6 and its upstream adapter MyD88 to limit polyubiquitination of TRAF6. Our studies in this thesis also added a new DUB to the list of deubiquitinases that can regulate IL-1R/TLR4 signaling, emphasizing the importance of controlling ubiquitination status in regulating NF-_B activation. Also, we reported a new co-receptor for Wnt3a-induced signaling, suggesting compensating roles for co-receptors in the control of Wnt signaling. Therefore, the studies in this thesis yield more perception in understanding how TGF-_/BMP, IL-1R/TLR4 and Wnt signaling pathways can be regulated depending on the cell type, cell localization and cell state. Show less
The main focus of this thesis is the study of the role of Wnt5 during central nervous system development in Drosophila. Wnt5, previously known as DWnt3, was identified as a gene required for proper... Show moreThe main focus of this thesis is the study of the role of Wnt5 during central nervous system development in Drosophila. Wnt5, previously known as DWnt3, was identified as a gene required for proper central nervous system development. The finding that a Wnt protein, traditionally associated with other developmental functions, is also required for axon guidance during late embryogenesis provided exciting new insights in the broad mechanisms that are used to establish the stereotypical neuronal connectivity of the central nervous system. Subsequent experiments described in this thesis reveal that, during embryonic development of the central nervous system, Wnt5 seems to be acting through an alternative pathway which involves src mediated signaling through the fractured tyrosine kinase RYK/Drl. Furthermore, we show that Wnt5 signaling through RYK/Drl is also required for proper muscle development suggesting a common signaling pathway required for muscle attachment site selection as well as axon guidance. Show less
