Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Wnt pathway”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources
PubMed2006

Wnt pathway is involved in pleomorphic adenomas induced by overexpression of PLAG1 in transgenic mice.

Pleomorphic adenoma gene 1 (PLAG1) was found frequently rearranged and activated in human salivary gland pleomorphic adenomas. It encodes a developmentally regulated transcription factor. Ectopic overexpression of PLAG1 has been proposed to play a crucial role in tumorigenesis of salivary gland pleomorphic adenomas. It was reported that PLAG1 can activate the transcription of insulin-like growth factor 2 (IGF2), functioning as a protooncogene. In this report, we show that the salivary gland tumors developed in PLAG1 transgenic mice share major histopathologic features with human pleomorphic adenomas. It was found that beta-catenin, the key component of Wnt signaling pathway, was upregulated at transcriptional level in tumors developed in 3 independent transgenic mouse lines. Immunohistochemical staining revealed that expression of beta-catenin as well as c-myc, downstream of beta-catenin in Wnt signaling pathway, was highly upregulated with overexpression of PLAG1 transgene in tumor and normal transgenic salivary gland tissues. Moreover, we found that PLAG1 can activate the transcription of mouse but not human beta-catenin in the 3T3 cells cotransfected with reporter constructs. Sequence analysis shows there are 4 PLAG1 consensus binding sites in mouse beta-catenin promoter region but not in human. Our findings provide the first in vivo evidence for the oncogenic activity of PLAG1 in pleomorphic adenoma tumorigenesis, reveal a valued animal model for human salivary gland tumors and suggest that Wnt signaling pathway may also contribute to the development of pleomorphic adenomas in transgenic mice.

3T3 Cells↗
PubMed2004

An autocrine mechanism for constitutive Wnt pathway activation in human cancer cells.

Autocrine Wnt signaling in the mouse mammary tumor virus model was the first identified mechanism of canonical pathway activation in cancer. In search of this transformation mechanism in human cancer cells, we identified breast and ovarian tumor lines with upregulation of the uncomplexed transcriptionally active form of beta-catenin without mutations afflicting downstream components. Extracellular Wnt antagonists FRP1 and DKK1 caused a dramatic downregulation of beta-catenin levels in these tumor cells associated with alteration of biological properties and increased expression of epithelial differentiation markers. Colorectal carcinoma cells with knockout of the mutant beta-catenin allele retained upregulated beta-catenin levels, which also could be inhibited by these Wnt antagonists. Together, these findings establish the involvement of autocrine Wnt signaling in human cancer cells.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2003

Cooperation between TGF-beta and Wnt pathways during chondrocyte and adipocyte differentiation of human marrow stromal cells.

UNLABELLED: Human marrow stromal cells have the potential to differentiate to chondrocytes or adipocytes. We show interactions between TGF-beta and Wnt signaling pathways during stimulation of chondrogenesis and inhibition of adipogenesis. Combining these signals may be useful in marrow stromal cell therapies. INTRODUCTION: Human bone marrow stromal cells (hMSCs) have the potential to differentiate to lineages of mesenchymal tissues, including cartilage, fat, bone, tendon, and muscle. Agents like transforming growth factor (TGF)-beta promote chondrocyte differentiation at the expense of adipocyte differentiation. In other processes, TGF-beta and Wnt/wingless signaling pathways play major roles in controling certain developmental events and activation of specific target genes. We tested whether these pathways interact during differentiation of chondrocytes and adipocytes in human marrow stromal cells. MATERIALS AND METHODS: Both a line of human marrow stromal cells (KM101) and freshly isolated hMSCs were studied. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and macroarrays were used for analysis of the modulation of TGF-beta1 on Wnt signaling-associated genes, chondrocyte differentiation genes, and TGFbeta/bone morphogenetic protein (BMP) signaling-associated genes in KM101 cells. Early passage hMSCs obtained from 42- and 58-year-old women were used for the effects of TGF-beta and/or Wnt (mimicked by LiCl) signals on chondrocyte and adipocyte differentiation in two-dimensional (2-D) cultures, 3-D pellet cultures, and collagen sponges. RESULTS: As indicated by macroarray, RT-PCR, and Western blot, TGF-beta activated genes in the TGF-beta/Smad pathway, upregulated Wnt2, Wnt4, Wnt5a, Wnt7a, Wnt10a, and Wnt co-receptor LRP5, and increased nuclear accumulation and stability of beta-catenin in KM101 cells. TGF-beta upregulated chondrocyte gene expression in KM101 cells and also stimulated chondrocyte differentiation and inhibited adipocyte differentiation in hMSCs, synergistically with Wnt signal. Finally, hMSCs cultured in 3-D collagen sponges were stimulated by TGF-beta1 to express aggrecan and collagen type II mRNA, whereas expression of lipoprotein lipase was inhibited. CONCLUSIONS: In summary, TGF-beta stimulated chondrocyte differentiation and inhibited adipocyte differentiation of hMSCs in vitro. The activation of both TGF-beta and Wnt signal pathways by TGF-beta, and synergy between TGF-beta and Wnt signals, supports the view that Wnt-mediated signaling is one of the mechanisms of TGF-beta's effects on chondrocyte and adipocyte differentiation of hMSCs.

Adipocytes↗
PubMed2001

Activation of the Wnt pathway interferes with serum response element-driven transcription of immediate early genes.

Mutational activation of the Wnt signaling pathway is a common early event in colorectal tumorigenesis, and the identification of target genes regulated by this pathway will provide a better understanding of tumor progression. Gene expression profiling on oligonucleotide microarrays revealed reduced expression of the immediate early genes fos and fosB following stimulation of cells by Wnt-1. Further analysis demonstrated that serum or 12-O-tetradecanoylphorbol-13-acetate activation of several immediate early genes including fos, fosB, junB, and egr1 was inhibited by Wnt signaling. Wnt signaling inhibited transcriptional activation driven by the serum response element without altering the activation of the extracellular signal-regulated kinase cascade or ternary complex formation at the fos serum response element promoter. The Wnt-mediated repression of c-Fos, FosB, and JunB expression was consistent with a decrease in their binding to an AP-1 promoter element and decreased target gene transcription. The expression of fos, fosB, junB, and egr1 was also repressed in human colon tumors relative to patient matched normal tissue. By contrast, the fos family member fra-1 was up-regulated in the human colon tumors, suggesting a compensatory mechanism for the reduction in fos and fosB expression. The results indicate that Wnt signaling can repress the expression of certain immediate early genes, and that this effect is consistent with changes in gene expression observed in human colorectal tumors.

Adenosine Triphosphate↗
PubMed2006

Human naive CD8 T cells down-regulate expression of the WNT pathway transcription factors lymphoid enhancer binding factor 1 and transcription factor 7 (T cell factor-1) following antigen encounter in vitro and in vivo.

The transcription factors lymphoid enhancer binding factor 1 (LEF1) and transcription factor 7 (TCF7) (T cell factor-1 (TCF-1)) are downstream effectors of the WNT signaling pathway, which is a critical regulator of T cell development in the thymus. In this study, we show that LEF1 and TCF7 (TCF-1) are not only expressed in thymocytes, but also in mature T cells. Our data demonstrate that Ag encounter in vivo and engagement of the TCR or IL-15 receptor in vitro leads to the down-regulation of LEF1 and TCF7 (TCF-1) expression in human naive CD8 T cells. We further show that resting T cells preferentially express inhibitory LEF1 and TCF7 (TCF-1) isoforms and that T cell activation changes the isoform balance in favor of stimulatory TCF7 (TCF-1) isoforms. Altogether, our study suggests that proteins involved in the WNT signaling pathway not only regulate T cell development, but also peripheral T cell differentiation.

Antigens↗
PubMed2000

A road to kidney tubules via the Wnt pathway.

Classical in vitro studies indicate that tubule induction in the kidney mesenchyme is mediated by cell-cell contacts between the inducer tissue and the metanephric mesenchyme. Induction is completed within the first 24 h, after which tubules will form because of stimulated cell proliferation, migration, and cell adhesion. Recent evidence has revealed an essential role for the secreted signals from the Wnt gene family. Of these, Wnt-4 is expressed in developing tubules and knocking out its function perturbed kidney development. More detailed studies demonstrated normal condensation, but tubules were missing. Subsequent experiments indicated that Wnt-4 is also a sufficient signal to trigger tubulogenesis. Cells that were engineered to express Wnt-4 not only induced tubulogenesis in the kidney mesenchyme of Wnt-4 mutant embryos, but also induced tubules in the wild type mesenchyme. With the transfilter induction assay, Wnt-4-mediated induction was completed within the first 24 h, depending on the presence of proteoglycans and cell-cell contacts between the interactants. In addition, Wnt-4 autoinduced expression of its own gene and a panel of other components of the Wnt signalling pathway, such as frizzleds and a candidate Wnt antagonist from the secreted frizzled-related protein family. Taken together, the data provide evidence of an essential role for Wnt signal transmission and transduction pathways in the induction of kidney tubules, and the findings have paved the way for detailed molecular studies.

Animals↗
PubMed2005

A possible role for the canonical Wnt pathway in endocrine cell development in chicks.

Wnt signalling is involved in many developmental processes such as proliferation, differentiation, cell fate decisions, and morphogenesis. However, little is known about Wnt signalling during pancreas development. Multiple Wnt ligands and Frizzled receptors are expressed in the embryonic mouse pancreas, the surrounding mesenchyme, and have also been detected in the chicken endoderm during development. The aim of this study was to investigate the role of canonical Wnt signalling on endocrine cell development by use of the in ovo electroporation of the chicken endoderm. Overexpression with a constitutive active form of beta-catenin in combination with Ngn3 resulted in reduced numbers of glucagon cells. dnLEF-1 or naked-1 did not alter endocrine cell differentiation when co-expressed with Ngn3, but dnLEF-1 appeared to have some potential for inhibiting delamination of Ngn3 cells. In addition, neuronal beta-III-tubulin, which had previously been considered a specific marker for neuronal cells, was observed in the pancreas and was upregulated in the electroporated Ngn3 cells and thus may be a new endocrine marker in the chicken.

Animals↗
PubMed2004

The proinvasive activity of Wnt-2 is mediated through a noncanonical Wnt pathway coupled to GSK-3beta and c-Jun/AP-1 signaling.

Inappropriate activation of the Wnt/APC/beta-catenin signaling pathways plays a critical role at early stages in a variety of human cancers. However, their respective implication in tumor cell invasion is still hypothetical. Here, we show that two activators of the canonical Wnt/beta-catenin transcription pathway, namely Dvl-2, the Axin 501-560 fragment binding glycogen synthase kinase -3beta (GSK-3beta), and the negative Wnt regulator wt-Axin did not alter cell invasion into type I collagen. In addition, both Dvl-2 and Axin 501-560 exerted a permissive action on the proinvasive activity of HGF and intestinal trefoil factor. Upstream activation of Wnt signaling by the Wnt-2 and Wnt-3a ligands, stable overexpression of Wnt-2, as well as GSK-3beta inhibition by lithium, SB216763, and GSK-3beta dominant negative forms (K85R and R96E) conferred the invasive phenotype through several proinvasive pathways. Induction of the matrix metalloprotease MMP-7 (matrilysin) gene and protein by Wnt-2 was abolished by inactivation of the AP-1 binding site in the promoter. Accordingly, invasion induced by Wnt-2 was prevented by soluble FRP-3 and FRP-1, sequestration of Gbetagamma subunits, depletion of the GSK-3beta protein by RNA interference, the c-Jun dominant negative mutant TAM67 and was not reversed by wt-Axin. Thus, the proinvasive activity of Wnt-2 is mediated by a noncanonical Wnt pathway using GSK-3beta and the AP-1 oncogene. Our data provide a potential clue for our understanding of the action and crosstalk between Wnt activators and other proinvasive pathways, in relation with matrix substrates and proteases in human cancers.

Animals↗
PubMed2007

Transcriptional cooperation between the transforming growth factor-beta and Wnt pathways in mammary and intestinal tumorigenesis.

Transforming growth factor-beta (TGF-beta) and Wnt ligands function in numerous developmental processes, and alterations of both signaling pathways are associated with common pathologic conditions, including cancer. To obtain insight into the extent of interdependence of the two signaling cascades in regulating biological responses, we used an oligonucleotide microarray approach to identify Wnt and TGF-beta target genes using normal murine mammary gland epithelial cells as a model. Combination treatment of TGF-beta and Wnt revealed a novel transcriptional program that could not have been predicted from single ligand treatments and included a cohort of genes that were cooperatively induced by both pathways. These included both novel and known components or modulators of TGF-beta and Wnt pathways, suggesting that mutual feedback is a feature of the coordinated activities of the ligands. The majority of the cooperative targets display increased expression in tumors derived from either Min (many intestinal neoplasia) or mouse mammary tumor virus (MMTV)-Wnt1 mice, two models of Wnt-induced tumors, with nine of these genes (Ankrd1, Ccnd1, Ctgf, Gpc1, Hs6st2, IL11, Inhba, Mmp14, and Robo1) showing increases in both. Reduction of TGF-beta signaling by expression of a dominant-negative TGF-beta type II receptor in bigenic MMTV-Wnt1/DNIIR mice increased mammary tumor latency and was correlated with a decrease in expression of Gpc1, Inhba, and Robo1, three of the TGF-beta/Wnt cooperative targets. Our results indicate that the TGF-beta and Wnt/beta-catenin pathways are firmly intertwined and generate a unique gene expression pattern that can contribute to tumor progression.

Adenoma↗
PubMed2004

TGF-beta targets the Wnt pathway components, APC and beta-catenin, as Mv1Lu cells undergo cell cycle arrest.

The highly coordinated interaction of TGF-beta and Wnt signaling pathways is critical for normal development. However, the effects of TGF-beta on APC and beta-catenin, two key mediators of Wnt signaling in epithelial cells, have been largely unknown. We determined the effect of TGF-beta on APC and beta-catenin expression in Mv1Lu, a nontransformed epithelial cell line, in which TGF-beta signaling causes a G(1) cell cycle arrest. We found that TGF-beta rapidly reduced APC protein levels through a post-transcriptional mechanism. Further, TGF-beta increased beta-catenin mRNA and protein levels, and increased beta-catenin nuclear accumulation. Finally, retrovirus-mediated overexpression of beta-catenin discernibly enhanced the ability of TGF-beta to induce a G(1) cell cycle arrest. This is the first report demonstrating that TGF-beta mimics the effect of Wnt signaling on beta-catenin in Mv1Lu cells, and that reduction of APC and nuclear accumulation of beta-catenin have cooperative effects on mechanisms that mediate TGF-beta-induced cell cycle arrest.

Adenomatous Polyposis Coli Protein↗
PubMed1999

MAP kinase and Wnt pathways converge to downregulate an HMG-domain repressor in Caenorhabditis elegans.

The signalling protein Wnt regulates transcription factors containing high-mobility-group (HMG) domains to direct decisions on cell fate during animal development. In Caenorhabditis elegans, the HMG-domain-containing repressor POP-1 distinguishes the fates of anterior daughter cells from their posterior sisters throughout development, and Wnt signalling downregulates POP-1 activity in one posterior daughter cell called E. Here we show that the genes mom-4 and lit-1 are also required to downregulate POP-1, not only in E but also in other posterior daughter cells. Consistent with action in a common pathway, mom-4 and lit-1 exhibit similar mutant phenotypes and encode components of the mitogen-activated protein kinase (MAPK) pathway that are homologous to vertebrate transforming-growth-factor-beta-activated kinase (TAK1) and NEMO-like kinase (NLK), respectively. Furthermore, MOM-4 and TAK1 bind related proteins that promote their kinase activities. We conclude that a MAPK-related pathway cooperates with Wnt signal transduction to downregulate POP-1 activity. These functions are likely to be conserved in vertebrates, as TAK1 and NLK can downregulate HMG-domain-containing proteins related to POP-1.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Cdx4 is a direct target of the canonical Wnt pathway.

There is considerable evidence that the Cdx gene products impact on vertebral patterning by direct regulation of Hox gene expression. Data from a number of vertebrate model systems also suggest that Cdx1, Cdx2 and Cdx4 are targets of caudalizing signals such as RA, Wnt and FGF. These observations have lead to the hypothesis that Cdx members serve to relay information from signaling pathways involved in posterior patterning to the Hox genes. Regulation of Cdx1 expression by RA and Wnt in the mouse has been well characterized; however, the means by which Cdx2 and Cdx4 are regulated is less well understood. In the present study, we present data suggesting that Cdx4 is a direct target of the canonical Wnt pathway. We found that Cdx4 responds to exogenous Wnt3a in mouse embryos ex vivo, and conversely, that its expression is down-regulated in Wnt3a(vt/vt) embryos and in embryos cultured in the presence of Wnt inhibitors. We also found that the Cdx4 promoter responds to Wnt signaling in P19 embryocarcinoma cells and have identified several putative LEF/TCF response elements mediating this effect. Consistent with these data, chromatin immunoprecipitation assays from either embryocarcinoma cells or from the tail bud of embryos revealed that LEF1 and beta-catenin co-localize with the Cdx4 promoter. Taken together, these results suggest that Cdx4, like Cdx1, is a direct Wnt target.

Animals↗
PubMed2007

WIF1, an inhibitor of the Wnt pathway, is rearranged in salivary gland tumors.

Chromosome rearrangements involving 12q13-15 are frequent among several tumors, including pleomorphic adenomas. The common molecular target for these aberrations is the HMGA2 gene, but various fusion partners of HMGA2 have been reported in tumors. Here we report the identification of the WNT inhibitory factor 1 (WIF1) gene as a novel HMGA2 fusion partner in a salivary gland pleomorphic adenoma. In normal salivary gland tissue WIF1 is expressed at a high level and HMGA2 is not expressed. However, in the pleomorphic adenoma expressing the HMGA2/WIF1 fusion transcript, we observed re-expression of HMGA2 wild-type transcripts and very low levels of WIF1 expression. These data suggest a possible synergistic effect between upregulation of HMGA2 and downregulation of WIF1. We screened 13 additional benign and malignant salivary gland tumors and detected WIF1 rearrangement in one out of two carcinomas ex-pleomorphic adenoma analyzed. In this malignant tumor, the rearrangement of one WIF1 allele coexists with loss of the other allele, a classic signature of a tumor suppressor gene. WIF1 is an antagonist of the Wnt signaling pathway, which plays a critical role in human cancer. In transgenic mouse models, Wnt activation leads to a high frequency of benign and malignant salivary gland tumors. To our knowledge, this is the first report suggesting that WIF1 is a recurrent target in human salivary gland oncogenesis and that downregulation of WIF1 plays a role in the development and/or progression of pleomorphic adenomas.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Expression of COX-2 and Wnt pathway genes in adenomas of familial adenomatous polyposis patients treated with meloxicam.

BACKGROUND: Familial adenomatous polyposis (FAP) is an autosomal, dominantly inherited predisposition to colorectal cancer caused by germline mutations within the adenomatous polyposis coli (APC) gene, a key member of the Wnt signalling pathway. A new class of non-steroidal anti-inflammatory drugs (NSAIDs), the specific cyclooxygenase 2 (COX-2) inhibitors, have recently been applied for the treatment of FAP patients. PATIENTS AND METHODS: The expressions of the Wnt members and targets APC, c-myc, cyclin D1 and COX-2, as measured by real-time quantitative RT-PCR, have been evaluated in fresh samples of normal colorectal mucosa and matched adenoma tissue of six unrelated FAP patients before and after treatment with meloxicam. RESULTS: A significant up-regulation of COX-2 in adenomas after treatment with meloxicam was found. Furthermore, in adenomas, a down-regulation of APC after treatment and a tight correlation of the expressions of the two Wnt targets, c-myc and cyclin D1, in both stages of treatment were observed. CONCLUSION: A feedback loop mediated by the peroxisome proliferator-activated receptor (PPAR) gamma is discussed as being responsible for the up-regulation of COX-2

Adenomatous Polyposis Coli↗
PubMed2001

Regulation of dharma/bozozok by the Wnt pathway.

The zebrafish homeobox gene dharma/bozozok (boz) is required for the formation and/or function of the Nieuwkoop center and the subsequent induction of the Spemann organizer. dharma is expressed soon after the midblastula transition in the dorsal blastomeres and the dorsal yolk syncytial layer (YSL). We found that the expression of dharma was upregulated or ectopically induced by misexpression of a Wnt protein and cytoplasmic components of the Wnt signaling pathway and downregulated by the expression of dominant-negative Tcf3. A 1.4-kbp fragment of the dharma promoter region contains consensus sequences for Tcf/Lef binding sites. This promoter region recapitulated the Wnt-dependent and dorsal dharma expression pattern when it was fused to luciferase or GFP. Deletion and point mutant analyses revealed that the Tcf/Lef binding sites were required to drive this expression pattern. These data established that dharma/boz functions between the dorsal determinants-mediated Wnt signals and the formation of the Nieuwkoop center.

Animals↗
PubMed1999

Domains of axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization.

Axin was identified as a regulator of embryonic axis induction in vertebrates that inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos indicated that Axin functioned downstream of glycogen synthase kinase 3beta (GSK3beta) and upstream of beta-catenin, and subsequent studies showed that Axin is part of a complex including these two proteins and adenomatous polyposis coli (APC). Here, we examine the role of different Axin domains in the effects on axis formation and beta-catenin levels. We find that the regulators of G-protein signaling domain (major APC-binding site) and GSK3beta-binding site are required, whereas the COOH-terminal sequences, including a protein phosphatase 2A binding site and the DIX domain, are not essential. Some forms of Axin lacking the beta-catenin binding site can still interact indirectly with beta-catenin and regulate beta-catenin levels and axis formation. Thus in normal embryonic cells, interaction with APC and GSK3beta is critical for the ability of Axin to regulate signaling via beta-catenin. Myc-tagged Axin is localized in a characteristic pattern of intracellular spots as well as at the plasma membrane. NH2-terminal sequences were required for targeting to either of these sites, whereas COOH-terminal sequences increased localization at the spots. Coexpression of hemagglutinin-tagged Dishevelled (Dsh) revealed strong colocalization with Axin, suggesting that Dsh can interact with the Axin/APC/GSK3/beta-catenin complex, and may thus modulate its activity.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Mutational activation of the RAS-RAF-MAPK and the Wnt pathway in small intestinal adenocarcinomas.

BACKGROUND: Adenocarcinomas of the small and the large intestine share risk factors and morphological features but both tumor types seem to follow different genetic pathways. The aim of this study on small intestinal carcinomas was to analyze alternative mechanisms of activation of pathways that are typically affected in colorectal cancer. METHODS: Twenty-one sporadic carcinomas were investigated for mutations in KRAS, BRAF, the beta-catenin gene CTNNB1, and the mutational cluster region of APC. Immunohistochemical analysis was performed with a monoclonal antibody for beta-catenin, the transcriptionally active downstream component of wnt signaling. RESULTS: Oncogene mutations were found in 13 (62%) small intestinal adenocarcinomas. Twelve tumors displayed a KRAS mutation, and a novel BRAF mutation at codon 603/604 was seen in one carcinoma without KRAS mutation. One tumor harbored a CTNNB1 mutation consisting of an insertion of 247 nucleotides deriving from chromosome 9. APC mutations were identified in 2 tumors. Immunohistochemistry demonstrated nuclear accumulation of beta-catenin in 5 carcinomas. These carcinomas included the tumor with a CTNNB1 mutation but not those with APC mutations. CONCLUSIONS: Our data show frequent activation of the RAS-RAF-MAPK pathway through mutations of either KRAS or, infrequently, BRAF. Activation of the wnt pathway through accumulation of beta-catenin may have a role in a subset of small intestinal adenocarcinomas but in contrast to colorectal carcinoma, accumulation of beta-catenin is generally not caused by inactivating APC or activating CTNNB1 mutations.

Adenocarcinoma↗
PubMed2005

Activation of canonical Wnt pathway promotes proliferation of retinal stem cells derived from adult mouse ciliary margin.

Adult retinal stem cells represent a possible cell source for the treatment of retinal degeneration. However, only a small number of stem cells reside in the ciliary margin. The present study aimed to promote the proliferation of adult retinal stem cells via the Wnt signaling pathway. Ciliary margin cells from 8-week-old mice were dissociated and cultured to allow sphere colony formation. Wnt3a, a glycogen synthase kinase (GSK) 3 inhibitor, fibroblast growth factor (FGF) 2, and a FGF receptor inhibitor were then applied in the culture media. The primary spheres were dissociated to prepare either monolayer or secondary sphere cultures. Wnt3a increased the size of the primary spheres and the number of Ki-67-positive proliferating cells in monolayer culture. The Wnt3a-treated primary sphere cells were capable of self-renewal and gave rise to fourfold the number of secondary spheres compared with nontreated sphere cells. These cells also retained their multilineage potential to express several retinal markers under differentiating culture conditions. The Wnt3a-treated cells showed nuclear accumulation of beta-catenin, and a GSK3 inhibitor, SB216763, mimicked the mitogenic activity of Wnt3a. The proliferative effect of SB216763 was attenuated by an FGF receptor inhibitor but was enhanced by FGF2, with Ki-67-positive cells reaching over 70% of the total cells. Wnt3a and SB216763 promoted the proliferation of retinal stem cells, and this was partly dependent on FGF2 signaling. A combination of Wnt and FGF signaling may provide a therapeutic strategy for in vitro expansion or in vivo activation of adult retinal stem cells.

Animals↗