Differential changes in Wnt7 and Dkk1 levels in astrocytes exposed to glutamate or TNFα.

Wnt signaling plays an important role in adult brain function, and its dysregulation has been implicated in the loss of neuronal homeostasis. Despite the existence of many studies on the participation of the Wnt pathway in adult neurons, its regulation in astrocytes has been scarcely explored. Several reports point to the presence of Wnt ligands in astrocytes and their possible impact on neuronal plasticity or neuronal death. We aimed to analyze the effect of the neurotransmitter glutamate and the inflammatory cytokine TNFα on the mRNA and protein levels of the canonical Wnt agonist Wnt7a and the antagonist Dkk1 in cultured astrocytes. Primary astrocyte cultures from rat cerebral cortices were exposed to glutamate or TNFα. Wnt7a and Dkk1 expression was analyzed by RT-qPCR and its protein abundance and distribution was assessed by immunofluorescence. We found high basal expression and protein levels of Wnt7a and Dkk1 in unstimulated astrocytes and overproduction of Dkk1 mRNA induced by the two stimuli. These results reveal the astrocytic source of the canonical Wnt ligands Wnt7a and Dkk1, whose levels are differentially regulated by glutamate and TNFα. Astrocytes are a significant source of Wnt ligands, the production of which can be differentially regulated under excitatory or proinflammatory conditions, thereby impacting neuronal function.

The signaling pathways activated by ROR1 in cancer.

The receptor tyrosine kinase orphan receptor 1 (ROR1) is a receptor for WNT5A and related Wnt proteins, that play an important role during embryonic development by regulating cell migration, cell polarity, neural patterning, and organogenesis. ROR1 exerts these functions by transducing signals from the Wnt secreted glycoproteins to the intracellular Wnt/PCP and Wnt/Ca++ pathways. Investigations in adult human cells, particularly cancer cells, have demonstrated that besides these two pathways, the WNT5A/ROR1 axis can activate a number of signaling pathways, including the PI3K/AKT, MAPK, NF-κB, STAT3, and Hippo pathways. Moreover, ROR1 is aberrantly expressed in cancer and was associated with tumor progression and poor survival by promoting cell proliferation, survival, invasion, epithelial to mesenchymal transition, and metastasis. Consequently, numerous therapeutic tools to target ROR1 are currently being evaluated in cancer patients. In this review, we will provide a detailed description of the signaling pathways regulated by ROR1 in cancer and their impact in tumor progression.

TIPE1 inhibits the growth of Ewing's sarcoma cells by suppressing Wnt/ß-catenin signaling.

BACKGROUND: Ewing's sarcoma is the second most common bone and soft tissue malignancy in children and adolescents. Tumor necrosis factor-α-induced protein 8-like 1 (TIPE1) functions as a tumor suppressor in several cancers. Activation of Wnt/ß-catenin signaling in subpopulations of tumor cells contributes to phenotypic heterogeneity and disease progression in Ewing's sarcoma. The exact role of TIPE1 in Ewing's sarcoma remains to be elucidated. PURPOSE: This study aimed to assess the expression and function of TIPE1 in Ewing's sarcoma. METHODS: TIPE1 expression in Ewing's sarcoma cells was determined by qPCR and western blotting. Furthermore, the Ewing's sarcoma cell line RD-ES was transfected with a lentivirus-based TIPE1 expression system to upregulate the expression of TIPE1. The Cell Counting Kit 8 was used to assess the effect of TIPE1 on cell proliferation. The effects of TIPE1 on cell migration and invasion was detected by Transwell assay. Flow cytometry was performed to detect apoptosis. RESULTS: Our results suggested lower TIPE1 expression in Ewing's sarcoma cell lines compared with normal osseous cells. TIPE1 remarkably inhibited the growth and proliferation of Ewing's sarcoma cell; TIPE1 also induced apoptosis and inhibited invasion in vitro. TIPE1 inhibited Ewing's sarcoma growth, motility, and survival through regulation of Wnt/ß-catenin signaling. CONCLUSIONS: Our results demonstrated the anti-tumor function of TIPE1 in Ewing's sarcoma and reveal a novel therapeutic target.

Stimulation of Osteoclast Formation by Oncostatin M and the Role of WNT16 as a Negative Feedback Regulator.

Oncostatin M (OSM), which belongs to the IL-6 family of cytokines, is the most potent and effective stimulator of osteoclast formation in this family, as assessed by different in vitro assays. Osteoclastogenesis induced by the IL-6 type of cytokines is mediated by the induction and paracrine stimulation of the osteoclastogenic cytokine receptor activator of nuclear factor κ-B ligand (RANKL), expressed on osteoblast cell membranes and targeting the receptor activator of nuclear factor κ-B (RANK) on osteoclast progenitor cells. The potent effect of OSM on osteoclastogenesis is due to an unusually robust induction of RANKL in osteoblasts through the OSM receptor (OSMR), mediated by a JAK-STAT/MAPK signaling pathway and by unique recruitment of the adapter protein Shc1 to the OSMR. Gene deletion of Osmr in mice results in decreased numbers of osteoclasts and enhanced trabecular bone caused by increased trabecular thickness, indicating that OSM may play a role in physiological regulation of bone remodeling. However, increased amounts of OSM, either through administration of recombinant protein or of adenoviral vectors expressing Osm, results in enhanced bone mass due to increased bone formation without any clear sign of increased osteoclast numbers, a finding which can be reconciled by cell culture experiments demonstrating that OSM can induce osteoblast differentiation and stimulate mineralization of bone nodules in such cultures. Thus, in vitro studies and gene deletion experiments show that OSM is a stimulator of osteoclast formation, whereas administration of OSM to mice shows that OSM is not a strong stimulator of osteoclastogenesis in vivo when administered to adult animals. These observations could be explained by our recent finding showing that OSM is a potent stimulator of the osteoclastogenesis inhibitor WNT16, acting in a negative feedback loop to reduce OSM-induced osteoclast formation.

Wnt1 and Wnt2b immunodetection of the regenerating tail and comparative ultrastructure of tail spinal cord in the Scincella tsinlingensis / Inmunodetección Wnt1 y Wnt2b de la cola en regeneración y ultraestructura comparativa de la médula espinal de la cola en Scincella tsinlingensis

SUMMARY: The Wnt pathway is essential for the initiation of lizard tail regeneration. The regenerated lizard tails exhibit obvious morphological differences compared to the original ones. The expression of Wnt1 and Wnt2b proteins in the regenerating tail of Scincella tsinlingensis was detected by immunohistochemistry and then comparatively analyzed for ultrastructural changes in the original and regenerated spinal cord. The ependymal layer of the original spinal cord was pseudostratified with multiciliated cells and primary monociliated cells, while the cells of the ependymal layer of the regenerated spinal cord were organized in a monolayer with a few biciliated cells. Immunolocalization indicated that Wnt1 and Wnt2b were mainly distributed in the dermis near the original tail stump, spinal cord, and clot-positive migratory cells during Stage I, 0-1 days post-amputation (dpa). Wnt1 and Wnt2b were predominantly detected in the epaxial and hypaxial musculature near the original tail stump, wound epithelium, and spinal cord in the original tail during Stage II, 1-7 dpa. Mesenchymal cells and wound epithelium showed immunostaining during Stage III and IV, 7-15 dpa. The ependymal tubes contained these signaling proteins during Stage V and VI, 20- 30 dpa. Labeling was mainly observed in nearby regenerative blood vessels, ependymal cells, epaxial and hypaxial musculature in the apical epithelial layer (AEC) after 45-160 dpa. These findings indicated that Wnt1 and Wnt2b proteins presented primarily in regenerating epidermis and nerve tissues were a critical signal for tail regeneration in S. tsinlingensis.

RESUMEN: La vía Wnt es esencial para el inicio de la regeneración de la cola del lagarto. Las colas de lagarto regeneradas exhiben diferencias morfológicas obvias en comparación con las originales. La expresión de las proteínas Wnt1 y Wnt2b en la cola en regeneración de Scincella tsinlingensis se detectó mediante inmunohistoquímica y luego se analizaron comparativamente los cambios ultraestructurales en la médula espinal original y regenerada. La capa ependimaria de la médula espinal original se pseudoestratificó con células multiciliadas y células monociliadas primarias, mientras que las células de la capa ependimaria de la médula espinal regenerada se organizaron en monocapa con algunas células bicilicadas. La inmunolocalización indicó que Wnt1 y Wnt2b se distribuyeron principalmente en la dermis cerca del muñón de la cola original, la médula espinal y las células migratorias positivas en el coágulo durante la Etapa I, 0-1 días después de la amputación (dpa). Wnt1 y Wnt2b se detectaron predominantemente en la musculatura epaxial e hipaxial cerca del muñón de la cola original, el epitelio de la herida y la médula espinal en la cola original durante la Etapa II, 1-7 dpa. Las células mesenquimales y el epitelio de la herida mostraron inmunomarcaje durante la Etapa III y IV, 7- 15 dpa. Los tubos ependimarios contenían estas proteínas de señalización durante la Etapa V y VI, 20-30 dpa. El marcaje se observó principalmente en vasos sanguíneos regenerativos cercanos, células ependimarias, musculatura epaxial e hipaxial en la capa epitelial apical (AEC) después de 45-160 dpa. Estos hallazgos indicaron que las proteínas Wnt1 y Wnt2b están presentes principalmente en la epidermis en regeneración y en los tejidos nerviosos y eran una señal crítica para la regeneración de la cola en S. tsinlingensis.

The multifaceted NF-kB: are there still prospects of its inhibition for clinical intervention in pediatric central nervous system tumors?

Despite advances in the understanding of the molecular mechanisms underlying the basic biology and pathogenesis of pediatric central nervous system (CNS) malignancies, patients still have an extremely unfavorable prognosis. Over the years, a plethora of natural and synthetic compounds has emerged for the pharmacologic intervention of the NF-kB pathway, one of the most frequently dysregulated signaling cascades in human cancer with key roles in cell growth, survival, and therapy resistance. Here, we provide a review about the state-of-the-art concerning the dysregulation of this hub transcription factor in the most prevalent pediatric CNS tumors: glioma, medulloblastoma, and ependymoma. Moreover, we compile the available literature on the anti-proliferative effects of varied NF-kB inhibitors acting alone or in combination with other therapies in vitro, in vivo, and clinical trials. As the wealth of basic research data continues to accumulate, recognizing NF-kB as a therapeutic target may provide important insights to treat these diseases, hopefully contributing to increase cure rates and lower side effects related to therapy.

Contribution of RAGE axis activation to the association between metabolic syndrome and cancer.

Far beyond the compelling proofs supporting that the metabolic syndrome represents a risk factor for diabetes and cardiovascular diseases, a growing body of evidence suggests that it is also a risk factor for different types of cancer. However, the involved molecular mechanisms underlying this association are not fully understood, and they have been mainly focused on the individual contributions of each component of the metabolic syndrome such as obesity, hyperglycemia, and high blood pressure to the development of cancer. The Receptor for Advanced Glycation End-products (RAGE) axis activation has emerged as an important contributor to the pathophysiology of many clinical entities, by fueling a chronic inflammatory milieu, and thus supporting an optimal microenvironment to promote tumor growth and progression. In the present review, we intend to highlight that RAGE axis activation is a crosswise element on the potential mechanistic contributions of some relevant components of metabolic syndrome into the association with cancer.

Differential Regulation of Wnt Signaling Components During Hippocampal Reorganization After Entorhinal Cortex Lesion.

Entorhinal cortex lesions have been established as a model for hippocampal deafferentation and have provided valuable information about the mechanisms of synapse reorganization and plasticity. Although several molecules have been proposed to contribute to these processes, the role of Wnt signaling components has not been explored, despite the critical roles that Wnt molecules play in the formation and maintenance of neuronal and synaptic structure and function in the adult brain. In this work, we assessed the reorganization process of the dentate gyrus (DG) at 1, 3, 7, and 30 days after an excitotoxic lesion in layer II of the entorhinal cortex. We found that cholinergic fibers sprouted into the outer molecular layer of the DG and revealed an increase of the developmental regulated MAP2C isoform 7 days after lesion. These structural changes were accompanied by the differential regulation of the Wnt signaling components Wnt7a, Wnt5a, Dkk1, and Sfrp1 over time. The progressive increase in the downstream Wnt-regulated elements, active-ß-catenin, and cyclin D1 suggested the activation of the canonical Wnt pathway beginning on day 7 after lesion, which correlates with the structural adaptations observed in the DG. These findings suggest the important role of Wnt signaling in the reorganization processes after brain lesion and indicate the modulation of this pathway as an interesting target for neuronal tissue regeneration.

Acute and chronic lithium treatment increases Wnt/ß-catenin transcripts in cortical and hippocampal tissue at therapeutic concentrations in mice.

Lithium activates Wnt/ß-catenin signaling leading to stabilization of free cytosolic ß-catenin. The aim of the present study is to evaluate the in vivo effect of acute and chronic lithium treatment on the expression of ß-catenin target genes, addressing its transcripts HIG2, Bcl-xL, Cyclin D1, c-myc, in cortical and hippocampal tissue from adult mice. Lithium doses were established to yield therapeutic working concentrations. In acute treatment, mice received a 300µL of a 350 mg/kg solution of LiCl by gavage, and were euthanized after 2 h, 6 h and 12 h. To determine the effect of chronic treatment, animals were continuously fed either with chow supplemented with 2 g/kg Li2CO3, or regular chow (controls), being euthanized after 30 days. All animals had access to drinking water and 0.9% saline ad libitum. After acute and chronic treatments samples of peripheral blood were obtained from the tail vein for each animal, and serum concentrations of lithium were determined. All transcripts were up-regulated in cortical and hippocampal tissues of lithium-treated mice, both under acute and chronic treatments. There was a positive correlation between serum lithium concentrations and the increment in the expression of all transcripts. This effect was observed in all time points of the acute treatment (i.e., 2, 6 and 12 hours) and also after 30 days. We conclude that Wnt/ß-catenin transcriptional response (HIG2, Bcl-xL, Cyclin D1 and c-myc) is up-regulated in the mouse brain in response to acute and chronic lithium treatment at therapeutic concentrations.

Wnt-5A/B Signaling in Hematopoiesis throughout Life.

Wnt signaling is well-known to play major roles in the hematopoietic system, from embryogenesis to aging and disease. In addition to the main ß-catenin-dependent pathway, it is now clear that Wnt5a and the structurally related Wnt5b are essential for hematopoiesis, bone marrow colonization and the final steps of hematopoietic stem cell (HSC) maturation via ß-catenin-independent signaling. Wnt5a and Wnt5b ligands prevent hematopoietic exhaustion (by maintaining quiescent, long-term HSCs), induce the proliferation of progenitors, and guide myeloid development, in addition to being involved in the development of aging-related alterations. The aim of this review is to summarize the current knowledge on these roles of Wnt5a and Wn5b signaling in the hematopoietic field.

Exogenous Expression of WNT7A in Leukemia-Derived Cell Lines Induces Resistance to Chemotherapeutic Agents.

BACKGROUND: Dysregulations of the WNT pathway are implicated in the malignant transformation of different types of neoplasia. WNT7A is expressed in normal peripheral lymphocytes, but is decreased in the tumoral counterpart. Furthermore, the treatment of leukemic cells with recombinant WNT7A decreases proliferation, suggesting its possible use as a therapeutic biomolecule. This study aimed to evaluate the concomitant action of WNT7A and different chemotherapeutic agents over proliferation and cell death of leukemia/ lymphoma derived cell lines. METHODS: Ectopic expression of WNT7A was induced in CEM and BJAB cell lines by using a lentiviral system. RNA expression was analyzed by microarrays and qPCR, and protein expression was determined by Western Blot. Cell proliferation was measured by cell counting, metabolic activity by WST-1 assay, cell death and DNA content by flow cytometry. RESULTS: WNT7A ectopic expression was shown to decrease cell proliferation, but the apoptosis rate of leukemic cells was not altered. Moreover, these cells acquired resistance to doxorubicin, vincristine and MG-132. Cell cycle analysis reveals a decrease in G1 and an increase in S and G2 phases with a further upregulation of senescence- associated genes. Microarray analysis reveals that most gene expression changes were related to cancer and metabolic associated pathways. All those changes appear to be independent of the WNT canonical pathway regulation. CONCLUSION: WNT7A negatively regulates cell proliferation in leukemic cell lines and promotes resistance to chemotherapeutic agents by inducing a senescence-like phenotype independently of the WNT canonical pathway.

WNT7A Expression is Downregulated in T Lymphocytes after T-Cell Receptor Activation Due to Histone Modifications and in T-ALL by DNA Methylation.

WNT signaling pathway regulates several processes involved in the homeostasis of normal cells. Its dysregulation is associated with pathological outcomes like cancer. We previously demonstrated that downregulation of WNT7A correlates with higher proliferation rates in acute lymphoblastic leukemia. However, the regulation of this gene in pathological and normal conditions remains unexplored. In this work, we aimed to analyze the transcriptional regulation of WNT7A in leukemic cells and in normal T lymphocytes after a proliferative stimulus. WNT7A expression was measured in blood cells and in T lymphocytes after phytohemagglutinin-L (PHA-L) treatment or T-cell receptor (TCR) activation by qPCR and Western blot. Promoter methylation was assessed using methylation-sensitive restriction enzymes, and histone modifications were determined by chromatin immunoprecipitation and qPCR. In T-cell acute lymphoblastic leukemia (T-ALL), WNT7A expression is silenced through DNA methylation of CpG island in the promoter region. In normal peripheral blood cells, WNT7A is mainly expressed by monocytes and T lymphocytes. TCR activation induces the downregulation of WNT7A in normal T lymphocytes by changes in histone methylation marks (H3K4me2/3) and histone deacetylases. A proliferative stimulus mediated by IL-2 keeps WNT7A expression at low levels but in the absence of IL-2, the expression of this gene tends to be restored. Furthermore, after TCR activation and WNT7A downregulation, target genes associated with the WNT canonical pathway were upregulated indicating an independent activity of WNT7A from the WNT canonical pathway. WNT7A expression is silenced by long-term DNA methylation in T-ALL-derived cells and downregulated by histone modifications after TCR activation in normal T lymphocytes.

Extracellular matrix stiffness and Wnt/ß-catenin signaling in physiology and disease.

The Wnt/ß-catenin signaling pathway plays fundamental roles during development, stem cell differentiation, and homeostasis, and its abnormal activation can lead to diseases. In recent years, it has become clear that this pathway integrates signals not only from Wnt ligands but also from other proteins and signaling routes. For instance, Wnt/ß-catenin signaling involves YAP and TAZ, which are transcription factors with crucial roles in mechanotransduction. On the other hand, Wnt/ß-catenin signaling is also modulated by integrins. Therefore, mechanical signals might similarly modulate the Wnt/ß-catenin pathway. However, and despite the relevance that mechanosensitive Wnt/ß-catenin signaling might have during physiology and diseases such as cancer, the role of mechanical cues on Wnt/ß-catenin signaling has received less attention. This review aims to summarize recent evidence regarding the modulation of the Wnt/ß-catenin signaling by a specific type of mechanical signal, the stiffness of the extracellular matrix. The review shows that mechanical stiffness can indeed modulate this pathway in several cell types, through differential expression of Wnt ligands, receptors and inhibitors, as well as by modulating ß-catenin levels. However, the specific mechanisms are yet to be fully elucidated.

Canonical and non-canonical Wnt signaling are simultaneously activated by Wnts in colon cancer cells.

The Wnt signaling pathway is a crucial regulator of the intestinal epithelium homeostasis and is altered in most colon cancers. While the role of aberrant canonical, ß-catenin-dependent Wnt signaling has been well established in colon cancer promotion, much less is known about the role played by noncanonical, ß-catenin-independent Wnt signaling in this type of cancer. This work aimed to characterize the noncanonical signal transduction pathway in colon cancer cells. To this end, we used the prototype noncanonical ligand, Wnt5a, in comparison with Wnt3a, the prototype of a canonical ß-catenin activating ligand. The analysis of the expression profile of Wnt receptors in colon cancer cell lines showed a clear increase in both level expression and variety of Frizzled receptor types expressed in colon cancer cells compared with non-malignant cells. We found that Wnt5a activates a typical Wnt/Ca++ - noncanonical signaling pathway in colon malignant cells, inducing the hyperphosphorylation of Dvl1, Dvl2 and Dvl3, promoting Ca++ mobilization as a result of phospholipase C (PLC) activation via pertussis toxin-sensitive G-protein, and inducing PLC-dependent cell migration. We also found that while the co-receptor Ror2 tyrosine kinase activity is not required for Ca++ mobilization-induced by Wnt5a, it is required for the inhibitory effects of Wnt5a on the ß-catenin-dependent transcriptional activity. Unexpectedly, we found that although the prototype canonical Wnt3a ligand was unique in stimulating the ß-catenin-dependent transcriptional activity, it also simultaneously activated PLC, promoted Ca++ mobilization, and induced Rho kinase and PLC-dependent cell migration. Our data indicate, therefore, that a Wnt ligand can activate at the same time the so-called Wnt canonical and noncanonical pathways inducing the formation of complex signaling networks to integrate both pathways in colon cancer cells.

Long noncoding RNA CASC2c inhibited cell proliferation in hepatocellular carcinoma by inactivated ERK1/2 and Wnt/ß-catenin signaling pathway.

PURPOSE: Long non-coding RNAs (lncRNAs) have been shown to play important roles in tumorigenesis, but their biological functions and the underlying molecular mechanisms remain unclear. Alternative splicing of five exons results in three transcript variants of cancer susceptibility 2 (CASC2): the lncRNAs CASC2a, CASC2b, and CASC2c. CASC2a/b have been found to have crucial regulatory functions in a number of malignancies, but few studies have examined the effects of CASC2c in cancers. The objective of the study was to investigate the role of CASC2c in the proliferation and apoptosis of hepatocellular carcinoma (HCC) cells. METHODS: This study first investigated the expression levels of CASC2c in tumor tissues, corresponding non-tumor tissues and cells using quantitative real-time polymerase chain reaction. The function and underlying molecular mechanism of CASC2c in human HCC were investigated in QGY-7703 cell line, as well as in gastric cancer (GC) cell and colorectal cancer (CRC) cell. RESULTS: In the present work, we observed that CASC2c was significantly down-regulated in HCC tissues and cells. Moreover, its overexpression remarkably inhibited the growth, migration, and invasion of HCC cells in vitro and promoted their apoptosis. Furthermore, we demonstrated that CASC2c overexpression decreased p-ERK1/2 levels in HCC, GC, and CRC cells. Interestingly, while overexpression of CASC2c decreased ß-catenin expression in HCC and GC cells, it increased that in CRC cells. CONCLUSION: The lncRNA-CASC2c has a vital role in tumorigenesis and cancer progression, and may serve as a biomarker or therapeutic target in cancer treatment via down-regulation of the ERK1/2 and Wnt/ß-catenin signaling pathways.

Canonical Wnt Signaling Modulates the Expression of Pre- and Postsynaptic Components in Different Temporal Patterns.

Wnt ligands play critical roles in neuronal development, synapse formation, synaptic activity, and plasticity. Synaptic plasticity requires molecular remodeling of synapses, implying the expression of key synaptic components. Some studies have linked Wnt signaling activity to changes in synaptic protein levels. However, the presynaptic and postsynaptic gene expression profiles of hippocampal neurons exposed to Wnt proteins have not been studied. Hence, we treated rat cultured hippocampal neurons with recombinant Wnt3a, lithium, and the Wnt inhibitor Dkk-1 for different treatment durations and measured the mRNA and protein levels of pre- and postsynaptic components. The ligand Wnt3a promoted the differential temporal expression of genes encoding presynaptic and postsynaptic proteins. Gene expression of the presynaptic proteins Rim1, piccolo (Pclo), Erc2, Ctbp1 and Rimbp2 increased in a specific temporal pattern. Simultaneously, the mRNA and protein levels of postsynaptic components showed a different temporal expression pattern, e.g., the mRNAs for postsynaptic scaffolding components such as postsynaptic density protein-95 (PSD-95/Dlg4), Homer1 and Shank1 were temporally regulated by both Wnt3a and lithium. On the other hand, the mRNA levels of the gene encoding the protein calcium/calmodulin-dependent protein kinase IV (Camk4), canonically upregulated by Wnt, were increased. Our results suggest that Wnt signaling orchestrates expressional changes in genes encoding presynaptic and postsynaptic components, probably as part of a synaptic plasticity mechanism in neurons.

Adult and iPS-derived non-parenchymal cells regulate liver organoid development through differential modulation of Wnt and TGF-ß.

BACKGROUND: Liver organoid technology holds great promises to be used in large-scale population-based drug screening and in future regenerative medicine strategies. Recently, some studies reported robust protocols for generating isogenic liver organoids using liver parenchymal and non-parenchymal cells derived from induced pluripotent stem cells (iPS) or using isogenic adult primary non-parenchymal cells. However, the use of whole iPS-derived cells could represent great challenges for a translational perspective. METHODS: Here, we evaluated the influence of isogenic versus heterogenic non-parenchymal cells, using iPS-derived or adult primary cell lines, in the liver organoid development. We tested four groups comprised of all different combinations of non-parenchymal cells for the liver functionality in vitro. Gene expression and protein secretion of important hepatic function markers were evaluated. Additionally, liver development-associated signaling pathways were tested. Finally, organoid label-free proteomic analysis and non-parenchymal cell secretome were performed in all groups at day 12. RESULTS: We show that liver organoids generated using primary mesenchymal stromal cells and iPS-derived endothelial cells expressed and produced significantly more albumin and showed increased expression of CYP1A1, CYP1A2, and TDO2 while presented reduced TGF-ß and Wnt signaling activity. Proteomics analysis revealed that major shifts in protein expression induced by this specific combination of non-parenchymal cells are related to integrin profile and TGF-ß/Wnt signaling activity. CONCLUSION: Aiming the translation of this technology bench-to-bedside, this work highlights the role of important developmental pathways that are modulated by non-parenchymal cells enhancing the liver organoid maturation.

Cancer as an Embryological Phenomenon and Its Developmental Pathways: A Hypothesis regarding the Contribution of the Noncanonical Wnt Pathway.

For gastrulation to occur in human embryos, a mechanism that simultaneously regulates many different processes, such as cell differentiation, proliferation, migration, and invasion, is required to consistently and effectively create a human being during embryonic morphogenesis. The striking similarities in the processes of cancer and gastrulation have prompted speculation regarding the developmental pathways involved in their regulation. One of the fundamental requirements for the developmental pathways in gastrulation and cancer is the ability to respond to environmental stimuli, and it has been proposed that the Kaiso and noncanonical Wnt pathways participate in the mechanisms regulating these developmental pathways. In particular, these pathways might also explain the notable differences in invasive capacity between cancers of endodermal and mesodermal origins and cancers of ectodermal origin. Nevertheless, the available information indicates that cancer is an abnormal state of adult human cells in which developmental pathways are reactivated in inappropriate temporal and spatial contexts.

Islet cells are the source of Wnts that can induce beta-cell proliferation in vitro.

Wnt proteins act mainly as paracrine signals regulating cell proliferation and differentiation. The canonical Wnt pathway has recently been associated with pancreas development and the onset of type 2 diabetes in rodent and human but the underlying mechanisms are still unclear. The aim of this work was threefold: (a) to screen for Wnt expressed by murine pancreas/islet cells, (b) to investigate whether the Wnt gene expression profile can be changed in hyperplastic islets from type 2 prediabetic mice (fed a high-fat diet), and (c) to verify whether soluble factors (namely Wnts) released by pancreatic islets affect insulin secretion and proliferation of a beta-cell line in vitro condition. The majority of the Wnt subtypes are expressed by islet cells, such as Wnts 2, 2b, 3, 3a, 4, 5a, 5b, 6, 7a, 7b, 8a, 8b, 9a, 9b, and 11, while in the whole pancreas homogenates were found the same subtypes, except Wnts 3, 6, 7a, and 7b. Among all the Wnts, the Wnts 3a and 5b showed a significantly increased gene expression in hyperplastic islets from prediabetic mice compared with those from control mice. Furthermore, we observed that coculture with hyperplastic or nonhyperplastic islets did not change the secretory function of the mouse insulinoma clone 6 (MIN6) beta cells but induced a significant increase in cell proliferation in this lineage, which was partially blocked by the IWR-1 and IWP-2 Wnt inhibitors. In conclusion, we demonstrated that murine pancreas/islet cells can secrete Wnts, and that islet-released Wnts may participate in the regulation of beta-cell mass under normal and prediabetic conditions.

6-Bromoindirubin-3'-oxime promotes osteogenic differentiation of canine BMSCs through inhibition of GSK3ß activity and activation of the Wnt/ß-catenin signaling pathway.

This study aimed to investigate how 6-bromoindirubin-3'-oxime (BIO) increases the osteogenic differentiation of canine bone mesenchymal stem cells (BMSCs) and the role of the Wnt/ß-catenin signaling pathway in this process. We mimicked the effect of Wnt by adding BIO to the culture medium of BMSCs and examined whether canonical Wnt signaling positively affects the differentiation of these cells into osteoblasts. Canine BMSCs were cultured with 0.5 and 1.0 µM BIO under osteogenic conditions and then differentiation markers were investigated. It was found that BIO significantly increased the activity of alkaline phosphatase (ALP), the number of ALP-positive cells, the mineralization level and calcium deposits. Moreover, cells cultured with 0.5 and 1.0 µM BIO exhibited detectable ß-catenin expression in their nuclei, and showed upregulated ß-catenin and glycogen synthase kinase 3 beta(GSK3ß) phosphorylation compared to untreated cells. In addition, BIO enhanced the mRNA expression of osteoblast differentiation markers such as ALP, runt-related transcription factor 2, collagen I, osteocalcin, and osteonectin. In conclusion, BIO upregulated GSK3ß phosphorylation and inhibited its activity, thereby activating the Wnt/ß-catenin signaling pathway and promoting the osteogenic differentiation of canine BMSCs. The effect of 1.0 µM BIO on BMSCs differentiation was stronger than that of 0.5 µM BIO.