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Kawano Y, Kypta RSecreted antagonists of the Wnt signalling pathway. J Cell Sci 116: 2627-2634
Abstract and Figures
The extracellular antagonists of the Wnt signalling pathway can be divided into two broad classes. Both classes of molecule prevent ligand-receptor interactions, but by different mechanisms: members of the first class, which include the sFRP (secreted Frizzled-related protein) family, WIF (Wnt inhibitory factor)-1 and Cerberus, primarily bind to Wnt proteins; the second class comprises certain members of the Dickkopf (Dkk) family, which bind to one subunit of the Wnt receptor complex. In addition, there are other protein interactions that contribute to Wnt antagonist function. Moreover, certain sFRPs and Dkks do not antagonise Wnt function, which suggests that these families have as-yet-undiscovered functions.
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... To further elucidate the transcriptomic landscape of the SC-like population, we compared it with the other ve supporting cell clusters, identifying 21 DEGs Table S3), including ATF3, a well-known TF involved in cellular stress responses 31 and a recently proposed candidate for leading SC proliferation and likely HC regeneration in the avian inner ear 32 . In addition, this analysis revealed that SC-like cells also expressed SFRP4 (Fig. 5c), the secreted frizzledrelated protein 4 that acts as an inhibitor of Wnt signalling through direct interaction, antagonizing both canonical and noncanonical Wnt pathways 33,34 , regulating cell growth and differentiation. We also found IL-32, LGALS3 (galectin-3), and ANX family genes, ANXA1 and ANXA2 among the upregulated genes. ...
The human utricle is a vestibular organ responsible for maintaining balance. Balance function commonly deteriorates with age. With the aging population expected to double to 2 billion by 2050 and no pharmaceutical or biological treatments currently available, balance disorders are a significant unmet medical need. The utricle is composed of sensory and non-sensory cells, which are closely related, and non-sensory cells have a limited capacity to regenerate sensory cells in a damaged balance organ and, therefore, are a relevant therapeutic target. Here, we show the cellular and transcriptional profiles of the adult human utricle and its response to damage by performing bulk and single-cell RNA-sequencing from patient-derived utricles. We discovered six transcriptionally distinct non-sensory cell types, including a novel population, demonstrating the heterogeneity of the adult human utricle. In addition, using an aminoglycoside damage paradigm, we determined the early transcriptional changes of the utricle after damage. Our findings demonstrate that this organ has the capacity to respond to ototoxic damage within 24 h and potentially initiate a regenerative response via an early-responding supporting cell population. This study represents a major step forward in inner ear regenerative medicine, and our results will serve as a foundation for preclinical studies, paving the way to therapeutic strategies for balance recovery.
... By blocking Wnt ligands from attaching to the Fz/LRP receptor complex, these proteins enable regulated suppression of the signaling pathway. These inhibitors are divided into two functional groups: the SFRP and DKK protein families (Kawano and Kypta 2003;Staal and Luis 2010). In this study, SFRP2, SFRP4, and SFRP5 gene expression levels in amniocyte cells exposed to 2100 MHz increased statistically significantly compared to the C, SH, and RF-1 groups (p < 0.001). ...
... At day 14 following bone injuries, the sfrp1/-mice displayed a marked reduction in the cartilage callus and an increase in intramembranous bone. Additionally, compared to wild-type mice, these mice showed earlier bone remodeling over the course of fracture repair process (Kawano and Kypta, 2003;Bodine et al., 2004). ...
Compared to an incisional skin or mucosal wound, a tooth extraction wound results in far more soft tissue loss. A blood clot instantly fills the gap left by the extracted tooth. An embryonic type of bone forms during the healing of extraction wounds, and mature bone only later replaces it. Osteocytes in embryonic bone, also known as coarse fibrillar bone or immature bone, differ from those in adult bone in terms of number, size, and irregular arrangement. This immature bone is more radiolucent than mature bone due to the higher cell density and the smaller volume of calcified intercellular material. The Wnt gene family contains genes that encode secreted signaling proteins that have good promise for promoting bone regeneration. However, we still have a limited understanding the interplay of the molecular elements of the Wnt pathway in signal transduction, from ligand detection on the cell surface to transcription of target genes in the nucleus. We discuss the function of Wnt signaling molecules in this review, in tissue repair following tooth extraction and present recent results about these molecules. Conclusions: Wnt signaling activity helps to hasten bone regeneration while bone healing is slowed down by mutations in LRP5/6 or β-catenin.
... SFRP1 has been classi ed as a tumor suppressor gene due to the loss of its expression in many human cancers 18 . Under physiological conditions, SFRP1 inactivates the canonical and non-canonical WNT/β-catenin pathway by directly binding to WNT proteins or the frizzled receptor 19 . Promoter methylation down-regulates the expression of SFRP1 in CRC, which relieves the negative regulation of Wnt/β-catenin, consequently altering the proliferation and differentiation of tumor cells 20 . ...
The methylation level of the SFRP1 gene promoter is closely related to colorectal cancer. 205 stool samples were enrolled and were divided into positive and negative groups in the study. MassArray was used to screen the level of methylation on the SFRP1 gene promoter in the two groups. A random forest model was established to analyze and screen methylation sites of characteristic expression. A further 60 fecal samples were collected and divided into positive and negative groups to verify the specificity and sensitivity of the characteristic methylation sites by RT-PCR. TCGA database was used to analyze the relationship between tumor immunity and methylation sites in CRC patients. 12 differentially expressed MassArray detected methylation sites on SFRP1 promoter in colorectal cancer or adenoma stool specimens compared to normal. Random forest regression models built using the R package randomForest analysis (OOB error rate is 39.29%) located in the OOB error fastest changing area and have a good performance (accuracy: 0.757, sensitivity: 0.857, specificity: 0.696). The cg15839448, cg17816908 and cg14548509 showed better on specificity and sensitivity. Further, RT-PCR analysis showed that SFRP1_CpG_16.17.18 (cg17816908) performed best between the three CpG sites. The AUC of the ROC curve is 0.791, which was higher than the other two CpG sites. And cg17816908 is negatively correlated with prognosis and immune checkpoints with immunotherapy sensitivity in CRC patients. Our study demonstrated that SFRP1_CpG_16.17.18 (cg17816908) has a good clinical performance as a prognosis marker of colorectal cancer, and may provide a reference to immunotherapy in the clinic.
To maintain an optimal body content of phosphorus throughout postnatal life, variable phosphate absorption from food must be finely matched with urinary excretion. This amazing feat is accomplished through synchronised phosphate transport by myriads of ciliated cells lining the renal proximal tubules. These respond in real time to changes in phosphate and composition of the renal filtrate and to hormonal instructions. How they do this has stimulated decades of research. New analytical techniques, coupled with incredible advances in computer technology, have opened new avenues for investigation at a sub-cellular level. There has been a surge of research into different aspects of the process. These have verified long-held beliefs and are also dramatically extending our vision of the intense, integrated, intracellular activity which mediates phosphate absorption. Already, some have indicated new approaches for pharmacological intervention to regulate phosphate in common conditions, including chronic renal failure and osteoporosis, as well as rare inherited biochemical disorders. It is a rapidly evolving field. The aim here is to provide an overview of our current knowledge, to show where it is leading, and where there are uncertainties. Hopefully, this will raise questions and stimulate new ideas for further research.
Wnt signaling plays a crucial role in embryonic development and homeostasis maintenance. Delicate and sensitive fine-tuning of Wnt signaling based on the proper timings and positions is required to balance cell proliferation and differentiation and maintain individual health. Therefore, homeostasis is broken by tissue hypoplasia or tumor formation once Wnt signal dysregulation disturbs the balance of cell proliferation. The well-known regulatory mechanism of Wnt signaling is the molecular reaction associated with the cytoplasmic accumulation of effector β-catenin. In addition to β-catenin, most Wnt effector proteins are also regulated by ubiquitin-dependent modification, both qualitatively and quantitatively. This review will explain the regulation of the whole Wnt signal in four regulatory phases, as well as the different ubiquitin ligases and the function of deubiquitinating enzymes in each phase. Along with the recent results, the mechanism by which RNF43 negatively regulates the surface expression of Wnt receptors, which has recently been well understood, will be detailed. Many RNF43 mutations have been identified in pancreatic and gastrointestinal cancers and examined for their functional alteration in Wnt signaling. Several mutations facilitate or activate the Wnt signal, reversing the RNF43 tumor suppressor function into an oncogene. RNF43 may simultaneously play different roles in classical multistep tumorigenesis, as both wild-type and mutant RNF43 suppress the p53 pathway. We hope that the knowledge obtained from further research in RNF43 will be applied to cancer treatment in the future despite the fully unclear function of RNF43.
Although KDM5C is one of the most frequently mutated genes in X-linked intellectual disability¹, the exact mechanisms that lead to cognitive impairment remain unknown. Here we use human patient-derived induced pluripotent stem cells and Kdm5c knockout mice to conduct cellular, transcriptomic, chromatin and behavioural studies. KDM5C is identified as a safeguard to ensure that neurodevelopment occurs at an appropriate timescale, the disruption of which leads to intellectual disability. Specifically, there is a developmental window during which KDM5C directly controls WNT output to regulate the timely transition of primary to intermediate progenitor cells and consequently neurogenesis. Treatment with WNT signalling modulators at specific times reveal that only a transient alteration of the canonical WNT signalling pathway is sufficient to rescue the transcriptomic and chromatin landscapes in patient-derived cells and to induce these changes in wild-type cells. Notably, WNT inhibition during this developmental period also rescues behavioural changes of Kdm5c knockout mice. Conversely, a single injection of WNT3A into the brains of wild-type embryonic mice cause anxiety and memory alterations. Our work identifies KDM5C as a crucial sentinel for neurodevelopment and sheds new light on KDM5C mutation-associated intellectual disability. The results also increase our general understanding of memory and anxiety formation, with the identification of WNT functioning in a transient nature to affect long-lasting cognitive function.
The Wnt/β-catenin pathway is an evolutionarily conserved signaling pathway that controls fundamental physiological and pathological processes by regulating cell proliferation and differentiation. The Wnt/β-catenin pathway enables liver homeostasis by inducing differentiation and contributes to liver-specific features such as metabolic zonation and regeneration. In contrast, abnormalities in the Wnt/β-catenin pathway promote the development and progression of hepatocellular carcinoma (HCC). Similarly, hepatoblastoma, the most common childhood liver cancer, is frequently associated with β-catenin mutations, which activate Wnt/β-catenin signaling. HCCs with activation of the Wnt/β-catenin pathway have unique gene expression patterns and pathological and clinical features. Accordingly, they are highly differentiated with retaining hepatocyte-like characteristics and tumorigenic. Activation of the Wnt/β-catenin pathway in HCC also alters the state of immune cells, causing “immune evasion” with inducing resistance to immune checkpoint inhibitors, which have recently become widely used to treat HCC. Activated Wnt/β-catenin signaling exhibits these phenomena in liver tumorigenesis through the expression of downstream target genes, and the molecular basis is still poorly understood. In this review, we describe the physiological roles of Wnt/b-catenin signaling and then discuss their characteristic changes by the abnormal activation of Wnt/b-catenin signaling. Clarification of the mechanism would contribute to the development of therapeutic agents in the future.
Background
The aim of this study was to analyse transcriptomic differences between primary and recurrent high-grade serous ovarian carcinoma (HGSOC) to identify prognostic biomarkers.
Methods
We analysed 19 paired primary and recurrent HGSOC samples using targeted RNA sequencing. We selected the best candidates using in silico survival and pathway analysis and validated the biomarkers using immunohistochemistry on a cohort of 44 paired samples, an additional cohort of 504 primary HGSOCs and explored their function.
Results
We identified 233 differential expressed genes. Twenty-three showed a significant prognostic value for PFS and OS in silico. Seven markers ( AHRR, COL5A2, FABP4, HMGCS2, ITGA5, SFRP2 and WNT9B ) were chosen for validation at the protein level. AHRR expression was higher in primary tumours ( p < 0.0001) and correlated with better patient survival ( p < 0.05). Stromal SFRP2 expression was higher in recurrent samples ( p = 0.009) and protein expression in primary tumours was associated with worse patient survival ( p = 0.022). In multivariate analysis, tumour AHRR and SFRP2 remained independent prognostic markers. In vitro studies supported the anti-tumorigenic role of AHRR and the oncogenic function of SFRP2.
Conclusions
Our results underline the relevance of AHRR and SFRP2 proteins in aryl-hydrocarbon receptor and Wnt-signalling, respectively, and might lead to establishing them as biomarkers in HGSOC.
Flavonoids are polyphenolic compounds produced by plants as secondary metabolites that are known to exhibit wide range of pharmaceutical properties. Flavonoids from different medicinal plants have been used in traditional medicine to treat several musculoskeletal disorders for centuries. Of the numerous flavonoids, baicalein from Oroxylum indicum has a well-documented protective effect in skeletal health. However, studies into its influence on the canonical Wnt/β-catenin signaling pathway for musculoskeletal disorders remain limited. With the results of our previous study, the current research investigated the molecular mechanism of baicalein to inhibit the interaction between LRP6 and sclerostin to activate the canonical Wnt/β-catenin signaling pathway. Molecular docking revealed that baicalein docks between LRP6 and sclerostin with a binding energy of −8.4 kcal/mol and interacts with key binding residues of both the proteins. The molecular dynamics simulations predicted the stability of baicalein through 100 ns with more conformational changes observed in sclerostin than LRP6 especially in and around the PNAIG motif of loop-2 region, hinting at a possible inhibitory effect of baicalein over sclerostin. The findings of this research could pave the way for novel drug design approaches while promoting the use of natural flavonoids as potential therapeutics for musculoskeletal disorders.
A gradient of Wnt/β-catenin signalling formed by posteriorising Wnts and anteriorising Wnt antagonists regulates anteroposterior (AP) patterning of the central nervous system (CNS) during Xenopus gastrulation. In this process, the secreted Wnt antagonist Dkk1 functions in the Spemann organiser and its anterior derivatives by blocking Wnt receptors of the lipoprotein receptor-related protein (LRP) 5 and 6 class. In addition to LRP6, Dkk1 interacts with another recently identified receptor class, the transmembrane proteins Kremen1 (Krm1) and Kremen2 (Krm2) to synergistically inhibit LRP6. We have investigated the role of Krm1 and Krm2 during early Xenopus embryogenesis. Consistent with a role in zygotic Wnt inhibition, overexpressed Krm anteriorises embryos and rescues embryos posteriorised by Wnt8. Antisense morpholino oligonucleotide (Mo) knockdown of Krm1 and Krm2 leads to deficiency of anterior neural development. In this process, Krm proteins functionally interact with Dkk1: (1) in axis duplication assays krm2 synergises with dkk1 in inhibiting Wnt/LRP6 signalling; (2) krm2 rescues microcephalic embryos induced by injection of inhibitory anti-Dkk1 antibodies; and (3) injection of krm1/2 antisense Mo enhances microcephaly induced by inhibitory anti-Dkk1 antibodies. The results indicate that Krm proteins function in a Wnt inhibition pathway regulating early AP patterning of the CNS.
Convergent extension movements are the main driving force of Xenopus gastrulation. A fine-tuned regulation of cadherin-mediated cell–cell adhesion is thought to be required for this process. Members of the Wnt family of extracellular glycoproteins have been shown to modulate cadherin-mediated cell–cell adhesion, convergent extension movements, and cell differentiation. Here we show that endogenous Wnt/β-catenin signaling activity is essential for convergent extension movements due to its effect on gene expression rather than on cadherins. Our data also suggest that XLEF-1 rather than XTCF-3 is required for convergent extension movements and that XLEF-1 functions in this context in the Wnt/β-catenin pathway to regulate Xnr-3. In contrast, activation of the Wnt/Ca2+ pathway blocks convergent extension movements, with potential regulation of the Wnt/β-catenin pathway at two different levels. PKC, activated by the Wnt/Ca2+ pathway, blocks the Wnt/β-catenin pathway upstream of β-catenin and phosphorylates Dishevelled. CamKII, also activated by the Wnt/Ca2+ pathway, inhibits the Wnt/β-catenin signaling cascade downstream of β-catenin. Thus, an opposing cross-talk of two distinct Wnt signaling cascades regulates convergent extension movements in Xenopus.
Convincing evidence has accumulated to identify the Frizzled proteins as receptors for the Wnt growth factors. In parallel,
a number of secreted frizzled-like proteins with a conserved N-terminal frizzled motif have been identified. One of these
proteins, Frzb-1, binds Wnt-1 and Xwnt-8 proteins and antagonizes Xwnt-8 signaling in Xenopus embryos. Here we report that Frzb-1 blocks Wnt-1 induced cytosolic accumulation of β-catenin, a key component of the Wnt
signaling pathway, in human embryonic kidney cells. Structure/function analysis reveals that complete removal of the frizzled
domain of Frzb-1 abolishes the Wnt-1/Frzb-1 protein interaction and the inhibition of Wnt-1 mediated axis duplication in Xenopus embryos. In contrast, removal of the C-terminal portion of the molecule preserves both Frzb-Wnt binding and functional inhibition
of Wnt signaling. Partial deletions of the Frzb-1 cysteine-rich domain maintain Wnt-1 interaction, but functional inhibition
is lost. Taken together, these findings support the conclusion that the frizzled domain is necessary and sufficient for both
activities. Interestingly, Frzb-1 does not block Wnt-5A signaling in a Xenopus functional assay, even though Wnt-5A coimmunoprecipitates with Frzb-1, suggesting that coimmunoprecipitation does not necessarily
imply inhibition of Wnt function.
Frizzled polypeptides are integral membrane proteins that recently were shown to function as receptors for Wnt signaling molecules.
Here, we report the identification of a novel, secreted 36-kDa protein that contains a region homologous to a putative Wnt-binding
domain of Frizzleds. This protein, called Frizzled-related protein (FRP), was first identified as a heparin-binding polypeptide
that copurified with hepatocyte growth factor/scatter factor in conditioned medium from a human embryonic lung fibroblast
line. Degenerate oligonucleotides, based on the NH2-terminal sequence of the purified protein, were used to isolate corresponding cDNA clones. These encoded a 313-amino acid
polypeptide, containing a cysteine-rich domain of ≈110 residues that was 30–40% identical to the putative ligand-binding domain
of Frizzled proteins. A 4.4-kb transcript of the FRP gene is present in many organs, both in the adult and during embryogenesis, and homologs of the gene are detectable in DNA
from several vertebrate species. In biosynthetic studies, FRP was secreted but, like Wnts, tended to remain associated with
cells. When coexpressed with several Wnt family members in early Xenopus embryos, FRP antagonized Wnt-dependent duplication of the embryonic dorsal axis. These results indicate that FRP may function
as an inhibitor of Wnt action during development and in the adult.
The Wnt proteins constitute a large family of extracellular signalling molecules that are found throughout the animal kingdom and are important for a wide variety of normal and pathological developmental processes
Frizzled polypeptides are integral membrane proteins that recently were shown to function as receptors for Wnt signaling molecules. Here, we report the identification of a novel, heparin-binding, 36 kDa Frizzled-related protein (FRP) that was purified from the conditioned medium of a human embryonic lung fibroblast line. Degenerate oligonucleotides, based on the amino-terminal sequence of the purified protein, were used to isolate corresponding cDNA clones. These encoded a 313-amino acid polypeptide, containing a cysteine-rich domain of ∼110 residues that was 30-40% identical to the putative ligand-binding domain of Frizzled proteins. A 4.4 kb transcript of the FRP gene is present in many organs, and homologs of the gene are detectable in DNA from several vertebrate species. In biosynthetic studies, FRP was secreted but, like Wnts, tended to remain associated with cells. When co-expressed with several Wnt family members in earlyXenopus embryos, FRP antagonized Wnt-dependent duplication of the embryonic dorsal axis. These results indicate that FRP may function as an inhibitor of Wnt action during development and in the adult.
Patterning of the pre-gastrula embryo and subsequent neural induction post-gastrulation are very complex and intricate processes of which little, until recently, has been understood. The earliest decision in neural development, the choice between epidermal or neural fates, is regulated by bone morphogenetic protein (BMP) signaling within the ectoderm. Inhibition of BMP signaling is sufficient for neural induction. Many secreted BMP inhibitors are expressed exclusively within the organizer of the Xenopus gastrula embryo and therefore are predicted to act as bona fide endogenous neural inducers. Other cell-autonomous inhibitors of the BMP pathway are more widely expressed, such as the inhibitory Smads, Smad6 and Smad7. In this report we describe the biological and biochemical characterization of 51-B6, a novel member of Cerberus/Dan family of secreted BMP inhibitors, which we identified in a screen for Smad7-induced genes. This gene is expressed maternally in an animal to vegetal gradient, and its expression levels decline rapidly following gastrulation. In contrast to known BMP inhibitors, 51-B6 is broadly expressed in the ectoderm until the end of gastrulation. The timing, pattern of expression, and activities of this gene makes it unique when compared to other BMP/TGFβ/Wnt secreted inhibitors which are expressed only zygotically and maintained post-gastrulation. We propose that a function of 51-B6 is to block BMP and TGFβ signals in the ectoderm in order to regulate cell fate specification and competence prior to the onset of neural induction. In addition, we demonstrate that 51-B6 can act as a neural inducer and induce ectopic head-like structures in neurula staged embryos. Because of this embryological activity, we have renamed this clone Coco, after the Spanish word meaning head.
Wnts are secreted glycoproteins implicated in diverse processes during embryonic patterning in metazoans. They signal through seven-transmembrane receptors of the Frizzled (Fz) family [1] to stabilise β-catenin [2]. Wnts are antagonised by several extracellular inhibitors including the product of the dickkopf1 (dkk1) gene, which was identified in Xenopus embryos and is a member of a multigene family. The dkk1 gene acts upstream of the Wnt pathway component dishevelled but its mechanism of action is unknown [3]. Although the function of Dkk1 as a Wnt inhibitor in vertebrates is well established [3–6], the effect of other Dkks on the Wnt/β-catenin pathway is unclear. Here, we report that a related family member, Dkk2, activates rather than inhibits the Wnt/β-catenin signalling pathway in Xenopus embryos. Dkk2 strongly synergised with Wnt receptors of the Fz family to induce Wnt signalling responses. The study identifies Dkk2 as a secreted molecule that is able to activate Wnt/β-catenin signalling. The results suggest that a coordinated interplay between inhibiting dkk1 and activating dkk2 can modulate Fz signalling.
Members of the vertebrate Wnt family have been subdivided into two functional classes according to their biological activities. Some Wnts signal through the canonical Wnt-1/wingless pathway by stabilizing cytoplasmic β-catenin. By contrast other Wnts stimulate intracellular Ca2+ release and activate two kinases, CamKII and PKC, in a G-protein-dependent manner. Moreover, putative Wnt receptors belonging to the Frizzled gene family have been identified that preferentially couple to the two prospective pathways in the absence of ectopic Wnt ligand and that might account for the signaling specificity of the Wnt pathways. As Ca2+ release was the first described feature of the noncanonical pathway, and as Ca2+ probably plays a key role in the activation of CamKII and PKC, we have named this Wnt pathway the Wnt/Ca2+ pathway.
The Spemann organizer in amphibian embryos is a tissue with potent head-inducing activity, the molecular nature of which is unresolved. Here we describe dickkopf-1 (dkk-1), which encodes Dkk-1, a secreted inducer of Spemann's organizer in Xenopus and a member of a new protein family. Injections of mRNA and antibody indicate that dkk-1 is sufficient and necessary to cause head induction. dkk-1 is a potent antagonist of Wnt signalling, suggesting that dkk genes encode a family of secreted Wnt inhibitors.