Role Of Wnt Signaling Pathway Research Articles

Wnt Pathway in Osteosarcoma, from Oncogenic to Therapeutic

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Although pathologic characteristics of this disease are clear and well established, much remains to be understood about this tumor, particularly at the molecular signaling level. Secreted signaling molecules of the Wnt family have been widely investigated and found to play a central role in controlling embryonic bone development, bone mass, and postnatal bone regeneration. A variety of studies also suggest that Wnt signaling pathway is closely associated with bone malignancies, including breast or prostate cancer induced bone metastasis, multiple myeloma, as well as osteosarcoma. Here, we provide an overview of the role of Wnt signaling pathway in osteosarcoma development and progression, highlighting the aberrant activation of Wnt pathway in this bone malignancy. We also discuss the potential therapeutic applications for the treatment of osteosarcoma targeting Wnt pathway.

Essential and Unexpected Role of Wnt Signalling Pathway in High Glucose-Induced Mesenchymal Progenitor Cell Differentiation

Essential and Unexpected Role of Wnt Signalling Pathway in High Glucose-Induced Mesenchymal Progenitor Cell Differentiation

Identification and Characterization of Wnt Signaling Pathway in Keloid Pathogenesis

Keloid is characterized by fibroblastic cell proliferation and abundant collagen synthesis. Numerous studies have shown that the Wingless type (Wnt) signaling pathways play key roles in various cellular functions including proliferation, differentiation, survival, apoptosis and migration. The aim of this study was to clarify the role of Wnt signaling pathway in keloid pathogenesis. Primary fibroblast cultures and tissue samples from keloid and normal appearing dermis were used. The expression of Wnt family members, frizzled (FZD)4 receptor, receptor tyrosine kinase-like orphan receptor (ROR)2 and the Wnt signaling downstream targets, glycogen synthase kinase (GSK)3-β and β-catenin were assessed using semi-quantitative RT-PCR, Western blot, or immunohistochemical methods. Of the Wnt family members, Wnt5a mRNA and protein levels were elevated in keloid fibroblasts (KF) as compared to normal fibroblasts (NF). A higher expression of β-catenin protein was also found in KF. No detectable levels of FZD4 receptor and ROR2 proteins were observed in both NF and KF. Functional analysis showed that treatment of NF and KF with recombinant Wnt5a peptide resulted in an increase in protein levels of total β-catenin and phosphorylated β-catenin at Ser33/37/Thr 41 but no significant change in phosphorylated β-catenin at Ser45/Thr 41 positions. In addition, the expression of total GSK3-β protein was not affected but its phosphorylated/inactivated form was increased in NF and KF. Our findings highlight a potential role for a Wnt/β-catenin canonical signaling pathway triggered by Wnt5a in keloid pathogenesis thereby providing a new molecular target for therapeutic modulations.

Involvement of a Wnt/β-catenin Canonical Signaling Pathway in Keloid Pathogenesis

Numerous studies have shown that the Wingless type (Wnt) signaling pathways play key roles in various cellular functions including proliferation, apoptosis and migration. The aim of this study was to clarify the role of Wnt signaling pathway in keloid pathogenesis. Primary fibroblast cultures and tissue samples from keloid (KF) and normal appearing dermis (NF) were used. The expression of Wnt family members, frizzled (FZD)4 receptor, ROR2 receptor and the Wnt signaling downstream targets, GSK3-β and β-catenin were assessed using semi-quantitative RT-PCR, Western blot, or immunohistochemical methods.

Hypermethylation of adenomatous polyposis coli gene promoter is associated with novel Wnt signaling pathway in gastric adenomas

Gastric adenomas (GAs) are considered as premalignant lesions of gastric adenocarcinoma. The role of Wnt signaling pathway in GAs is rarely identified. In the present study, we aimed to determine whether Wnt signaling plays a role in the pathogenesis of GAs, and to clarify the mechanism of Wnt signaling in GAs. The study investigated the relationship between clinicopathological characteristics, Helicobacter pylori (Hp) infection, adenomatous polyposis coli (APC) promoter methylation, APC and β-catenin immunohistochemistry expression and mutation status, compared with 38 gastric adenoma and periadenomatous tissues (PTs). The abnormal expression of β-catenin in PTs, low-grade adenomas (LGAs) and high-grade adenomas (HGAs) was 0%, 9.09% and 81.25%. For APC, immunoreactive score (IRS) was 5.50 ± 0.5 in PTs, 3.59 ± 1.4 in LGAs and 1.8 ± 2.0 in HGAs. The scores in LGAs and HGAs were significantly lower than those in PTs (P = 0.000). IRS reflected significantly reduced expression of APC in HGAs (P = 0.002). The absent expression of APC had a correlation with the expression of β-catenin (P = 0.000). Four LGAs (18.18%) and nine HGAs (56.25%) had methylation of APC. APC promoter methylation correlated with the grade (P = 0.014) and the expression of β-catenin and APC (P = 0.000). Genes mutation was detected in only two adenomas (5.3%). The presence of Hp in HGAs (43.8%) was significantly higher than in LGAs (13.6%) (P = 0.038). But there was no statistical correlation to growth pattern, size, APC hypermethylation and gene mutation. Hypermethylation of APC promoter, instead of mutations involving APC and β-catenin, may play a role in the development and progression of GAs contributing to moderate activation of Wnt signaling. Helicobacter pylori may accelerate the progress of gastric adenoma, but the pathogenesis needs further research.

Wnt signaling pathway and osteosarcoma

The Wnt pathway plays a crucial role in skeletal development and is indispensable for determination of OS cell lines. In recent years, experimental evidences on Wnt signaling pathway in OS cell lines and OS animal models, and that of Wnt signaling pathway as a diagnosis and prognosis marker of OS suggested that Wnt signaling pathway plays an important role in the progression, invasion and metastasis of OS. In addition, the strategy and safety evaluation to target Wnt to treat OS are underway. Exploring the significant role of Wnt signaling pathway in OS may aid in personalizing therapeutics to increase patient survival. Key words: Osteosarcoma; Wnt signaling pathway

The role of Wnt signaling pathway in osteogenic differentiation of BMSCs stimulated by simvastatin

The role of Wnt signaling pathway in osteogenic differentiation of BMSCs stimulated by simvastatin

Abstract B86: JS-K, a nitric oxide-donating prodrug, modulates β-catenin/TCF signaling in leukemic Jurkat cells through S-nitrosylation

Abstract Introduction: Leukemia is the most common form of cancer in children and acute lymphoblastic leukemia (ALL) is the primary cause of cancer-related mortality. The deregulation of theWnt signaling cascade and its components has been implicated in T-cell ALL as well as in B-cell chronic lymphocytic leukemia (CLL). The protein β-catenin has a central role in Wnt signaling pathway and may promote leukemia cell proliferation and differentiation. β-catenin is expressed in T- ALL cells, tumor lines of hematopoietic origin and primary leukemia cells but is undetectable in normal peripheral blood T cells. Among the leukemic cell lines, β-catenin is expressed in high levels in Jurkat T-cells. JS-K [O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1yl]diazen-1-ium-1,2-diolate] is a prodrug of the diazeniumdiolate class that releases NO through a reaction catalyzed by GST utilizing glutathione. This agent has been shown to inhibit the growth of various cancer cell lines. This report highlights the effects of JSK on the growth properties of human Jurkat T-acute lymphoblastic leukemia cells, the β-catenin signaling pathway, and protein S-nitrosylation. Methods: JS-K: synthesized by us. Cell growth: MTT; cell cycle phase distribution: flow cytometry; apoptosis: subdiploid (sub-G0/G1) peak in DNA content histograms. Transient transfection: luciferase reporter constructs TOPflash or FOPflash. Luciferase activity: kit per manufacturer's instructions. Protein S-nitrosylation: Biotin switch assay (Jaffrey et al, Nature Cell Biology, 2001, 3, 193–197); β-catenin and biotinylated protein levels: immunoblotting. Results: JS-K inhibited the growth of Jurkat cells, IC50s of 12 ± 3 µM and 7 ± 1 µM at 24 h and 48 hr, respectively. This effect was due, in part, to a dose-dependent induction of apoptosis and alterations in the distribution of the cells in the cell cycle, arrest was in G0/G1. JS-K dose-dependently inhibited β-catenin/TCF-4 signaling by reporter assays, and reduced β-catenin levels in the nucleus and cytoplasm. JS-K treatment for 24 hr at 0.5 × IC50, 1 × IC50 and 2 × IC50 caused an increase in total S-nitrosylated proteins and also caused a reduction in S-nitrosylated β-catenin expression. These effects were reversed by carboxy-PTIO (an NO scavenger). Conclusions: These findings establish a strong inhibitory effect of JS-K in human Jurkat T-acute lymphoblastic leukemia cells, an effect that is through inhibition of Wnt/β-catenin/TCF-4 signaling and through induction of apoptosis modulated by protein S-nitrosylation. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B86.

Role of the Wnt signaling pathway in bone and tooth

Signaling by the Wnt plays a central role in many processes during embryonic development and adult homeostasis. At least 19 types of Wnts, several families of secreted antagonists and multiple receptors have been identified. Two distinct Wnt signaling pathways, the canonical pathway and the noncanonical pathway have been described. Functional studies and experimental analysis of relevant animal models confirmed the effects of Wnt on regulation of developing mineralized tissue formation and adult homeostasis. In osteoblasts, the canonical Wnt pathway modulates differentiation, proliferation and mineralization, while it blocks apoptosis and osteoclastogenesis by increasing osteoprotegerin. Functional crosstalk between Wnt and bone morphogenetic protein signaling during osteoblastic differentiation has been reported. Recently, non-canonical Wnt signaling was shown to play a role in bone formation. The Wnt signaling pathway also plays an important role not only in tooth formation but also in differentiation and proliferation of cementoblasts and odontoblasts in the tooth. This present review provides an overview of progress in elucidating the role of Wnt signaling pathways in bone and tooth and the resulting possibilities for therapeutic potential.

Hypermethylation of APC promoter 1A is associated with moderate activation of Wnt signalling pathway in a subset of colorectal serrated adenomas

The role of Wnt signalling pathway in serrated adenomas (SAs) remains to be identified. The aim of this study was to determine whether Wnt signalling plays a role in the pathogenesis of SAs, and to clarify the mechanism of Wnt signalling activation in SAs. This study investigated immunoreactivities of adenomatous polyposis coli (APC) and beta-catenin, mutations of APC and beta-catenin genes, methylation status of APC promoter 1A in 12 SAs, and compared the findings with normal colorectal mucosa, hyperplastic polyps, traditional adenomas (TAs) and colorectal cancers (CRCs). APC expression was moderately decreased in SAs. Cytoplasmic accumulation of beta-catenin was demonstrated in 41.7% (5/12) of SAs, but membranous immunoreactivity of beta-catenin was lost in only 8.3% (1/12) of SAs. No beta-catenin mutation was detected in any of 12 SAs, and only one SA was found to be positive for APC gene mutation. Complete methylation of APC promoter 1A was found in 41.7% (5/12) of SAs, but in no TAs or CRCs. Hypermethylation of APC promoter 1A, instead of mutations involving APC and beta-catenin, contributes to moderate activation of Wnt signalling in a subset of SAs.

Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11

Recently, various approaches for controlling the embryonic stem (ES) cell microenvironment have been developed for regulating cellular fate decisions. It has been reported that the lineage specific differentiation could be affected by the size of ES cell colonies and embryoid bodies (EBs). However, much of the underlying biology has not been well elucidated. In this study, we used microengineered hydrogel microwells to direct ES cell differentiation and determined the role of WNT signaling pathway in directing the differentiation. This was accomplished by forming ES cell aggregates within microwells to form different size EBs. We determined that cardiogenesis was enhanced in larger EBs (450 microm in diameter), and in contrast, endothelial cell differentiation was increased in smaller EBs (150 microm in diameter). Furthermore, we demonstrated that the EB-size mediated differentiation was driven by differential expression of WNTs, particularly noncanonical WNT pathway, according to EB size. The higher expression of WNT5a in smaller EBs enhanced endothelial cell differentiation. In contrast, the increased expression of WNT11 enhanced cardiogenesis. This was further validated by WNT5a-siRNA transfection assay and the addition of recombinant WNT5a. Our data suggest that EB size could be an important parameter in ES cell fate specification via differential gene expression of members of the noncanonical WNT pathway. Given the size-dependent response of EBs to differentiate to endothelial and cardiac lineages, hydrogel microwell arrays could be useful for directing stem cell fates and studying ES cell differentiation in a controlled manner.

Potential role of Wnt signaling pathway in epidermal cell dedifferentiation under traumatic microenvironment

Objective To investigate the inductive role of traumatic microenvironment in dedifferentiation of epidermal cells and explore the potential role of Wnt signaling pathway in this biological process. Methods The sheets of human foreskin were digested overnight after removal of adipose tissue, and then the epidermis was separated from the dermis. The separated epidermis sheets were repeatedly adhered to type Ⅳ collagen and flushed to remove the epidermal stem cells. The obtained epidermis sheets were transplanted onto the full-thickness skin wounds on the back of BALB/c nude mice, five days after which the cell lineage was evaluated by immunohistochemistry and the expressions of Wnts and downstream components in the grafted epidermal sheets examined by RT-PCR and Western blot. Results The cells in the basal layer of full-thickness epidermal sheets were positive for CK19 and β1 integrin and negative for CK10. While the cells in uhrathin epidermal sheets treated with type Ⅳ collagen were fully positive for CK10. Five days after transplantation of the ultrathin epidermal sheets, cells negative for CK10 but positive for CK19 and β1 integrin emerged at the wound-neighboring side of the skin grafts. At the same time, the expressions of Wnt-10b, Wnt-4 and Wnt-7a mRNA were increased by about 3.1-fold, 2.2-fold and 1.4-fold independently after transplantation. Furthermore, the expressions of β-catenin and β-catenin target genes (cyclin D1 and c-myc) were elevated by about 3-fold, 1.5-fold and 2-fold respectively in the grafted epidermal sheets (P < 0.01). Conclusion Traumatic microenvironment can induce epidermal cell dedifferentiation, when the Wnt/β -catenin signaling pathway may play an important role. Key words: Wounds and injuries; Epidermis; Dedifferentiation; Signal transduction; Wnt/β-catenin signaling pathway

Role of Smad- and Wnt-Dependent Pathways in Embryonic Cardiac Development

The development of the heart is essential for embryogenesis and precedes development of other organs. However, the mechanisms involved in embryonic cardiac development are ill-defined. Recent evidence suggests that Smad and Wnt signaling pathways are important in stem cell fate determination and their commitment to cardiovascular differentiation. We have previously reported that bone morphogenetic proteins (BMP)-2, -5, and -7 and fibroblast growth factors (FGF)-2 and -4 secreted from the adjoining endodermal cells favor cardiac differentiation in murine embryonic stem (ES) cells. Here, we demonstrate that BMP-2, -5, and -7 stimulate receptor-activated Smad1, 5, and 8, which in turn causes oligomerization of Smad4 in the nucleus. We further delineate the role of Wnt signaling pathway as evidenced by induction of Wnt3 and Wnt8b, stimulation of FRP-1, inhibition of GSK-B, accumulation of cytosolic beta-catenin, and transcription of target genes, including c-myc and cyclin-D1. We also ascertained the specificity of BMP- and Wnt-evoked activation of signaling cascades. Our data are consistent with the hypothesis that BMP-dependent activation of transcription factors including GATA-4, Nkx2.5, and MEF-2C augments cardiac differentiation mediated by cooperative control of Smad and Wnt signaling pathways. Our results provide a solid foundation for further study of the biochemistry of cardiac differentiation from stem cells.

A regulatory role of Wnt signaling pathway in the hematopoietic differentiation of murine embryonic stem cells

One of the most important issues in stem cell research is to understand the regulatory mechanisms responsible for their differentiation. An extensive understanding of mechanism underlying the process of differentiation is crucial in order to prompt stem cells to perform a particular function after differentiation. To elucidate the molecular mechanisms responsible for the hematopoietic differentiation of embryonic stem cells (ESCs), we investigated murine ES cells for the presence of hematopoietic lineage markers as well as Wnt signaling pathway during treatments with different cytokines alone or in combination with another. Here we report that Wnt/β-catenin signaling is down-regulated in hematopoietic differentiation of murine ES cells. We also found that differentiation induced by the interleukin-3, interleukin-6, and erythropoietin combinations resulted in high expression of CD3e, CD11b, CD45R/B220, Ly-6G, and TER-119 in differentiated ES cells. A high expression of β-catenin was observed in two undifferentiated ES cell lines. Gene and protein expression analysis revealed that the members downstream of Wnt in this signaling pathway including β-catenin, GSK-3β, Axin, and TCF4 were significantly down-regulated as ES cells differentiated into hematopoietic progenitors. Our results show that the Wnt/β-catenin signaling pathway plays a role in the hematopoietic differentiation of murine ESCs and also may support β-catenin as a crucial factor in the maintenance of ES cells in their undifferentiated state.

M1 muscarinic receptor activation protects neurons from β-amyloid toxicity. A role for Wnt signaling pathway

Amyloid-beta-peptide (Abeta) deposits are one of the hallmark features of Alzheimer's disease. Signal transduction alterations are implicate in the neuronal responses to Abeta, which include neurotransmitter systems and pathways involved in the maintenance of the nervous system. In this context, we have recently found that Abeta-neurotoxicity triggers a loss of Wnt signaling. We report here that M1-acetylcholine-muscarinic-receptor (mAChR) activation protects neurons from Abeta-toxicity. Concomitant with this effect, a modulation of the Wnt signaling was observed. M1 mAChR activation inhibits glycogen-synthase-kinase-3beta (GSK-3beta) activity, stabilizes cytoplasmic and nuclear beta-catenin, and induces the expression of the Wnt target genes engrailed and cyclin-D1, reverting the switch off of the Wnt pathway caused by Abeta-toxicity. Neurons from mice that overexpress GSK-3beta allow us to establish that M1 mAChR stimulation leads to GSK-3beta inactivation. We conclude that the cross-talk between the muscarinic signaling and Wnt components underlie the neuroprotective effect of the M1 mAChR activation.

Antisense Tcf inhibits the neoplastic growth of liver cancer cells.

T cell transcription factors are nuclear effectors of the Wnt signaling transduction pathway and play crucial roles in embryonic and malignant development. Our previous study showed increased expression level of Tcf mRNA in liver cancer. In the present paper, antisense Tcf RNA was used to explore the possible therapeutic effect on liver cancer cells by interrupting the abnormal Wnt pathway. Antisense expression vectors containing the conserved sequence of Tcf cDNA were constructed and transfected into a human liver cancer cell line SMMC-7721. Tumorigenic potential was determined by cellular growth assay and tumor growth in nude mice. The stable transfection of anti-sense Tcf in SMMC-7721 cells significantly reduced Tcf expression at both mRNA and protein levels compared with parental and mock-transfected cells. Antisense-mediated suppression of Tcf inhibited the in vitro proliferation and in vivo tumor formation ability. Furthermore, the apoptosis rate of antisense transfected cells was significantly higher than that of control, indicating that antisense RNA suppressed malignant growth by induction of apoptosis. Our studies demonstrate the critical role of Wnt signaling pathway in the neoplastic growth of liver cancer cells and suggest that inhibition of Tcf activity with antisense Tcf RNA may be a potential new gene therapy method for liver cancer.