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Pharmacological modulation of the Wnt/β-catenin signaling pathway holds promises for both basic research and therapeutic applications. In this issue of Cell Chemical Biology, Kschonsak et al. 1 engineered knotted peptides that promote Wnt signaling by targeting ZNRF3 and serve as pharmacological tools for studying Wnt biology and supporting organoid growth., Competing Interests: Declaration of interests Stephane Angers is a founder, shareholder, and board member of AntlerA Therapeutics. S.A. is also an inventor on patents describing Frizzled antibody agonists (US16/969,909; US17/846,846)., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Signaling by bone morphogenetic proteins (BMPs) plays pivotal roles in embryogenesis, adult tissue homeostasis, and disease. Recent studies revealed that the well-established WNT agonist R-spondin 2 (RSPO2) is also a BMP receptor (BMP receptor type 1A) antagonist, with roles in early Xenopus embryogenesis and human acute myeloid leukemia (AML). To uncouple the BMP antagonist function from the WNT agonist function and to promote development of AML therapeutics, here we identified a 10-mer peptide (RW) derived from the thrombospondin 1 domain of RSPO2, which specifically prevents binding between RSPO2 and BMP receptor type 1A without altering WNT signaling. We also show that a corresponding RW dendrimer (RW d ) exhibiting improved half-life relieves inhibition of BMP receptor signaling by RSPO2 in human AML cells, reduces cell growth, and induces differentiation. Moreover, microinjection of RW d in Xenopus embryos ventralizes the dorsoventral embryonic patterning by upregulating BMP signaling without affecting WNT signaling. Our study corroborates the function of RSPO2 as a BMP receptor antagonist and provides a proof of concept for pharmacologically uncoupling BMP antagonist from WNT agonist functions of RSPO2 using the inhibitor peptide RW d with enhanced target selectivity and limited side effects., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Bovine embryonic stem cells (ESC) have features associated with the primed pluripotent state including low expression of one of the core pluripotency transcription factors, NANOG. It has been reported that NANOG expression can be upregulated in porcine ESC by treatment with activin A and the WNT agonist CHIR99021. Accordingly, it was tested whether expression of NANOG and another pluripotency factor SOX2 could be stimulated by activin A and the WNT agonist CHIR99021. Immunoreactive NANOG and SOX2 were analyzed for bovine ESC lines derived under conditions in which activin A and CHIR99021 were added singly or in combination. Activin A enhanced NANOG expression but also reduced SOX2 expression. CHIR99021 depressed expression of both NANOG and SOX2. In a second experiment, activin A enhanced blastocyst development while CHIR99021 treatment impaired blastocyst formation and reduced number of blastomeres. Activin A treatment decreased blastomeres in the blastocyst that were positive for either NANOG or SOX2 but increased those that were CDX2+ and that were GATA6+ outside the inner cell mass. CHIR99021 reduced SOX2+ and NANOG+ blastomeres without affecting the number or percent of blastomeres that were CDX2+ and GATA6+. Results indicate activation of activin A signaling stimulates NANOG expression during self-renewal of bovine ESC but suppresses cells expressing pluripotency markers in the blastocyst and increases cells expressing CDX2. Actions of activin A to promote blastocyst development may involve its role in promoting trophectoderm formation. Furthermore, results demonstrate the negative role of canonical WNT signaling in cattle for pluripotency marker expression in ESC and in formation of the inner cell mass and epiblast during embryonic development. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The Wnt signaling pathway is intricately connected with bone mass regulation in humans and rodent models. We designed an antibody-based platform that generates potent and selective Wnt mimetics. Using this platform, we engineer bi-specific Wnt mimetics that target Frizzled and low-density lipoprotein receptor-related proteins and evaluate their effects on bone accrual in murine models. These synthetic Wnt agonists induce rapid and robust bone building effects, and correct bone mass deficiency and bone defects in various disease models, including osteoporosis, aging, and long bone fracture. Furthermore, when these Wnt agonists are combined with antiresorptive bisphosphonates or anti-sclerostin antibody therapies, additional bone accrual/maintenance effects are observed compared to monotherapy, which could benefit individuals with severe and/or acute bone-building deficiencies. Our data support the continued development of Wnt mimetics for the treatment of diseases of low bone mineral density, including osteoporosis.

Lymphotoxin β-receptor (LTβR) signalling promotes lymphoid neogenesis and the development of tertiary lymphoid structures 1,2 , which are associated with severe chronic inflammatory diseases that span several organ systems 3-6 . How LTβR signalling drives chronic tissue damage particularly in the lung, the mechanism(s) that regulate this process, and whether LTβR blockade might be of therapeutic value have remained unclear. Here we demonstrate increased expression of LTβR ligands in adaptive and innate immune cells, enhanced non-canonical NF-κB signalling, and enriched LTβR target gene expression in lung epithelial cells from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke. Therapeutic inhibition of LTβR signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue, induced regeneration of lung tissue, and reverted airway fibrosis and systemic muscle wasting. Mechanistically, blockade of LTβR signalling dampened epithelial non-canonical activation of NF-κB, reduced TGFβ signalling in airways, and induced regeneration by preventing epithelial cell death and activating WNT/β-catenin signalling in alveolar epithelial progenitor cells. These findings suggest that inhibition of LTβR signalling represents a viable therapeutic option that combines prevention of tertiary lymphoid structures 1 and inhibition of apoptosis with tissue-regenerative strategies.

Acute liver failure (ALF) is known for extremely high mortality rate, the result of widespread damage of hepatocytes. Orthotopic liver transplantation is the only effective therapy but its application is limited by the scarcity of donor organs. Given the importance in the liver biology of Wnt/beta-catenin signaling pathway, we hypothesized that its stimulation could enhance hepatocyte regeneration and attenuate the course of thioacetamide (TAA)-induced ALF in Lewis rats. Chronic treatment with Wnt agonist was started either immediately after hepatotoxic insult ("early treatment") or when signs of ALF had developed ("late treatment"). Only 23 % of untreated Lewis rats survived till the end of experiment. They showed marked increases in plasma alanine aminotransferase (ALT) activity and bilirubin and ammonia (NH3) levels; plasma albumin decreased significantly. "Early" and "late" Wnt agonist treatment raised the final survival rate to 69 % and 63 %, respectively, and normalized ALT, NH3, bilirubin and albumin levels. In conclusion, the results show that treatment with Wnt agonist attenuates the course of TAA-induced ALF in Lewis rats, both with treatment initiated immediately after hepatotoxic insult and in the phase when ALF has already developed. Thus, the pharmacological stimulation of Wnt/beta-catenin signaling pathway can present a new approach to ALF treatment.

Inhibitors of Apoptosis Protein (IAP) genes participate in processes like apoptosis, proliferation, innate immunity, inflammation, cell motility, differentiation and in malignancies. Here we reveal 25 IAP genes in the tunicate Botryllus schlosseri's genome and their functions in two developmental biology phenomena, a new mode of whole body regeneration (WBR) induced by budectomy, and blastogenesis, the four-staged cycles of botryllid ascidian astogeny. IAP genes that were specifically upregulated during these developmental phenomena were identified, and protein expression patterns of one of these genes, IAP28, were followed. Most of the IAP genes upregulation recorded at blastogenetic stages C/D was in concert with the upregulation at 100 μM H 2 O 2 apoptotic-induced treatment and in parallel to expressions of AIF1, Bax, Mcl1, caspase 2 and two orthologues of caspase 7. Wnt agonist altered the takeover duration along with reduced IAP expressions, and displacement of IAP28 + phagocytes. WBR was initiated solely at blastogenetic stage D, where zooidal absorption was attenuated and regeneration centers were formed either from remains of partially absorbed zooids or from deformed ampullae. Subsequently, bud-bearing zooids developed, in concert with a massive IAP28-dependent phagocytic wave that eliminated the old zooids, then proceeded with the establishment of morphologically normal-looking colonies. IAP4, IAP14 and IAP28 were also involved in WBR, in conjunction with the expression of the pro-survival PI3K-Akt pathway. IAPs function deregulation by Smac mimetics resulted in severe morphological damages, attenuation in bud growth and differentiation, and in destabilization of colonial coordination. Longtime knockdown of IAP functions prior to the budectomy, resulted in colonial death., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The present study aimed to investigate the role and mechanisms of microRNA (miR)‑33b in endometriosis (Ems). Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), MTT assays, flow cytometry, caspase‑3/9 activity assays and western blotting were performed in the present study. Initially, miR‑33b expression in an Ems rat model was investigated by RT‑qPCR and was demonstrated to be upregulated in Ems tissue samples of rats compared with the control group. In addition, miR‑33b upregulation inhibited cell growth and enhanced apoptosis in an Ems model (primary cell cultures) compared with the control group. In addition, miR‑33b up‑regulation reduced Wnt/β‑catenin signaling pathway and suppressed zinc finger E‑box‑binding homeobox 1 (ZEB1) protein expression in the in vitro Ems model (primary cell cultures) compared with the control group. Furthermore, small interfering‑ZEB1 ameliorated the effects of miR‑33b downregulation on Ems cell growth in the in vitro Ems model. Additionally, a Wnt agonist reduced the effects of miR‑33b upregulation on Ems cell growth in the in vitro Ems model. In conclusion, the present study demonstrated that upregulation of miR‑33b may promote Ems through Wnt/β‑catenin by ZEB1 expression.

Previously, HLY78, a lycorine derivative, was identified as the first Wnt/β-catenin signaling agonist through binding to the DAX domain of Axin, a scaffold of Wnt/β-catenin complex. In this study, to obtain more potent Wnt/β-catenin agonist, the structure optimization of HLY78 was carried out by design and synthesis of six phenanthridine derivatives, which afforded five active ones. In particular, 8,9-bis((1,3-dimethyl-1H-pyrazol)methoxy)-5-ethyl-4-methyl-5,6-dihydrophenanthridine showed the most potent activity (0.15/μM) that was increased nearly 30 times as that of the lead HLY78. These compounds may be valuable in future pharmacological or biological studies., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Real-time, quantitative measurement of muscle progenitor cell (myoblast) differentiation is an important tool for skeletal muscle research and identification of drugs that support skeletal muscle regeneration. While most quantitative tools rely on sacrificial approach, we developed a double fluorescent tagging approach, which allows for dynamic monitoring of myoblast differentiation through assessment of fusion index and nuclei count. Fluorescent tagging of both the cell cytoplasm and nucleus enables monitoring of cell fusion and the formation of new myotube fibers, similar to immunostaining results. This labeling approach allowed monitoring the effects of Myf5 overexpression, TNFα, and Wnt agonist on myoblast differentiation. It also enabled testing the effects of surface coating on the fusion levels of scaffold-seeded myoblasts. The double fluorescent labeling of myoblasts is a promising technique to visualize even minor changes in myogenesis of myoblasts in order to support applications such as tissue engineering and drug screening.

Background/aims: Interferon consensus sequence-binding protein 8 (IRF8) belongs to a family of interferon (IFN) regulatory factors that modulates various important physiological processes including carcinogenesis. As reported by others and our group, IRF8 expression is silenced by DNA methylation in both human solid tumors and hematological malignancies. However, the role of IRF8 in lung carcinoma remains elusive. In this study, we determined IRF8 epigenetic regulation, biological functions, and the signaling pathway involved in non-small cell lung cancer (NSCLC)., Methods: IRF8 expression were determined by Q- PCR. MSP and A+T determined promotor methylation. MTS, clonogenic, Transwell assay, Flow cytometry, three-dimensional culture and AO/EB stain verified cell function. In vivo tumorigenesis examed the in vivo effects. By Chip-QPCR, RT-PCR, Western blot and Immunofluorescence staining, the mechanisms were studied., Results: IRF8 was significantly downregulated in lung tumor tissues compared with adjacent non-cancerous tissues. Furthermore, methylation-specific PCR analyses revealed that IRF8 methylation in NSCLC was a common event, and demethylation reagent treatment proved that downregulation of IRF8 was due to its promoter CpG hypermethylation. Clinical data showed that the IRF8 methylation was associated with tumor stage, lymph node metastasis status, patient outcome, and tumor histology. Exogenous expression of IRF8 in the silenced or downregulated lung cancer cell lines A549 and H1299 at least partially restored the sensitivity of lung cancer cells to apoptosis, and arrested cells at the G0/G1 phase. Cell viability, clonogenicity, and cell migration and invasive abilities were strongly inhibited by restored expression of IRF8. A three-dimensional culture system demonstrated that IRF8 changed the cells to a more spherical phenotype. Moreover, ectopic expression of IRF8 enhanced NSCLC chemosensitivity to cisplatin. Furthermore, as verified by Chip-qPCR, immunofluorescence staining, and western blotting, IRF8 bound to the T-cell factor/lymphoid enhancer factor (TCF /LEF) promoter, thus repressing β-catenin nuclear translocation and its activation. IRF8 significantly disrupted the effects of Wnt agonist, bml284, further suggesting its involvement in the Wnt/β-catenin pathway., Conclusion: IRF8 acted as a tumor suppressor gene through the transcriptional repression of β-catenin-TCF/LEF in NSCLC. IRF8 methylation may serve as a potential biomarker in NSCLC prognosis., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Hematopoietic stem cells play the vital role of maintaining appropriate levels of cells in blood. Therefore, regulation of their fate is essential for their effective therapeutic use. Here we report the role of polyunsaturated fatty acids (PUFAs) in regulating hematopoiesis which has not been explored well so far. Mice were fed daily for 10 days with n-6/n-3 PUFAs, viz. linoleic acid (LA), arachidonic acid (AA), alpha-linolenic acid and docosahexanoic acid (DHA) in four separate test groups with phosphate-buffered saline fed mice as control set. The bone marrow cells of PUFA-fed mice showed a significantly higher hematopoiesis as assessed using side population, Lin-Sca-1 + ckit+, colony-forming unit (CFU), long-term culture, CFU-spleen assay and engraftment potential as compared to the control set. Thrombopoiesis was also stimulated in PUFA-fed mice. A combination of DHA and AA was found to be more effective than when either was fed individually. Higher incorporation of PUFAs as well as products of their metabolism was observed in the bone marrow cells of PUFA-fed mice. A stimulation of the Wnt, CXCR4 and Notch1 pathways was observed in PUFA-fed mice. The clinical relevance of this study was evident when bone marrow-transplanted recipient mice, which were fed with PUFAs, showed higher engraftment of donor cells, suggesting that the bone marrow microenvironment may also be stimulated by feeding with PUFAs. These data indicate that oral administration of PUFAs in mice stimulates hematopoiesis and thrombopoiesis and could serve as a valuable supplemental therapy in situations of hematopoietic failure., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Wnt proteins modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD) and the co-receptors LRP5 and LRP6 to regulate cell fate decisions and the growth and repair of several tissues. The 19 mammalian Wnt proteins are cross-reactive with the 10 FZD receptors, and this has complicated the attribution of distinct biological functions to specific FZD and Wnt subtype interactions. Furthermore, Wnt proteins are modified post-translationally by palmitoylation, which is essential for their secretion, function and interaction with FZD receptors. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation and the functional importance of FZD subtypes, and the use of Wnt proteins as therapeutic agents. Here we develop surrogate Wnt agonists, water-soluble FZD-LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. Similar to WNT3A, these Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion, enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells, and support the growth of a broad range of primary human organoid cultures. In addition, the surrogates can be systemically expressed and exhibit Wnt activity in vivo in the mouse liver, regulating metabolic liver zonation and promoting hepatocyte proliferation, resulting in hepatomegaly. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine.

In androgenetic alopecia, androgens impair dermal papilla-induced hair follicle stem cell (HFSC) differentiation inhibiting Wnt signaling. Wnt agonists/antagonists balance was analyzed after dihydrotestosterone (DHT) stimulation in androgen-sensitive dermal papilla cells (DPC) cultured as spheroids or monolayer. In both culture conditions, DHT stimulation downregulated Wnt5a and Wnt10b mRNA while the Wnt antagonist Dkk-1 was upregulated. Notably, tissue architecture of DPC-spheroids lowers Dkk-1 and enhances Wnt agonists' basal expression; probably contributing to DPC inductivity. The role of Wnt agonists/antagonists as mediators of androgen inhibition of DPC-induced HFSC differentiation was evaluated. Inductive DPC-conditioned medium supplemented with DKK-1 impaired HFSC differentiation mimicking androgens' action. This effect was associated with inactivation of Wnt/β-catenin pathway in differentiating HFSC by both DPC-conditioned media. Moreover, addition of WNT10b to DPC-medium conditioned with DHT, overcame androgen inhibition of HFSC differentiation. Our results identify DKK1 and WNT10b as paracrine factors which modulate the HFSC differentiation inhibition involved in androgen-driven balding., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Alzheimer's disease (AD) is the most common neurodegenerative disorder and the most frequent cause of dementia in the aged population. According to the amyloid hypothesis, the amyloid-β (Aβ) peptide plays a key role in the pathogenesis of AD. Aβ is generated from the amyloidogenic processing of amyloid precursor protein and can aggregate to form oligomers, which have been described as a major synaptotoxic agent in neurons. Dysfunction of Wnt signaling has been linked to increased Aβ formation; however, several other studies have argued against this possibility. Herein, we use multiple experimental approaches to confirm that the inhibition of Wnt signaling promoted the amyloidogenic proteolytic processing of amyloid precursor protein. We also demonstrate that inhibiting Wnt signaling increases the production of the Aβ 42 peptide, the Aβ 42 /Aβ 40 ratio, and the levels of Aβ oligomers such as trimers and tetramers. Moreover, we show that activating Wnt signaling reduces the levels of Aβ 42 and its aggregates, increases Aβ 40 levels, and reduces the Aβ 42 /Aβ 40 ratio. Finally, we show that the protective effects observed in response to activation of the Wnt pathway rely on β-catenin-dependent transcription, which is demonstrated experimentally via the expression of various 'mutant forms of β-catenin'. Together, our findings indicate that loss of the Wnt signaling pathway may contribute to the pathogenesis of AD., (© 2016 International Society for Neurochemistry.)

Human milk contains growth factors that maintain intestinal mucosal homeostasis, but the molecular mechanisms behind how these growth factors regulate gene transcription are largely unknown. In this study, IEC-6 (rat intestinal epithelial cells) cells were used as a model to study cell differentiation mediated by transforming growth factor-β2 (TGF-β2), the most abundant growth factor in human milk. We focused on the transcription factor early growth response-1 (EGR-1), as we found a robust and rapid response in our initial transcription factor screen. Immunoblotting and immunofluorescent assays confirmed the phenotype change upon TGF-β2 treatment and EGR-1 stimulation in the nucleus, with maximum expression occurring at 1 h. Chromatin immunoprecipitation sequencing was performed to map genome-wide EGR-1 binding sites on more than 1800 genes, widely involved in processes such as gene expression, transcription, membrane invagination and metabolism. In particular, more than 15 Wnt signaling pathway genes have EGR-1 binding sites; among them, Axin1 was the limiting factor, ensuring proper β-catenin accumulation in the cytoplasm. We further used chromatin immunoprecipitation quantitative PCR to validate that EGR-1 binds to the region of -636/-454 bp and -454/-200 bp of the Axin1 promoter and functionally activates gene expression. The effect of TGF-β2 on maintaining small intestinal cell homeostasis was partially explained by Axin1 activation through EGR-1., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: In drug discovery research, cell-based phenotypic screening is an essential method for obtaining potential drug candidates. Revealing the mechanism of action is a key step on the path to drug discovery. However, elucidating the target molecules of hit compounds from phenotypic screening campaigns remains a difficult and troublesome process. Simple and efficient methods for identifying the target molecules are essential., Results: 2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) was identified as a senescence inducer from a phenotypic screening campaign. The compound is widely used as a Wnt agonist, although its target molecules remain to be clarified. To identify its target proteins, we compared a series of cellular assay results for the compound with our pathway profiling database. The database comprises the activities of compounds from simple assays of cellular reporter genes and cellular proliferations. In this database, compounds were classified on the basis of statistical analysis of their activities, which corresponded to a mechanism of action by the representative compounds. In addition, the mechanisms of action of the compounds of interest could be predicted using the database. Based on our database analysis, the compound was anticipated to be a tubulin disruptor, which was subsequently confirmed by its inhibitory activity of tubulin polymerization., Conclusion: These results demonstrate that tubulin is identified for the first time as a target molecule of the Wnt-activating small molecule and that this might have misled the conclusions of some previous studies. Moreover, the present study also emphasizes that our pathway profiling database is a simple and potent tool for revealing the mechanisms of action of hit compounds obtained from phenotypic screenings and off targets of chemical probes.

The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3β (GSK-3β) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3β interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3β redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3β at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3β in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3β. In vitro activity assays demonstrated that GSK-3β phosphorylation mediated by PKCζ enhanced GSK-3β activity. We mapped Ser147 of GSK-3β as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3β activity, resulting in β-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3β basal activity. Thus, we found that PKCζ phosphorylates GSK-3β at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3β activity in opposite manners in normal and malignant colon cells., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The recent development of 3D-liver stem cell cultures (hepatic organoids) opens up new avenues for gene and/or stem cell therapy to treat liver disease. To test safety and efficacy, a relevant large animal model is essential but not yet established. Because of its shared pathologies and disease pathways, the dog is considered the best model for human liver disease. Here we report the establishment of a long-term canine hepatic organoid culture allowing undifferentiated expansion of progenitor cells that can be differentiated toward functional hepatocytes. We show that cultures can be initiated from fresh and frozen liver tissues using Tru-Cut or fine-needle biopsies. The use of Wnt agonists proved important for canine organoid proliferation and inhibition of differentiation. Finally, we demonstrate that successful gene supplementation in hepatic organoids of COMMD1-deficient dogs restores function and can be an effective means to cure copper storage disease., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Promoting mesenchymal stem cell (MSC) proliferation has numerous applications in stem cell therapies, particularly in the area of regenerative medicine. In order for cell-based regenerative approaches to be realized, MSC proliferation must be achieved in a controlled manner without compromising stem cell differentiation capacities. Here we demonstrate that 6-bromoindirubin-3'-oxime (BIO) increases MSC β-catenin activity 106-fold and stem cell-associated gene expression ~33-fold, respectively, over untreated controls. Subsequently, BIO treatment increases MSC populations 1.8-fold in typical 2D culture conditions, as well as 1.3-fold when encapsulated within hydrogels compared to untreated cells. Furthermore, we demonstrate that BIO treatment does not reduce MSC multipotency where MSCs maintain their ability to differentiate into osteoblasts, chondrocytes and adipocytes using standard conditions. Taken together, our results demonstrate BIO's potential utility as a proliferative agent for cell transplantation and tissue regeneration., (Copyright © 2013 John Wiley & Sons, Ltd.)

Both the Wnt/β-catenin signaling pathway and small GTPases of the ADP-ribosylation factors (ARF) family play important roles in regulating cell development, homeostasis and fate. The previous report of QS11, a small molecule Wnt synergist that binds to ARF GTPase-activating protein 1 (ARFGAP1), suggests a role for ARFGAP1 in the Wnt/β-catenin pathway. However, direct inhibition of enzymatic activity of ARFGAP1 by QS11 has not been established. Whether ARFGAP1 is the only target that contributes to QS11's Wnt synergy is also not clear. Here we present structure-activity relationship (SAR) studies of QS11 analogs in two assays: direct inhibition of enzymatic activity of purified ARFGAP1 protein and cellular activation of the Wnt/β-catenin pathway. The results confirm the direct inhibition of ARFGAP1 by QS11, and also suggest the presence of other potential cellular targets of QS11., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Pathological pain is one of the most common neurological complications in patients with HIV-1/AIDS. However, the pathogenic process is unclear. Our recent studies show that Wnt5a is upregulated in the spinal cord dorsal horn (SDH) of the patients with HIV who develop pain and that HIV-1 gp120, a potential causal factor of the HIV-associated pain, rapidly upregulates Wnt5a in the mouse SDH. Using a mouse model, we show here that a specific Wnt5a antagonist, Box-5, attenuated gp120-induced mechanical allodynia. Conversely, a Wnt5a agonist, Foxy5, facilitated the allodynia. To elucidate the molecular mechanism by which Wnt5a regulates gp120-induced allodynia, we tested the role of the JNK/TNF-α pathway. We observed that the JNK-specific inhibitor SP600125 blocked either gp120- or Foxy5-induced allodynia. Similarly, the TNF-α-specific antagonist Enbrel also reversed either gp120- or Foxy5-induced allodynia. These data suggest that JNK and TNF-α mediate the biological effects of Wnt5a in regulating gp120-induced allodynia. To investigate the cellular mechanism, we performed extracellular single-unit recording from SDH neurons in anesthetized mice. Both Box-5 and SP600125 negated gp120-induced potentiation of SDH neuron spiking evoked by mechanical stimulation of the hind paw. Furthermore, while Foxy5 potentiated spike frequency of SDH neurons, either SP600125 or Enbrel blocked the potentiation. The data indicate that Wnt5a potentiates the activity of SDH neurons through the JNK-TNF-α pathway. Collectively, our findings suggest that Wnt5a regulates the pathogenesis of gp120-induced pain, likely by sensitizing pain-processing SDH neurons through JNK/TNF-α signaling.

It is desirable to obtain unlimited supplies of endothelial cells for research and therapeutics. However, current methods of deriving endothelial cells from humans suffer from issues, such as limited supplies, contamination from animal substances, and lengthy/complicated procedures. In this article we developed a way to differentiate human iPS and ES cells to highly pure endothelial cells in 5 days. The chemically defined system is robust, easy to perform, and free of animal substances. Using the system, we verified that combined TGFβ and canonical Wnt agonists are essential and sufficient for iPS/ES cell-to-mesoderm transition. Besides, VEGF-KDR signaling alone is required for endothelial formation at high density while supplementation with FGF allows for colonial endothelial differentiation. Finally, anti-adsorptive agents could enrich the endothelial output by allowing selective attachment of the endothelial precursors. The system was validated to work on multiple iPS/ES cells lines to produce endothelial cells capable of forming capillary-like structures in vitro and integrating into host vasculature in vivo. In sum, the simple yet robust differentiation system permits the unlimited supply of human endothelial cells. The defined and animal substance-free nature of the system is compatible with clinical applications and characterization of endothelial differentiation in an unbiased manner.

Wnt signaling plays an important role in regulating the activity of cancer stem cells (CSCs) in a variety of cancers. In this study, we explored the role of Wnt signaling in the lung cancer stem cells (LCSCs). LCSCs were obtained by sphere culture, for which human lung adenocarcinoma cell line SPC-A1 was treated with IGF, EGF and FGF-10. The stemness of LCSCs was confirmed by immunofluorescence, and pathway analysis was performed by functional genome screening and RT-PCR. The relationship between the identified signaling pathway and the expression of the stemness genes was explored by agonist/antagonist assay. Moreover, the effects of different signaling molecule inhibitors on sphere formation, cell viability and colony formation were also analyzed. The results showed that LCSCs were successfully generated as they expressed pluripotent stem cell markers Nanog and Oct 4, and lung distal epithelial markers CCSP and SP-C, by which the phenotype characterization of stem cells can be confirmed. The involvement of Wnt pathway in LCSCs was identified by functional genome screening and verified by RT-PCR. The expression of Wnt signaling components was closely related to the expression of the Nanog and Oct 4. Furthermore, targeting Wnt signaling pathway by using different signaling molecule inhibitors can exert anticancer effects. In conclusion, Wnt signaling pathway is involved in the stemness regulation of LCSCs and might be considered as a potential therapeutic target in lung adenocarcinoma.

Renal ischemia-reperfusion (IR) injury (IRI) after shock states or transplantation causes tissue damage and delayed graft function, respectively. The Wnt/β-catenin signaling pathway plays a critical role in nephrogenesis. We therefore hypothesized that pharmacological activation of the Wnt/β-catenin signaling by the Wnt agonist, a synthetic pyrimidine, could protect kidneys from IRI. Adult male rats were subjected to bilateral clamping of the renal pedicles with microvascular clips for 60 min, followed by reperfusion. The Wnt agonist (5 mg/kg body weight) or vehicle (20% dimethyl sulfoxide in saline) was administered intravenously 1 h before ischemia. Blood and renal tissues were collected 24 h after IR for evaluation. Renal IR caused a significant reduction of β-catenin and its downstream target gene cyclin D1 by 65% and 39%, respectively, compared with the sham, whereas the Wnt agonist restored them to sham levels. The number and intensity of cells staining with the proliferation marker Ki67 in ischematized kidneys were enhanced by the Wnt agonist. The integrity of the renal histological architecture in the Wnt agonist group was better preserved than the vehicle group. The Wnt agonist significantly lowered serum levels of creatinine, aspartate aminotransferase, and lactate dehydrogenase and inhibited the production of interleukin 6 and interleukin 1β and myeloperoxidase activities. Lastly, the Wnt agonist reduced inducible nitric oxide synthase, nitrotyrosine proteins, and 4-hydroxynonenal in the kidneys by 60%, 47%, and 21%, respectively, compared with the vehicle. These results indicate that the Wnt agonist improves renal regeneration and function while attenuating inflammation and oxidative stress in the kidneys after IR. Thus, pharmacologic stimulation of the Wnt/β-catenin signaling provides a beneficial effect on the prevention of renal IRI.

With more than 1.4 billion overweight or obese adults worldwide, obesity and progression of the metabolic syndrome are major health and economic challenges. To address mechanisms of obesity, adipose tissue-derived mesenchymal stem cells (ADSCs) are being studied to detail the molecular mechanisms involved in adipogenic differentiation. Activation of the Wnt signalling pathway has inhibited adipogenesis from precursor cells. In our study, we examined this anti-adipogenic effect in further detail stimulating Wnt with lithium chloride (LiCl) and 6-bromo indirubin 3'oxime (BIO). We also examined the effect of Wnt inhibition using secreted frizzled-related protein 4 (sFRP4), which we have previously shown to be pro-apoptotic, anti-angiogenic, and anti-tumorigenic. Wnt stimulation in LiCl and BIO-treated ADSCs resulted in a significant reduction (2.7-fold and 12-fold respectively) in lipid accumulation as measured by Oil red O staining while Wnt inhibition with sFRP4 induced a 1.5-fold increase in lipid accumulation. Furthermore, there was significant 1.2-fold increase in peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα), and 1.3-fold increase in acetyl CoA carboxylase protein levels. In contrast, the expression of adipogenic proteins (PPARγ, C/EBPα, and acetyl CoA carboxylase) were decreased significantly with LiCl (by 1.6, 2.6, and 1.9-fold respectively) and BIO (by 7, 17, and 5.6-fold respectively) treatments. These investigations demonstrate interplay between Wnt antagonism and Wnt activation during adipogenesis and indicate pathways for therapeutic intervention to control this process.

Ketoacids (KA) are known to improve muscle mass among patients with chronic kidney disease (CKD) on a low-protein diet (CKD-LPD), but the mechanism of its preventive effects on muscle atrophy still remains unclear. Since muscle atrophy in CKD may be attributable to the down-regulation of the Wnt7a/Akt/p70S6K pathway and the activation of the ubiquitin-proteasome system (UPS) and the apoptotic signalling pathway, a hypothesis can readily be drawn that KA supplementation improves muscle mass by up-regulating the Wnt7a/Akt/p70S6K pathway and counteracting the activation of the UPS and caspase-3-dependent apoptosis in the muscle of CKD-LPD rats. Rats with 5/6 nephrectomy were randomly divided into three groups, and fed with either 22 % protein (normal-protein diet; NPD), 6 % protein (LPD) or 5 % protein plus 1 % KA for 24 weeks. Sham-operated rats with NPD intake were used as the control. The results demonstrated that KA supplementation improved protein synthesis and increased related mediators such as Wnt7a, phosphorylated Akt and p70S6K in the muscle of CKD-LPD rats. It also inhibited protein degradation, withheld the increase in ubiquitin and its ligases MAFbx (muscle atrophy F-box) and MuRF1 (muscle ring finger-1) as well as attenuated proteasome activity in the muscle of CKD-LPD rats. Moreover, KA supplementation gave rise to a reduction in DNA fragment, cleaved caspase-3 and 14 kDa actin fragment via the down-regulation of the Bax:Bcl-2 ratio in the muscle of CKD-LPD rats. The beneficial effects unveiled herein further consolidate that KA may be a better therapeutic strategy for muscle atrophy in CKD-LPD.

Canonical Wnt/β-catenin signaling has been implicated in multiple developmental events including the regulation of proliferation, cell fate, and differentiation. In the inner ear, Wnt/β-catenin signaling is required from the earliest stages of otic placode specification through the formation of the mature cochlea. Within the avian inner ear, the basilar papilla (BP), many Wnt pathway components are expressed throughout development. Here, using reporter constructs for Wnt/β-catenin signaling, we show that this pathway is active throughout the BP (E6-E14) in both hair cells (HCs) and supporting cells. To characterize the role of Wnt/β-catenin activity in developing HCs, we performed gain- and loss-of-function experiments in vitro and in vivo in the chick BP and zebrafish lateral line systems, respectively. Pharmacological inhibition of Wnt signaling in the BP and lateral line neuromasts during the periods of proliferation and HC differentiation resulted in reduced proliferation and decreased HC formation. Conversely, pharmacological activation of this pathway significantly increased the number of HCs in the lateral line and BP. Results demonstrated that this increase was the result of up-regulated cell proliferation within the Sox2-positive cells of the prosensory domains. Furthermore, Wnt/β-catenin activation resulted in enhanced HC regeneration in the zebrafish lateral line following aminoglycoside-induced HC loss. Combined, our data suggest that Wnt/β-catenin signaling specifies the number of cells within the prosensory domain and subsequently the number of HCs. This ability to induce proliferation suggests that the modulation of Wnt/β-catenin signaling could play an important role in therapeutic HC regeneration., (Copyright © 2013 Wiley Periodicals, Inc.)

Hematopoietic stem cells (HSCs) are a rare population of somatic stem cells that have the ability to regenerate the entire mature blood system in a hierarchical way for the duration of an adult life. Adult HSCs reside in the bone marrow niche. Different niche cell types and molecules regulate the balance of HSC dormancy and activation as well as HSC behavior in both normal and malignant hematopoiesis. Here, we describe the interplay of HSCs and their niche, in particular the involvement of the Wnt signaling pathway. Although the prevailing notion has been that malignant transformation of HSCs is the main cause of leukemia, evidence is mounting that disruption of niche regulation by transformed hematopoietic cells, which may overexpress Wnt signaling or intrinsic stromal defects in gene expression, is at least a collaborative factor in leukemogenesis. Thus, insights into the normal and altered functions of niche components will help to obtain a better understanding of normal and malignant hematopoiesis and how environmental factors affect these processes., (© 2014 New York Academy of Sciences.)

Cellular microenvironmental conditions coordinate to regulate stem cell populations and their differentiation. Mesenchymal precursor cells (MPCs), which have significant potential for a wide range of therapeutic applications, can be expanded or differentiated into osteo- chondro- and adipogenic lineages. The ability to establish, screen, and control aspects of the microenvironment is paramount if we are to elucidate the complex interplay of signaling events that direct cell fate. Whilst modulation of Wnt signaling may be useful to direct osteogenesis in MPCs, there is still significant controversy over how the Wnt signaling pathway influences osteogenesis. In this study, we utilised a full-factorial microbioreactor array (MBA) to rapidly, combinatorially screen several Wnt modulatory compounds (CHIR99021, IWP-4 and IWR-1) and characterise their effects upon osteogenesis. The MBA screening system showed excellent consistency between donors and experimental runs. CHIR99021 (a Wnt agonist) had a profoundly inhibitory effect upon osteogenesis, contrary to expectations, whilst the effects of the IWP-4 and IWR-1 (Wnt antagonists) were confirmed to be inhibitory to osteogenesis, but to a lesser extent than observed for CHIR99021. Importantly, we demonstrated that these results were translatable to standard culture conditions. Using RT-qPCR of osteogenic and Wnt pathway markers, we showed that CHIR exerted its effects via inhibition of ALP and SPP1 expression, even though other osteogenic markers (RUNX2, MSX2, DLX, COL1A1) were upregulated. Lastly, this MBA platform, due to the continuous provision of medium from the first to the last of ten serially connected culture chambers, permitted new insight into the impacts of paracrine signaling on osteogenic differentiation in MPCs, with factors secreted by the MPCs in upstream chambers enhancing the differentiation of cells in downstream chambers. Insights provided by this cell-based assay system will be key to better understanding signaling mechanisms, as well as optimizing MPC growth and differentiation conditions for therapeutic applications.

Aims: Cigarette smoking is one of the high risk factors of adult chronic periodontitis and nicotine is the well established toxic substance in cigarette. However, the mechanism of nicotine induced periodontitis is still unknown. Here we studied whether nicotine impaired the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) through activating α7 nicotinic acetylcholine receptor (α7 nAChR)., Methods: hPDLSCs with multi differentiation potential and surface makers for mesenchymal stem cells were harvested by limiting dilution technique. The level of mineralized nodule formation was assessed by alizarin red S staining. Expression level of ostegenic related genes and proteins were detected by real-time PCR and western blot analysis. The expression of α7 nAChR and its downstream signaling pathway were examined by western blot. The role of the receptor and related signaling pathway in nicotine impairing the osteogenic potential of hPDLSCs were also studied in different levels., Results: Nicotine deteriorated the ostegenic differentiation of hPDLSCs in a dose dependent manner. Activation of α7 nAChR by nicotine treatment activated wnt/β-catenin signaling pathway, leading to osteogenic deficiency of hPDLSCs. Blockage of α7 nAChR and wnt pathway inhibitor treatment rescued nicotine induced osteogenic differentiation deficiency., Conclusions: These data suggested that nicotine activated α7 nAChR expressed on PDLSCs and further activated wnt signaling downstream, thus deteriorating the osteogenic potential of PDLSCs. The impairment of osteogenic differentiation of PDLSCs by nicotine might lead to cigarette smoking related periodontitis.

Signaling by the Wnt family of secreted glycoproteins plays important roles in embryonic development and adult homeostasis. Wnt signaling is modulated by a number of evolutionarily conserved inhibitors and activators. Wnt inhibitors belong to small protein families, including sFRP, Dkk, WIF, Wise/SOST, Cerberus, IGFBP, Shisa, Waif1, APCDD1, and Tiki1. Their common feature is to antagonize Wnt signaling by preventing ligand-receptor interactions or Wnt receptor maturation. Conversely, the Wnt activators, R-spondin and Norrin, promote Wnt signaling by binding to Wnt receptors or releasing a Wnt-inhibitory step. With few exceptions, these antagonists and agonists are not pure Wnt modulators, but also affect additional signaling pathways, such as TGF-β and FGF signaling. Here we discuss their interactions with Wnt ligands and Wnt receptors, their role in developmental processes, as well as their implication in disease.

Objectives were to evaluate the role of canonical WNT signaling in development of the preimplantation embryo. Signaling was activated with 2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP) and inhibited with Dickkopf-related protein 1 (DKK1). Treatment of bovine embryos with AMBMP at day 5 after insemination decreased development to the blastocyst stage at day 7 and reduced numbers of trophectoderm and inner cell mass cells. At high concentrations, AMBMP caused disorganization of the inner cell mass. DKK1 blocked actions of AMBMP but did not affect development in the absence of AMBMP. Examination of gene expression in day 6 morulae by microarray revealed expression of 16 WNT genes and other genes involved in WNT signaling; differences in relative expression were confirmed by PCR for 7 genes. In conclusion, the preimplantation embryo possesses a functional WNT signaling system and activation of the canonical pathway can inhibit embryonic development.

The Wnt/β-catenin signaling pathway is well characterized in stem cell biology and plays a critical role in liver development, regeneration, and homeostasis. We hypothesized that pharmacologic activation of Wnt signaling protects against hepatic ischemia/reperfusion (I/R) injury through its known proliferative and antiapoptotic properties. Sprague-Dawley rats underwent 70% hepatic ischemia by microvascular clamping of the hilum of the left and median lobes of the liver for 90 min, followed by reperfusion. Wnt agonist (2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine, 5 mg/kg body weight) or vehicle (20% dimethyl sulfoxide in saline) in 0.5 mL was injected i.p. 1 h before ischemia or infused i.v. over 30 min right after ischemia. Blood and tissue samples from the pretreated groups were collected 24 h after reperfusion, and a survival study was performed. Hepatic expression of β-catenin and its downstream target gene Axin2 were decreased after I/R, whereas Wnt agonist restored their expression to sham levels. Wnt agonist blunted I/R-induced elevations of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase and significantly improved the microarchitecture of the liver. The cell proliferation determined by Ki67 immunostaining significantly increased with Wnt agonist treatment, and inflammatory cascades were dampened in Wnt agonist-treated animals, as demonstrated by attenuations in interleukin 6, myeloperoxidase, inducible nitric oxide synthase, and nitrotyrosine. Wnt agonist also significantly decreased the amount of apoptosis, as evidenced by decreases in both TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining as well as caspase 3 activity levels. Finally, the 10-day survival rate was increased from 27% in the vehicle group to 73% in the pretreated Wnt agonist group and 55% in the Wnt agonist postischemia treatment group. Thus, we propose that direct Wnt/β-catenin stimulation may represent a novel therapeutic approach in the treatment of hepatic I/R.

In a recent issue of Nature, Hao et al. report an unexpected link between the secreted stem cell factor/Wnt agonist R-spondin and Wnt receptors through the transmembrane ZNRF3 protein, a RING finger ubiquitin ligase. ZNRF3 acts to turn over Frizzled and LRP6 receptors. R-spondin binds to ZNRF3, in addition to transmembrane LGR4/5 receptors, to antagonize degradation of the Wnt receptors by ZNRF3, thereby resulting in increased Frizzled and LRP6 levels and a greater Wnt response.

Embryonic stem cells (ESCs) - undifferentiated cells originating from preimplantation stage embryos - have prolonged self-renewal capacity and are pluripotent. Activation of the canonical Wnt pathway is implicated in maintenance of and exit from the pluripotent state. Recent findings demonstrate that the essential mediator of canonical Wnt signaling, β-catenin, is dispensable for ESC maintenance; however, its activation inhibits differentiation through derepression of T cell factor 3 (Tcf3)-bound genes. Wnt agonists are useful in deriving ESCs from recalcitrant mouse strains and the rat and in nuclear reprogramming of somatic stem cells. We discuss recent advances in our understanding of the role of canonical Wnt signaling in the regulation of ESC self-renewal and how its manipulation can improve pluripotent ESC derivation and maintenance., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Dorsal-ventral specification in the amphibian embryo is controlled by β-catenin, whose activation in all dorsal cells is dependent on maternal Wnt11. However, it remains unknown whether other maternally secreted factors contribute to β-catenin activation in the dorsal ectoderm. Here, we show that maternal Xenopus Norrin (xNorrin) promotes anterior neural tissue formation in ventralized embryos. Conversely, when xNorrin function is inhibited, early canonical Wnt signaling in the dorsal ectoderm and the early expression of the zygotic neural inducers Chordin, Noggin, and Xnr3 are severely suppressed, causing the loss of anterior structures. In addition, xNorrin potently inhibits BMP- and Nodal/Activin-related functions through direct binding to the ligands. Moreover, a subset of Norrin mutants identified in humans with Norrie disease retain Wnt activation but show defective inhibition of Nodal/Activin-related signaling in mesoderm induction, suggesting that this disinhibition causes Norrie disease. Thus, xNorrin is an unusual molecule that acts on two major signaling pathways, Wnt and TGF-β, in opposite ways and is essential for early neuroectoderm specification., Competing Interests: The authors have declared that no competing interests exist.

Aims/hypothesis: The wingless-type MMTV integration site (WNT) pathway mediates multiple physiological and pathological processes, such as inflammation, angiogenesis and fibrosis. The aim of this study was to investigate whether canonical WNT signalling plays a role in the pathogenesis of diabetic nephropathy., Methods: Expression of WNT ligands and frizzled receptors in the canonical WNT pathway in the kidney was compared at the mRNA level using real-time RT-PCR between Akita mice, streptozotocin-induced diabetic rats and db/db mice and their respective non-diabetic controls. Renal function was evaluated by measuring the urine albumin excretion. Human renal proximal tubular epithelial cells were treated with high-glucose medium and 4-hydroxynonenal (HNE). Levels of β-catenin, connective tissue growth factor and fibronectin were determined by western blot analysis., Results: Some of the WNT ligands and frizzled receptors showed increased mRNA levels in the kidneys of Akita mice, streptozotocin-induced diabetic rats and db/db mice compared with their non-diabetic controls. Renal levels of β-catenin and WNT proteins were upregulated in these diabetic models. Lowering the blood glucose levels by insulin attenuated the activation of WNT signalling in the kidneys of Akita mice. In cultured human renal proximal tubular epithelial cells, both high glucose and HNE activated WNT signalling. Inhibition of WNT signalling with a monoclonal antibody blocking LDL-receptor-related protein 6 ameliorated renal inflammation and fibrosis and reduced proteinuria in Akita mice., Conclusions/interpretation: The WNT pathway is activated in the kidneys of models of both type 1 and 2 diabetes. Dysregulation of the WNT pathway in diabetes represents a new pathogenic mechanism of diabetic nephropathy and renders a new therapeutic target.

The recent development of high-throughput sequencing technologies and the availability of whole genome sequences of a variety of living organisms, including that of humans, have led to an enormous push in the quest for a comprehensive inquiry for the function of each and every gene discovered in different model organisms. A major conclusion from the sequencing projects was that while forward genetics had been extremely successful in identifying key genes/components of many biological processes, such as signal transduction cascades, the function(s) of the majority of genes in the genome remains a mystery. In this article, we discuss the use of a variety of high-throughput postgenomic tools, including functional genomics, proteomics, and chemical genetics that are being implemented in an exhaustive molecular dissection of a key evolutionarily conserved signal transduction pathway, namely the Wnt/wingless (wg) pathway and its associated signaling network., (Copyright © 2011 John Wiley & Sons, Inc.)

In addition to its role in cellular development and proliferation, there are emerging in vitro data implicating the Wnt/β-catenin pathway in synaptic plasticity. Yet in vivo studies have not examined whether Wnt activity is required for learning and memory. In the amygdala during fear memory formation, we found that many Wnt-signaling genes were dynamically regulated, with an immediate decrease, followed by an eventual normalization during memory consolidation. This rapid decrease in Wnt mRNA was confirmed with individual quantitative PCR and in situ hybridization. We then manipulated Wnt signaling with a specific peptide antagonist (Dkk-1) or agonist (Wnt1) injected stereotaxically into the adult amygdala during fear learning. We found that neither manipulation had an effect on locomotion, anxiety, fear acquisition, or fear expression. However, both Wnt modulators prevented long-term fear memory consolidation without affecting short-term memory. Dkk-1 and Wnt infusions had destabilizing, but opposite, effects on the requisite β-catenin/cadherin dynamic interactions that occur during consolidation. These data suggest that dynamic modulation of Wnt/β-catenin signaling during consolidation is critical for the structural basis of long-term memory formation.

WNT signaling activity is involved in the regulation of many cellular functions, including proliferation, migration, cell fate specification, maintenance of pluripotency and induction of tumorigenicity. Here we summarize recent progress towards understanding the regulation of canonical WNT/β-catenin signaling activity through feedback regulatory loops involving the ligands, agonists and antagonists, the availability of intracellular pools of active β-catenin and the cross-regulation of the WNT activity by β-catenin independent pathway. We also review recent findings on the role of WNT/β-catenin signaling in tissue lineage differentiation during embryogenesis and the maintenance and self renewal of embryo-derived stem cells in vitro., (© 2011 The Authors. Development, Growth & Differentiation © 2011 Japanese Society of Developmental Biologists.)

Axin proteins are key negative regulators of the canonical Wnt signal transduction pathway. Although Axin2 null mice are viable, we identified an unusual ENU-induced recessive allele of Axin2, canp, that causes midgestation lethality in homozygotes. We show that the Axin2(canp) mutation is a V26D substitution in an invariant N-terminal sequence motif and that the Axin2(canp) protein is more stable than wild type. As predicted for an increased level of a negative regulator, the Axin2(canp) mutation leads to decreased Wnt signaling in most tissues, and this can account for most of the morphological phenotypes of Axin2(canp) mutants. In contrast, there is a paradoxical increase in canonical Wnt activity in the late primitive streak of all Axin2(canp) mutant embryos that is associated with the formation of an ectopic tail in some mutants. Treatment of wild-type embryos with an inhibitor of Tankyrase that stabilizes Axin proteins also causes inhibition of Wnt signaling in anterior regions of the embryo and a gain of Wnt signaling in the primitive streak. The results indicate that although increased stability of Axin2 leads to a loss of canonical Wnt signaling in most tissues, stabilized Axin2 enhances Wnt pathway activity in a specific progenitor population in the late primitive streak.

E26 transforming sequence-related gene (ERG) is a transcription factor involved in normal hematopoiesis and is dysregulated in leukemia. ERG mRNA overexpression was associated with poor prognosis in a subset of patients with T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Herein, a genome-wide screen of ERG target genes was conducted by chromatin immunoprecipitation-on-chip (ChIP-chip) in Jurkat cells. In this screen, 342 significant annotated genes were derived from this global approach. Notably, ERG-enriched targets included WNT signaling genes: WNT11, WNT2, WNT9A, CCND1 and FZD7. Furthermore, chromatin immunoprecipitation (ChIP) of normal and primary leukemia bone marrow material also confirmed WNT11 as a target of ERG in six of seven patient samples. A larger sampling of patient diagnostic material revealed that ERG and WNT11 mRNA were co-expressed in 80% of AML (n=30) and 40% in T-ALL (n=30) bone marrow samples. Small interfering RNA (siRNA)-mediated knockdown of ERG confirmed downregulation of WNT11 transcripts. Conversely, in a tet-on ERG-inducible assay, WNT11 transcripts were co-stimulated. A WNT pathway agonist, 6-bromoindirubin-3-oxime (BIO), was used to determine the effect of cell growth on the ERG-inducible cells. The addition of BIO resulted in an ERG-dependent proliferative growth advantage over ERG-uninduced cells. Finally, ERG induction prompted morphological transformation whereby round unpolarized K562 cells developed elongated protrusions and became polarized. This morphological transformation could effectively be inhibited with BIO and with siRNA knockdown of WNT11. In conclusion, ERG transcriptional networks in leukemia converge on WNT signaling targets. Specifically, WNT11 emerged as a direct target of ERG. Potent ERG induction promoted morphological transformation through WNT11 signals. The findings in this study unravel new ERG-directed molecular signals that may contribute to the resistance of current therapies in acute leukemia patients with poor prognosis characterized by high ERG mRNA expression.

Glycogen synthase kinase 3β (GSK3β) is an essential integrating molecule for multiple proliferation and differentiation signals that regulate cell fate. Here, we have examined the effects of inhibiting GSK3β on the development of oligodendrocytes (OLs) from their oligodendrocyte precursors (OP) in vivo by injection into the lateral ventricle of postnatal mice and ex vivo in organotypic cultures of isolated intact rodent optic nerve. Our results show that a range of GSK3β inhibitors (ARA-014418, lithium, indirubin, and L803-mt) increase OPs and OLs and promote myelination. Inhibition of GSK3β stimulates OP proliferation and is prosurvival and antiapoptotic. The effects of GSK3β inhibition in OPs is via the canonical Wnt signaling pathway by stimulating nuclear translocation of β-catenin. However, direct comparison of the effects of Wnt3a and GSK3β inhibition in optic nerves shows that they have opposing actions on OLs, whereby GSK3β inhibition strikingly increases OL differentiation, whereas Wnt3a inhibits OL differentiation. Notably, GSK3β inhibition overrides the negative effects of Wnt3a on OLs, indicating novel GSK3β signaling mechanisms that negatively regulate OL differentiation. We identify that two mechanisms of GSK3β inhibition are to stimulate cAMP response element binding (CREB) and decrease Notch1 signaling, which positively and negatively regulate OL differentiation and myelination, respectively. A key finding is that GSK3β inhibition has equivalent effects in the adult and stimulates the regeneration of OLs and remyelination following chemically induced demyelination. This study identifies GSK3β as a profound negative regulator of OL differentiation that contributes to inefficient regeneration of OLs and myelin repair in demyelination., (Copyright © 2011 Wiley-Liss, Inc.)

Graft-versus-host disease (GVHD) is a major complication of allogeneic bone marrow transplantation (BMT), and damage to the gastrointestinal (GI) tract plays a critical role in amplifying systemic disease. Intestinal stem cells (ISCs) play a pivotal role not only in physiological tissue renewal but also in regeneration of the intestinal epithelium after injury. In this study, we have discovered that pretransplant conditioning regimen damaged ISCs; however, the ISCs rapidly recovered and restored the normal architecture of the intestine. ISCs are targets of GVHD, and this process of ISC recovery was markedly inhibited with the development of GVHD. Injection of Wnt agonist R-spondin1 (R-Spo1) protected against ISC damage, enhanced restoration of injured intestinal epithelium, and inhibited subsequent inflammatory cytokine cascades. R-Spo1 ameliorated systemic GVHD after allogeneic BMT by a mechanism dependent on repair of conditioning-induced GI tract injury. Our results demonstrate for the first time that ISC damage plays a central role in amplifying systemic GVHD; therefore, we propose ISC protection by R-Spo1 as a novel strategy to improve the outcome of allogeneic BMT.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration of cognitive abilities, accumulation of the amyloid-beta-peptide (Abeta) and synaptic alterations. Treatment with lithium has been shown to provide neuroprotection against several insults, including protection against Abeta neurotoxicity in vitro. Rosiglitazone, a peroxisome proliferator activated receptor-gamma agonist, has been shown to attenuate Abeta-peptide neurotoxic effects, including the inflammatory response of microglia and astrocytes. Both types of drugs activate Wnt signaling, a pathway that has been shown to be related to AD. In this study, a double transgenic mouse model, which coexpresses APPswe and the exon 9 deletion of the presenilin 1 (PSEN1) gene, was used to examine, in vivo, the effect of lithium and rosiglitazone on Abeta neurotoxicity. Mice were tested for spatial memory, and their brain samples were used for histochemical and biochemical analysis. In this study, we report that both drugs significantly reduced (1) spatial memory impairment induced by amyloid burden; (2) Abeta aggregates and Abeta oligomers; and (3) astrocytic and microglia activation. They also prevented changes in presynaptic and postsynaptic marker proteins. Finally, both drugs activate Wnt signaling shown by the increase in beta-catenin and by the inhibition of the glycogen synthase kinase-3beta. We conclude that lithium and rosiglitazone, possibly by the activation of the Wnt signaling pathway, reduce various AD neuropathological markers and may be considered as potential therapeutic agents against the disease.

A series of (hetero)arylpyrimidines agonists of the Wnt-beta-catenin cellular messaging system have been prepared. These compounds show activity in U2OS cells transfected with Wnt-3a, TCF-luciferase, Dkk-1 and tk-Renilla. Selected compounds show minimal GSK-3beta inhibition indicating that the Wnt-beta-catenin agonism activity most likely comes from interaction at Wnt-3a/Dkk-1. Two examples 1 and 25 show in vivo osteogenic activity in a mouse calvaria model. One example 1 is shown to activate non-phosphorylated beta-catenin formation in bone., (Copyright 2009 Elsevier Ltd. All rights reserved.)

A high-throughput screening campaign to discover small molecule leads for the treatment of bone disorders concluded with the discovery of a compound with a 2-aminopyrimidine template that targeted the Wnt beta-catenin cellular messaging system. Hit-to-lead in vitro optimization for target activity and molecular properties led to the discovery of (1-(4-(naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine (5, WAY-262611). Compound 5 has excellent pharmacokinetic properties and showed a dose dependent increase in the trabecular bone formation rate in ovariectomized rats following oral administration.