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Negative Regulation of Wnt/β-catenin Signaling is blocked by Wnt Signaling Component Protein Dishevelled1 Binding Aptamer (WD-Aptamer)
Background:
When Wnt binds to the N-terminal of Frizzled, a conformational change occurs in the C-terminal of Frizzled, which binds to Dishevelled1 (Dvl1), a Wnt signaling component protein. When Dvl1 binds to the C-terminal of Frizzled, the concentration of β-catenin increases and it enters the nucleus to transmit cell proliferation signals. CXXC-type zinc finger protein 5 (CXXC5) binds to the Frizzled binding site of Dvl1 and interferes with Dvl1-Frizzled binding. Therefore, blocking CXXC5-Dvl1 binding may induce Wnt signal transduction.
Materials and methods:
We used WD-aptamer, a DNA aptamer that specifically binds to Dvl1 and interferes with CXXC5-Dvl1 interaction. We confirmed the penetration of WD-aptamer into human hair follicle dermal papilla cells (HFDPCs) and measured β-catenin expression following treatment with WD-aptamer in HFDPCs, wherein Wnt signaling was activated by Wnt3a. In addition, MTT assay was performed to investigate the effect of WD-aptamer on cell proliferation.
Results:
WD-aptamer penetrated the cell, affected Wnt signaling, and increased β-catenin expression, which plays an important role in signaling. Additionally, WD-aptamer induced HFDPC proliferation.
Conclusion:
CXXC5-associated negative feedback of Wnt/β-catenin signaling can be regulated by interfering with CXXC5-Dvl1 interaction.
Wnt/β-catenin signaling plays important roles in cutaneous wound healing and dermal fibrosis. However, its regulatory mechanism has not been fully elucidated, and a commercially available wound-healing agent targeting this pathway is desirable but currently unavailable. We found that CXXC-type zinc finger protein 5 (CXXC5) serves as a negative feedback regulator of the Wnt/β-catenin pathway by interacting with the Dishevelled (Dvl) protein. In humans, CXXC5 protein levels were reduced in epidermal keratinocytes and dermal fibroblasts of acute wounds. A differential regulation of β-catenin, α-smooth muscle actin (α-SMA), and collagen I by overexpression and silencing of CXXC5 in vitro indicated a critical role for this factor in myofibroblast differentiation and collagen production. In addition, CXXC5(-/-) mice exhibited accelerated cutaneous wound healing, as well as enhanced keratin 14 and collagen synthesis. Protein transduction domain (PTD)-Dvl-binding motif (DBM), a competitor peptide blocking CXXC5-Dvl interactions, disrupted this negative feedback loop and activated β-catenin and collagen production in vitro. Co-treatment of skin wounds with PTD-DBM and valproic acid (VPA), a glycogen synthase kinase 3β (GSK3β) inhibitor which activates the Wnt/β-catenin pathway, synergistically accelerated cutaneous wound healing in mice. Together, these data suggest that CXXC5 would represent a potential target for future therapies aimed at improving wound healing.
© 2015 Lee et al.
The positive roles of the Wnt/β-catenin pathway in osteoblast differentiation and bone mineral density (BMD) maintenance have been clearly demonstrated in both animal experiments and clinical investigations. CXXC finger protein 5 (CXXC5), a recently identified negative regulator of the Wnt/β-catenin pathway, showed altered cellular localization and function, which were dependent on the cell type in previous studies. However, the in vivo function of CXXC5 has not been clearly investigated yet. Here, we characterized CXXC5 as a negative regulator of osteoblast differentiation and bone formation. Deficiency of CXXC5 resulted in elevated BMD in mice without any severe gross developmental abnormalities. CXXC5 exerted a negative-feedback effect on the Wnt/β-catenin pathway via Wnt-dependent binding to Dishevelled (Dvl) during osteoblast differentiation. Suppression of the Dvl-CXXC5 interaction using a competitor peptide resulted in the activation of the Wnt/β-catenin pathway and osteoblast differentiation, and accelerated thickness growth of ex vivo-cultured calvariae. Overall, CXXC5 is a negative-feedback regulator induced by Wnt/β-catenin signaling that inhibits osteoblast differentiation and bone formation via interaction with Dvl.Cell Death and Differentiation advance online publication, 30 January 2015; doi:10.1038/cdd.2014.238.
With many advantages over other therapeutic agents such as monoclonal antibodies, aptamers have recently emerged as a novel and powerful class of ligands with excellent potential for diagnostic and therapeutic applications. Typically generated through Systematic Evolution of Ligands by EXponential enrichment (SELEX), aptamers have been selected against a wide range of targets such as proteins, phospholipids, sugars, nucleic acids, as well as whole cells. DNA/RNA aptamers are single-stranded DNA/RNA oligonucleotides (with a molecular weight of 5-40 kDa) that can fold into well-defined 3D structures and bind to their target molecules with high affinity and specificity. A number of strategies have been adopted to synthesize aptamers with enhanced in vitro/in vivo stability, aiming at potential therapeutic/diagnostic applications in the clinic. In cardiovascular diseases, aptamers can be developed into therapeutic agents as anti-thrombotics, anti-coagulants, among others. This review focuses on aptamers that were selected against various molecular targets involved in cardiovascular diseases: von Willebrand factor (vWF), thrombin, factor IX, phospholamban, P-selectin, platelet-derived growth factor, integrin α(v)β(3), CXCL10, vasopressin, among others. With continued effort in the development of aptamer-based therapeutics, aptamers will find their niches in cardiovascular diseases and significantly impact clinical patient management.
Nucleic acid-based aptamers have been shown as high-affinity ligands and potential antagonists of disease-associated proteins. Aptamers, isolated from combinatorial libraries by an iterative in vitro selection process, discriminate between closely related targets and are characterized by high specificity and low toxicity thus representing a valid alternative to antibodies to target specific proteins of biomedical interest. Moreover, they are non-immunogenic and can be easily stabilized by chemical modifications thus expanding their therapeutic potential. Here, we will focus on the structural and functional features of aptamers that have entered the clinical development pipeline together with those aptamers holding great potential as therapeutics in preclinical studies. The future perspectives of aptamers as therapeutics will be discussed as well.
Mutations in the adenomatous polyposis coli (APC) tumour-suppressor gene occur in most human colon cancers. Loss of functional APC protein results in the accumulation of beta-catenin. Mutant forms of beta-catenin have been discovered in colon cancers that retain wild-type APC genes, and also in melanomas, medulloblastomas, prostate cancer and gastric and hepatocellular carcinomas. The accumulation of beta-catenin activates genes that are responsive to transcription factors of the TCF/LEF family, with which beta-catenin interacts. Here we show that beta-catenin activates transcription from the cyclin D1 promoter, and that sequences within the promoter that are related to consensus TCF/LEF-binding sites are necessary for activation. The oncoprotein p21ras further activates transcription of the cyclin D1 gene, through sites within the promoter that bind the transcriptional regulators Ets or CREB. Cells expressing mutant beta-catenin produce high levels of cyclin D1 messenger RNA and protein constitutively. Furthermore, expression of a dominant-negative form of TCF in colon-cancer cells strongly inhibits expression of cyclin D1 without affecting expression of cyclin D2, cyclin E, or cyclin-dependent kinases 2, 4 or 6. This dominant-negative TCF causes cells to arrest in the G1 phase of the cell cycle; this phenotype can be rescued by expression of cyclin D1 under the cytomegalovirus promoter. Abnormal levels of beta-catenin may therefore contribute to neoplastic transformation by causing accumulation of cyclin D1.
Bone morphogenetic proteins such as BMP4 are essential for proper development of telencephalic forebrain structures and induce differentiation of telencephalic neural stem cells into a variety of cellular fates, including astrocytic, neuronal, and mesenchymal cells. Little is yet understood regarding the mechanisms that underlie the spatiotemporal differences in progenitor response to BMP4. In a screen designed to identify novel targets of BMP4 signaling in telencephalic neural stem cells, we found the mRNA levels of the previously uncharacterized factor CXXC5 reproducibly up-regulated upon BMP4 stimulation. In vivo, CXXC5 expression overlapped with BMP4 adjacent to Wnt3a expression in the dorsal regions of the telencephalon, including the developing choroid plexus. CXXC5 showed partial homology with Idax, a related protein previously shown to interact with the Wnt-signaling intermediate Dishevelled (Dvl). Indeed CXXC5 and Dvl co-localized in the cytoplasm and interacted in co-immunoprecipitation experiments. Moreover, fluorescence resonance energy transfer (FRET) experiments verified that CXXC5 and Dvl2 were located in close spatial proximity in neural stem cells. Studies of the functional role of CXXC5 revealed that overexpression of CXXC5 or exposure to BMP4 repressed the levels of the canonical Wnt signaling target Axin2, and CXXC5 attenuated Wnt3a-mediated increase in TOPflash reporter activity. Accordingly, RNA interference of CXXC5 attenuated the BMP4-mediated decrease in Axin2 levels and facilitated the response to Wnt3a in neural stem cells. We propose that CXXC5 is acting as a BMP4-induced inhibitor of Wnt signaling in neural stem cells.
Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 10(10) random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.
The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c-MYC oncogene is identified as a target gene in this signaling pathway. Expression of c-MYC was shown to be repressed by wild-type APC and activated by beta-catenin, and these effects were mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.
beta-Catenin plays a dual role in the cell: one in linking the cytoplasmic side of cadherin-mediated cell-cell contacts to the actin cytoskeleton and an additional role in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated beta-catenin levels in colorectal cancer caused by mutations in beta-catenin or by the adenomatous polyposis coli molecule, which regulates beta-catenin degradation, result in the binding of beta-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. Here, we show that the cyclin D1 gene is a direct target for transactivation by the beta-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Inhibitors of beta-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin suppressed cyclin D1 promoter activity in colon cancer cells. Cyclin D1 protein levels were induced by beta-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain. Increased beta-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression into the cell cycle.
Tight control of cell-cell communication is essential for the generation of a normally patterned embryo. A critical mediator of key cell-cell signaling events during embryogenesis is the highly conserved Wnt family of secreted proteins. Recent biochemical and genetic analyses have greatly enriched our understanding of how Wnts signal, and the list of canonical Wnt signaling components has exploded. The data reveal that multiple extracellular, cytoplasmic, and nuclear regulators intricately modulate Wnt signaling levels. In addition, receptor-ligand specificity and feedback loops help to determine Wnt signaling outputs. Wnts are required for adult tissue maintenance, and perturbations in Wnt signaling promote both human degenerative diseases and cancer. The next few years are likely to see novel therapeutic reagents aimed at controlling Wnt signaling in order to alleviate these conditions.
Aptamers are single-stranded nucleic acid molecules that bind to and inhibit proteins and are commonly produced by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers undergo extensive pharmacological revision, which alters affinity, specificity, and therapeutic half-life, tailoring each drug for a specific clinical need. The first therapeutic aptamer was described 25 years ago. Thus far, one aptamer has been approved for clinical use, and numerous others are in preclinical or clinical development. This review presents a short history of aptamers and SELEX, describes their pharmacological development and optimization, and reviews potential treatment of diseases including visual disorders, thrombosis, and cancer.
Dishevelled (Dvl) plays a crucial role in Wnt signaling by interacting with membrane-bound receptors and downstream molecules through its PDZ domain. CXXC5 is one of the key molecules that interacts with Dvl and negatively regulates the Wnt/β-catenin pathway in osteoblast differentiation. Recently, the Dvl-CXXC5 interaction has been identified as an excellent target for osteoporosis treatment. Therefore, it is desirable to have detailed structural information for the Dvl-CXXC5 interaction. Although solution structures of the Dvl1 PDZ domain have been reported, a high-resolution crystal structure would provide detailed sidechain information that is essential for drug development. Here, we determined the first crystal structure of the Dvl-1 PDZ domain at a resolution of 1.76 Å, and compared it with its previously reported solution structure. The Dvl1 PDZ domain crystal belonged to the space group H32 with unit-cell parameters a = b = 72.837, c = 120.616, α = ß = 90.00, γ = 120.00. The crystal structure of Dvl1 PDZ shared its topology with the previously reported structure determined by nuclear magnetic resonance (NMR); however, the crystal structure was quite different from the solution structure in both the secondary structural region and the ligand-binding pocket. Molecular modeling based on NMR and X-ray crystallographic data yielded detailed information about the Dvl1/CXXC5 interaction, which will be useful for designing inhibitors.
Background:
Recent studies have suggested that vascular endothelial growth factor (VEGF) is an important stimulus for the growth of new blood vessels in the eye. Anti-VEGF therapy is thus a potential treatment for exudative macular degeneration and diabetic retinopathy.
Methods:
Previously described animal models of vascular leakage and ocular neovascularization, including the Miles assay, the rat corneal angiogenesis model, and the mouse retinopathy of prematurity (ROP) model, were used to study this drug. After these studies, a phase IA single ascending dose study of intravitreal injections of the drug was performed in 15 patients with subfoveal choroidal neovascularization secondary to exudative age-related macular degeneration (AMD).
Results:
The Miles assay model showed almost complete attenuation of VEGF-mediated vascular leakage following addition of EYE001, and the corneal angiogenesis model also showed a significant reduction in neovascularization with EYE001. The ROP model showed inhibition of 80% of the retinal neovascularization compared with controls (P = 0.0001). The phase IA safety study of patients with exudative AMD showed no significant safety issues related to the drug. Ophthalmic evaluation revealed that 80% of patients showed stable or improved vision 3 months after treatment and that 27% of eyes demonstrated a three-line or greater improvement in vision on the Early Treatment for Diabetic Retinopathy Study chart at this time.
Conclusion:
Anti-VEGF therapy is a promising new avenue for the treatment of neovascular diseases of the eye, including exudative macular degeneration and diabetic retinopathy. Preclinical data from studies with EYE001 support clinical evaluation of its efficacy in such diseases. This report is the first to describe administration of anti-VEGF therapy in humans for exudative macular degeneration and shows the safety of such therapy for single injections. Further clinical studies are necessary to determine the safety of multiple intravitreal injections of EYE001 and larger studies are needed to prove the efficacy of this novel, potentially therapeutic agent for neovascular AMD.
Aptamers, a promising new class of therapeutics, are single-stranded oligonucleotides generated via an in vitro selection process that bind to and inhibit the activity of target proteins in a manner similar to therapeutic antibodies. In order to enhance the drug-like character of aptamers, oligonucleotide libraries containing modified nucleotides are increasingly being used for selection. Principally, the choice of modifications aims to increase aptamer potency by enhancing nuclease-resistance, or increasing target affinity by providing more target recognition functionality or generating more stable aptamer structures.
Signaling by the Wnt family of secreted glycolipoproteins via the transcriptional coactivator beta-catenin controls embryonic development and adult homeostasis. Here we review recent progress in this so-called canonical Wnt signaling pathway. We discuss Wnt ligands, agonists, and antagonists, and their interactions with Wnt receptors. We also dissect critical events that regulate beta-catenin stability, from Wnt receptors to the cytoplasmic beta-catenin destruction complex, and nuclear machinery that mediates beta-catenin-dependent transcription. Finally, we highlight some key aspects of Wnt/beta-catenin signaling in human diseases including congenital malformations, cancer, and osteoporosis, and discuss potential therapeutic implications.
High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.
We developed a quartz crystal biosensor designed to detect concentrations and ligand affinity parameters of free unlabeled proteins in real time. Using a model system with human IgE as the analyte and single-stranded DNA aptamers or an anti-IgE antibody as immobilized ligands, we could demonstrate that aptamers were equivalent to antibodies in terms of specificity and sensitivity. Both receptor types selectively detected 0.5 nmol/L of IgE. In addition, the aptamer receptors tolerated repeated affine layer regeneration after ligand binding and recycling of the biosensor with little loss of sensitivity. Because of the small size and nonprotein nature of the aptamers, they were immobilized in a dense, well-oriented manner, thus extending the linear detection range to 10-fold higher concentrations of IgE. In addition to demonstrating for the first time that an aptamer-based biosensor can specifically and quantitatively detect an analyte in various complex protein mixes, the aptamer-ligand proved to be relatively heat resistant and stable over several weeks. Since aptamers consist of nucleic acids, well-established chemistry can be applied to produce optimized affine layers on biosensors that may be developed to specifically detect proteins in solution for analysis of proteomes.
Since its discovery in the early 1990s, aptamer technology has progressed tremendously. Automated selection procedures now allow rapid identification of DNA and RNA sequences that can target a broad range of extra- and intracellular proteins with nanomolar affinities and high specificities. The unique binding properties of nucleic acids, which are amenable to various modifications, make aptamers perfectly suitable for different areas of biotechnology. Moreover, the approval of an aptamer for vascular endothelial growth factor by the US Food and Drug Administration highlights the potential of aptamers for therapeutic applications. This review summarizes recent developments and demonstrates that aptamers are valuable tools for diagnostics, purification processes, target validation, drug discovery, and even therapeutic approaches.
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