A dual role of the Wnt signaling pathway during aging in C aenorhabditis elegans
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Wnt signaling is a major and highly conserved developmental pathway that guides many important events during embryonic and larval development. In adulthood, misregulation of Wnt signaling has been implicated in tumorigenesis and various age-related diseases. These effects occur through highly complicated cell-to-cell interactions mediated by multiple Wnt-secreted proteins. While they share a high degree of sequence similarity, their function is highly diversified. Although the role of Wnt ligands during development is well studied, very little is known about the possible actions of Wnt signaling in natural aging. In this study, Caenorhabditis elegans serves, for the first time, as a model system to determine the role of Wnt ligands in aging. Caenorhabditis elegans has five Wnt proteins, mom-2, egl-20, lin-44, cwn-1, and cwn-2. We show that all five Wnt ligands are expressed and active past the development stages. The ligand mom-2/Wnt plays a major detrimental role in longevity, whereas the function of lin-44/Wnt is beneficial for long life. Interestingly, no evidence was found for Wnt signaling being involved in cellular or oxidative stress responses during aging. Our results suggest that Wnt signaling regulates aging-intrinsic genetic pathways, opening a new research direction on the role of Wnt signaling in aging and age-related diseases.Keywords:
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Bone remodeling, which plays an important role in maintaining bone homeostasis and its structural adaptation to mechanical environment, is regulated by various kinds of molecular signals. Wnt signaling is one of the most important factors in bone remodeling, particularly in bone formation. To reveal the Wnt signaling and its inhibition mechanisms, we measured the interaction between Wnt signal receptor, LRP5, and its signaling molecules, Wnt3a, Dkk1, and Sclerostin, by using Atomic Force Microscope (AFM). In this study, we calculated the dissociation constants of Wnt3a/LRP5, sclerostin/LRP5, and Dkk1/LRP5, and it was proved that the dissociation constant of Dkk1/LRP5 is about 10-times smaller than that of sclerostin/LRP5, that is, the bond life time of Dkk1/LRP5 was about 10-times longer than that of sclerostin/LRP5. As the results, we quantitatively verified a previous suggestion that Dkk1 may function as a main regulator of Wnt signaling, and that sclerostin, which is more selective in its activity and restricted in its expression, may function as a more refined regulator of Wnt signaling.
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Author(s): Sebastian, Aimy | Advisor(s): Loots, Gabriela G | Abstract: AbstractHigh-throughput Analysis of WNT Signaling Pathway in OsteoblastsbyAimy SebastianDoctor of PhilosophyUniversity of California, Merced, 2016Dr. Suzanne S. Sindi, ChairDr. David H. ArdellDr. Gabriela G. LootsDr. Jennifer O. ManilayMultiple signaling pathways have been shown to regulate bone development and metabolism, and the WNT signaling pathway is emerging as one of the most crucial contributors. Several WNT ligands, receptors and WNT antagonists are expressed in bone and play a role in maintaining postnatal bone homeostasis. However, specific functions of individual WNT pathway members in bone are only beginning to be elucidated. Investigating the role of WNT signaling in bone development and metabolism will provide important implications for the treatment of fractures and bone thinning disorders such as osteoporosis and osteopenia. The focus of my thesis is to elucidate the functions of three out of nineteen WNT ligands and WNT co-receptors LRP5 and LRP6 in osteoblasts (bone forming cells).In this thesis, I investigated the role of WNT ligands WNT3A, WNT5A and WNT16 in osteoblasts to identify the target genes regulated by these WNTs and to understand the molecular mechanism by which these WNTs regulate bone metabolism. Gene expression analysis of neonatal osteoblasts treated with recombinant WNTs identified more than 1000 genes regulated by WNT signaling in osteoblasts and suggested that WNT3A and WNT16 positively regulate early stages of osteoblast differentiation and inhibit osteoblast maturation/mineralization.I also studied the role of WNT co-receptors LRP5 and LRP6 in mediating canonical WNT signaling. LRP5 and LRP6 are two WNT co-receptors that have been linked to bone development and metabolism. Both LRP5 and LRP6 are required for normal postnatal bone homeostasis. However, their specific roles are not well understood. To determine the roles of LRP5 and 6 in mediating canonical WNT signaling, osteoblasts lacking Lrp5, Lrp6 and both Lrp5 and 6 were treated with recombinant WNT3A. The RNA isolated from all WNT3A treated samples were sequenced and analyzed to identify genes regulated through LRP5 and LRP6 and genes that do not require LRP5/6 for WNT3A induced transcriptional regulation. This study revealed that LRP6 plays a dominant role in mediating WNT3A signaling in osteoblasts.Canonical WNTs such as WNT3A regulate target gene expression by activating TCF/LEF family transcription factors. These transcription factors bind to promoters and/or enhancers of target genes to induce gene transcription. To identify direct targets of canonical WNT signaling, using ChIP-seq, I identified TCF/LEF binding sites near WNT3A targets. More than 80% WNT3A targets had TCF/LEF binding sites in their promoter and/or enhancers. This study also identified more than 500 putative WNT inducible enhancers in osteoblasts. A subset of predicted WNT inducible enhancers was validated experimentally to confirm WNT3A inducible enhancer activity.My findings expand our current understanding of the role of WNT signaling pathway in regulating osteoblast differentiation and function, as well as contribute to the knowledge of the WNT signaling pathway itself. The WNT target genes identified in this study may be further explored for their therapeutic potential in treating osteoporosis and other bone disorders.
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Dysregulation of Wnt signaling is implicated in multiple ocular disorders. The roles of Wnt co-receptors LRP5 and LRP6 in Wnt signaling regulation remain elusive, as most retinal cells express both of the co-receptors. To address this question, LRP5 and LRP6 were individually knocked-out in a human retinal pigment epithelium cell line using the CRISPR-Cas9 technology. Wnt signaling activity induced by various Wnt ligands was measured using wild-type and the KO cell lines. The results identified three groups of Wnt ligands based on their co-receptor specificity: 1) activation of Wnt signaling only through LRP6, 2) through both LRP5 and LRP6 and 3) predominantly through LRP5. These results indicate that LRP5 and LRP6 have differential roles in Wnt signaling regulation.
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During development and homeostasis, precise control of Wnt/β-catenin signaling is in part achieved by secreted and membrane proteins that negatively control activity of the Wnt co-receptors Lrp5 and Lrp6. Lrp4 is related to Lrp5/6 and is implicated in modulation of Wnt/β-catenin signaling, presumably through its ability to bind to the Wise (Sostdc1)/sclerostin (Sost) family of Wnt antagonists. To gain insights into the molecular mechanisms of Lrp4 function in modulating Wnt signaling, we performed an array of genetic analyses in murine tooth development, where Lrp4 and Wise play important roles. We provide genetic evidence that Lrp4 mediates the Wnt inhibitory function of Wise and also modulates Wnt/β-catenin signaling independently of Wise. Chimeric receptor analyses raise the possibility that the Lrp4 extracellular domain interacts with Wnt ligands, as well as the Wnt antagonists. Diverse modes of Lrp4 function are supported by severe tooth phenotypes of mice carrying a human mutation known to abolish Lrp4 binding to Sost. Our data suggest a model whereby Lrp4 modulates Wnt/β-catenin signaling via interaction with Wnt ligands and antagonists in a context-dependent manner.
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Wnt signaling plays an important role not only in development and morphogenesis of embryos but in pathogenesis of various disorders including cancers. Analyses of the mutations in LDL receptor-related protein 5 (LRP5), a Wnt co-receptor, revealed that the Wnt signaling pathway is important for bone formation. Glucocorticoids suppress the canonical Wnt signaling pathway by such mechanisms as the enhancement the expression of dickkopf-1 (Dkk-1) and secreted frizzled-related protein 1 (sFRP1), and activation of the glycogen synthase kinase-3beta (GSK-3beta) activity. The inhibition of the canonical Wnt signal by glucocorticoid may be involved in the pathogenesis of glucocorticoid-induced osteoporosis.
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The nematode Caenorhabditis elegans is a favorite model for the study of aging. A wealth of genetic and genomic studies show that metabolic regulation is a hallmark of life-span modulation. A recent study in BMC Biology identifying metabolic signatures for longevity suggests that amino-acid pools may be important in longevity. See research article http://www.biomedcentral.com/1741-7007/8/14 .
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Wnt-beta-catenin signaling controls critical events in metazoan development, and its dysregulation leads to cancers and developmental disorders. Binding of a Wnt ligand to its transmembrane co-receptors Frizzled (Fz) and low-density lipoprotein (LDL) receptor-related protein (LRP) 5 or LRP6 inhibits the degradation of the transcriptional coactivator beta-catenin, which translocates to the nucleus to regulate gene expression. The secreted protein Dickkopf1 (Dkk1) inhibits Wnt signaling by binding to LRP5 and LRP6 and blocking their interaction with Wnt and Fz. Kremen 1 and 2 (Krm1 and 2, collectively termed Krms) are single-pass transmembrane Dkk1 receptors that synergize with Dkk1 to inhibit Wnt signaling by promoting the endocytosis of LRP5 and LRP6. A study now suggests that Krms, in the absence of Dkk1, potentiate Wnt signaling by maintaining LRP5 and LRP6 at the plasma membrane. It is proposed that the absence or presence of Dkk1 determines whether Krms will activate or inhibit Wnt-beta-catenin signaling, respectively. Here, we speculate that the proposed context-dependent positive and negative roles for Krms could promote biphasic Wnt signaling in response to a shallow gradient of Dkk1, resulting in the generation of precise and robust borders between cells during development. Identification of a context-dependent role for Krms in Wnt-beta-catenin signaling offers insight into the mechanism of Wnt signaling and has important developmental implications.
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