Wnt-signaling is a key regulator of stem cell homeostasis, extensively studied in the intestinal crypt and other metazoan tissues. Yet, there is hardly any data available on the presence of Wnt-signaling components in the adult enteric nervous system (ENS) in vivo.
Therefore, we employed RNAscope HiPlex-assay, a novel and more sensitive in situ hybridization technology. By amplifying target specific signals, this technique enables the detection of low abundance, tightly regulated RNA content as is the case for Wnt-signaling components. Additionally, we compared our data to previously published physiological single cell RNA and RiboTag-based RNA sequencing analyses of enteric gliosis using data-mining approaches.
Our descriptive analysis shows that several components of the multidi-mensional regulatory network of the Wnt-signaling pathway are present in the murine ENS. The transport and secretion protein for Wnt-ligands Wntless as well as canonical (Wnt3a and Wnt2b) and non-canonical Wnt-ligands (Wnt5a, Wnt7a, Wnt8b and Wnt11) are detectable within submucosal and myenteric plexus. Further, corresponding Frizzled receptors (Fzd1, Fzd3, Fzd6, and Fzd7) and regulatory signaling mediators like R-Spondin/DKK ligands are present in the ENS of the small and large intestine. Further, data mining approaches revealed, that several Wnt-related molecules are expressed by enteric glial cell clusters and are dynamically regulated during the inflammatory manifestation of enteric gliosis.
Our results suggest, that canonical and non-canonical Wnt-signaling has a much broader impact on the mature ENS and its cellular homeostasis in health and inflammation, than previously anticipated.

Dickkopf1 (DKK1) was originally identified as a secreted protein that antagonizes Wnt signaling. Although DKK1 is essential for the developmental process, its functions in postnatal and adult life are unclear. However, evidence is accumulating that DKK1 is involved in tumorigenesis in a manner unrelated to Wnt signaling. In addition, recent studies have revealed that DKK1 may control immune reactions, although the relationship of this to Wnt signaling is unknown. Other DKK family members, DKK2-4, are likely to have their own functions. Here, we review the possible novel functions of DKKs. We summarize the characteristics of receptors of DKKs and the signaling mechanisms through DKKs and their receptors, provide evidence showing that DKKs are involved in tumor aggressiveness independently of Wnt signaling, and emphasize promising cancer therapies targeting DKKs and receptors. Lastly, we discuss various physiological and pathological processes controlled by DKKs.

The Wnt signaling pathway regulates crucial aspects such as cell fate determination, cell polarity and organogenesis during embryonic development. Wnt pathway deregulation is a hallmark of several cancers such as lung, gastric and liver cancer, and has been reported to be altered in others. Despite the general agreement reached by the scientific community on the oncogenic potential of the central components of the pathway, the role of the antagonist proteins remains less clear. Deregulation of the pathway may be caused by overexpression or downregulation of a wide range of antagonist proteins. Although there is growing information related to function and regulation of Dickkopf (DKK) proteins, their pharmacological potential as cancer therapeutics still has not been fully developed. This review provides an update on the role of DKK proteins in cancer and possible potential as therapeutic targets for the treatment of cancer; available compounds in pre-clinical or clinical trials are also reviewed.

Background: The Wnt/β catenin pathway promotes bone mineralization stimulating proliferation, differentiation, and survival of osteoblasts; it also inhibits osteoclast differentiation and osteocyte activity. Sclerostin (SOST) and Dickkopf 1 (DKK1) are Wnt/β catenin pathway inhibitors. Genetic variability in the expression of SOST and DKK1 might be involved in the development of postmenopausal osteoporosis (OP). Aim: To determine whether the SOST rs851056 and DKK1 rs1569198 polymorphisms are associated with OP in Mexican-Mestizo postmenopausal women. Materials and Methods: Two hundred and eighty Mexican-Mestizo postmenopausal women were assessed for their bone mineral density by dual-energy X-ray absorptiometry (DXA). Patients were classified as OP or non-OP. Genomic DNA was extracted from peripheral blood leukocytes. Genetic polymorphisms were analyzed by quantitative polymerase chain reaction using TaqMan probes. Results: The frequency of OP was 40% among the study population. Osteoporotic patients were older (p < 0.001), had a higher frequency of smoking (p = 0.01), and lower body mass index (p < 0.001) compared with the non-osteoporotic patients. The genotypic frequencies of the rs851056 locus of the SOST gene were GG 19%, GC 45%, and CC 35%, whereas the genotypic frequencies of the rs1569198 locus of the DKK1 gene were GG 15%, GA 40%, and AA 44%. In relation to rs851056 locus of the SOST gene, no differences were observed between the OP and non-OP cohorts in the frequencies of the GC polymorphism (48.7% vs. 43.1%). Similarly, analyses of the DKK1 rs1569198 does not demonstrate differences in the GA genotypic frequencies between the OP and non-OP cohorts (42.5% vs. 38.9%). Conclusion: Polymorphisms SOST rs851056 and DKK1 rs1569198 polymorphisms are not associated with OP in Mexican-Mestizo postmenopausal women.

Wnt molecules are a class of cysteine-rich secreted glycoproteins that participate in various developmental events during embryogenesis and adult tissue homeostasis. Since its discovery in 1982, the roles of Wnt signaling have been established in various key regulatory systems in biology. Wnt signals exert pleiotropic effects, including mitogenic stimulation, cell fate specification, and differentiation. The Wnt signaling pathway in humans has been shown to be involved in a wide variety of disorders including colon cancer, sarcoma, coronary artery disease, tetra-amelia, Mullerian duct regression, eye vascular defects, and abnormal bone mass. The canonical Wnt pathway functions by regulating the function of the transcriptional coactivator β-catenin, whereas noncanonical pathways function independent of β-catenin. Although the role of Wnt signaling is well established in epithelial malignancies, its role in mesenchymal tumors is more controversial. Some studies have suggested that Wnt signaling plays a pro-oncogenic role in various sarcomas by driving cell proliferation and motility; however, others have reported that Wnt signaling acts as a tumor suppressor by committing tumor cells to differentiate into a mature lineage. Wnt signaling pathway also plays an important role in regulating cancer stem cell function. In this review, we will discuss Wnt signaling pathway and its role in osteosarcoma.

Endocytosis has been proposed to modulate cell signaling activities. However, the role of endocytosis in embryogenesis, which requires coordination of multiple signaling inputs, has remained less understood. We previously showed that mouse embryos lacking a small guanosine triphosphate (GTP)-binding protein Rab7 implicated in endocytic flow are defective in gastrulation. Here, we investigate how subcellular defects associated with Rab7 deficiency are related to the observed developmental defects. Rab7-deficient embryos fail to organize mesodermal tissues due to defects in Wnt-β-catenin signaling. Visceral endoderm (VE)-specific ablation of Rab7 results in patterning defects similar to systemic Rab7 deletion. Rab7 mutants accumulate the Wnt antagonist Dkk1 in the extracellular space and in intracellular compartments throughout the VE epithelium. These data indicate that Rab7-dependent endocytosis regulates the concentration and availability of extracellular Dkk1, thereby relieving the epiblast of antagonism. This intercellular mechanism therefore organizes distinct spatiotemporal patterns of canonical Wnt activity during the peri-gastrulation stages of embryonic development.

Sarcomas represent an extensive group of divergent malignant diseases, with the only common characteristic of being derived from mesenchymal cells. As such, sarcomas are by definition very heterogeneous, and this heterogeneity does not manifest only upon intertumoral comparison on a bulk tumor level but can be extended to intratumoral level. Whereas part of this intratumoral heterogeneity could be understood in terms of clonal genetic evolution, an essential part includes a hierarchical relationship between sarcoma cells, governed by both genetic and epigenetic influences, signals that sarcoma cells are exposed to, and intrinsic developmental programs derived from sarcoma cells of origin. The notion of this functional hierarchy operating within each tumor implies the existence of sarcoma stem cells, which may originate from mesenchymal stem cells, and indeed, mesenchymal stem cells have been used to establish several crucial experimental sarcoma models and to trace down their respective stem cell populations. Mesenchymal stem cells themselves are heterogeneous, and, moreover, there are alternative possibilities for sarcoma cells of origin, like neural crest-derived stem cells, or mesenchymal committed precursor cells, or - in rhabdomyosarcoma - muscle satellite cells. These various origins result in substantial heterogeneity in possible sarcoma initiation. Genetic and epigenetic changes associated with sarcomagenesis profoundly impact the biology of sarcoma stem cells. For pediatric sarcomas featuring discrete reciprocal translocations and largely stable karyotypes, the translocation-activated oncogenes could be crucial factors that confer stemness, principally by modifying transcriptome and interfering with normal epigenetic regulation; the most extensively studied examples of this process are myxoid/round cell liposarcoma, Ewing sarcoma, and synovial sarcoma. For adult sarcomas, which have typically complex and unstable karyotypes, stemness might be defined more operationally, as a reflection of actual assembly of genetically and epigenetically conditioned stemness factors, with dedifferentiated liposarcoma providing a most thoroughly studied example. Alternatively, stemness can be imposed by tumor microenvironment, as extensively documented in osteosarcoma. In spite of this heterogeneity in both sarcoma initiation and underlying stemness biology, some of the molecular mechanisms of stemness might be remarkably similar in diverse sarcoma types, like abrogation of classical tumor suppressors pRb and p53, activation of Sox-2, or inhibition of canonical Wnt/β-catenin signaling. Moreover, even some stem cell markers initially characterized for their stem cell enrichment capacity in various carcinomas or leukemias seem to function quite similarly in various sarcomas. Understanding the biology of sarcoma stem cells could significantly improve sarcoma patient clinical care, leading to both better patient stratification and, hopefully, development of more effective therapeutic options.

Wnt signaling is essential for the differentiation of airway epithelial cells during development. Here, we examined the role of Wnt signaling during redifferentiation of ciliated airway epithelial cells in vitro at the air liquid interface as a model of airway epithelial repair. Phases of proliferation and differentiation were defined. Markers of squamous metaplasia and epithelial ciliation were followed while enhancing β-catenin signaling by blocking glycogen synthase kinase 3β with SB216763 and shRNA as well as inhibiting canonical WNT signaling with apical application of Dickkopf 1 (Dkk1). Our findings indicate that enhanced β-catenin signaling decreases the number of ciliated cells and causes squamous changes in the epithelium, whereas treatment with DDk1 leads to an increased number of ciliated cells.

Mouse F9 cells differentiate into primitive endoderm (PrE) following the activation of the canonical WNT-β-catenin pathway. The upregulation of Wnt6 and activation of β-catenin-TCF-LEF-dependent transcription is known to accompany differentiation, but the Frizzled (FZD) receptor responsible for transducing the WNT6 signal is not known. Eight of the 10 Fzd genes were found to be expressed in F9 cells, with Fzd7 being the most highly expressed, and chosen for further analysis. To alter steady-state Fzd7 levels and test the effect this has on differentiation, siRNA and overexpression approaches were used to knock-down and ectopically express the Fzd7 message, respectively. siRNA knock-down of Fzd7 resulted in reduced DAB2 levels, and the overexpression activated a TCF-LEF reporter, but neither approach affected differentiation. Our focus turned to how canonical WNT6 signaling was attenuated to allow PrE cells to form parietal endoderm (PE). Dkk1, encoding a WNT antagonist, was examined and results showed that its expression increased in F9 cells treated with retinoic acid (RA) or overexpressing Wnt6. F9 cells overexpressing human DKK1 or treated with DKK1-conditioned medium and then treated with RA failed to differentiate, indicating that a negative feedback loop involving WNT6 and DKK1 attenuates canonical WNT-β-catenin signaling, thereby allowing PE cells to differentiate.

OBJECTIVE: Dickkopf-3 (Dkk3) is a non-canonical member of the Dkk family of Wnt antagonists and its upregulation has been reported in microarray analysis of cartilage from mouse models of osteoarthritis (OA). In this study we assessed Dkk3 expression in human OA cartilage to ascertain its potential role in chondrocyte signaling and cartilage maintenance.
METHODS: Dkk3 expression was analysed in human adult OA cartilage and synovial tissues and during chondrogenesis of ATDC5 and human mesenchymal stem cells. The role of Dkk3 in cartilage maintenance was analysed by incubation of bovine and human cartilage explants with interleukin-1β (IL1β) and oncostatin-M (OSM). Dkk3 gene expression was measured in cartilage following murine hip avulsion. Whether Dkk3 influenced Wnt, TGFβ and activin cell signaling was assessed in primary human chondrocytes and SW1353 chondrosarcoma cells using qRT-PCR and luminescence assays.
RESULTS: Increased gene and protein levels of Dkk3 were detected in human OA cartilage, synovial tissue and synovial fluid. DKK3 gene expression was decreased during chondrogenesis of both ATDC5 cells and humans MSCs. Dkk3 inhibited IL1β and OSM-mediated proteoglycan loss from human and bovine cartilage explants and collagen loss from bovine cartilage explants. Cartilage DKK3 expression was decreased following hip avulsion injury. TGFβ signaling was enhanced by Dkk3 whilst Wnt3a and activin signaling were inhibited.
CONCLUSIONS: We provide evidence that Dkk3 is upregulated in OA and may have a protective effect on cartilage integrity by preventing proteoglycan loss and helping to restore OA-relevant signaling pathway activity. Targeting Dkk3 may be a novel approach in the treatment of OA.