Wnt Activity In Cells Research Articles

Repression of MAP3K1 expression and JNK activity by canonical Wnt signaling

Morphogenesis is a complex and highly coordinated process orchestrated by temporal spatial activity of developmental pathways. How the different pathways interact to guide the developmental program remains an intriguing and open question. MAP3K1-JNK and Wnt are signaling pathways crucial for embryonic eyelid closure, an epithelial morphogenetic event conserved in mammals. Here we used a mouse model of eyelid development and genetic and biochemistry tools to investigate the relationships between the two pathways. We found that Wnt activation repressed MAP3K1 expression. Using Axin-LacZ reporter mice, spatial Wnt activity was detected in the leading edge of the developing eyelid. Conditional knockout of Wntless (Wls) in ocular surface ectoderm blocked eyelid formation, and significantly increased MAP3K1 expression in eyelid cells at the nasal canthus region. Conversely, knockout of Dkk2, encoding a canonical Wnt antagonist, resulted in an increase of Wnt activity in cells at the upper eyelid margin near the nasal canthus. Up-regulation of Wnt signaling in the Dkk2-knockout embryos corresponded to down-regulation of MAP3K1 expression. In vitro data showed that Wnt3a treatment decreased MAP3K1 promoter activity, whereas activation of Wnt by lithium chloride inhibited MAP3K1 expression, and attenuated MAP3K1-mediated JNK activity. Our data identify a unique signal crosstalk between Wnt signaling and the MAP3K1-JNK pathway in epithelial morphogenesis.

The effect of plakophilin-2 gene mutations on the activity of canonical WNT signaling

Plakophilin-2 is a desmosomal protein encoded by PKP2 gene. Desmosomal proteins are usually considered as structural proteins with the main function of maintaining intercellular interactions. Genetic studies revealed that mutations in desmosomal genes could lead to arrhythmogenic right ventricular cardiomyopathy, a heart disease characterized by substitution of cardiomyocytes by adipose and fibrotic tissue predominantly in the right ventricle. Wnt signaling is a signal transduction pathway presumably involved in the development of pathology. The purpose of this study was to investigate Wnt activity in cells with wild-type and mutant PKP2 gene during adipogenic and cardiomyogenic differentiation. We used multipotent mesenchymal stromal cells and iPS cells generated from a patient carrying PKP2 gene mutation. We found that cells with mutant PKP2 exhibited lower transcriptional activity of β-catenin, a key player of Wnt signaling, as well as attenuated expression of its targets SOX2 and SOX9. These findings suggest a signaling role of plakophilin2 by regulating Wnt activity

Abstract A65: Characterization of CBP/p300-mediated butyrate-induced Wnt activity in colonic microadenoma cells.

Abstract Objectives of the study: We wished to (a) compare gene expression profiles between a colonic microadenoma cell line (LT97) and a metastatic colorectal cancer (CRC) cell line (SW620) in the presence and absence of butyrate, (b) determine whether the sensitivity of LT97 cells to the growth suppressive effects of butyrate correlates to a high-fold upregulation of Wnt signaling in these cells, and (c) evaluate how modulation of CBP/p300-mediated Wnt signaling influences the ability of butyrate to upregulate Wnt activity in LT97 cells. Methods: RNA was isolated from mock treated or butyrate treated LT97 microadenoma cells or SW620 metastatic CRC cells, followed by full human genome microarray analysis (Genus Biosystems). Colonic cells were transfected with Wnt reporter plasmids and treated with ICG-001 (which inhibits CBP-mediated Wnt activity) or co-transfected with a p300 expression vector or with an expression vector for KLF4, which inhibits CBP/p300 association with beta-catenin. Reporter assays, western blotting, and caspase assays were performed. Results: We demonstrate that LT97 colonic microadenoma cells differ substantially in gene expression profiles compared to a metastatic CRC cell line (SW620), both in the presence and absence of butyrate. Further, we demonstrate that the high degree of sensitivity to the growth repressive effects of butyrate of LT97 cells correlates to a marked butyrate-induced hyperactivation of Wnt activity in that cell line. Further, the induction of Wnt activity by butyrate in LT97 cells is sensitive to treatment with ICG-001, overexpression of CBP or p300, or blockade of CBP/p300-mediated Wnt activity by the transcription factor KLF4. Conclusions: Microadenoma cells, reflecting very early stage colonic neoplasia, are highly sensitive to the effects of butyrate on Wnt signaling, in a manner dependent upon CBP/p300. The findings generated from this study may lead to approaches to modulate the action of CBP/p300-mediated Wnt signaling transcriptional programs for therapeutic or chemopreventive effect. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A65. Citation Format: Darina L. Lazarova, Christopher Chiaro, Terrence Wong, Michael Bordonaro. Characterization of CBP/p300-mediated butyrate-induced Wnt activity in colonic microadenoma cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A65.

Abstract 5031: R-spondin potentiates Wnt/β-catenin signaling in Ewing sarcoma cells.

Abstract Proper regulation of Wnt signaling is highly context-dependent and critical for growth and cellular maintenance. It is well-established that dysregulation of the canonical Wnt/β-catenin pathway contributes to the development and progression of numerous malignancies. Despite this, there has been little evidence to support a role for canonical Wnt signaling in Ewing sarcoma (ES) pathogenesis. Recent studies have shown that when members of the leucine-rich G-protein coupled receptor (LGR) family, namely LGR4 and LGR5, are bound by their ligands, R-spondins, they potentiate Wnt/β-catenin activity. Importantly, recent studies from our lab have shown that some ES tumors and cell lines express high levels of LGR5. In addition, R-spondin is highly expressed in the developing bone, which is the primary site of ES in children. Since ES cells do not exhibit canonical Wnt/β-catenin activity in standard culture conditions, we hypothesized that cues from the tumor microenvironment may be required for activation of this pathway. In particular, we hypothesized that R-spondins would potentiate Wnt activity in LGR-expressing ES cells. To test this hypothesis, ES cells were cultured in the presence of WNT3A and/or R-spondin 2 (Rspo2). Immunocytochemistry was used to determine β-catenin nuclear localization, and Wnt transcriptional activity was determined by flow cytometry and luciferase activity using cell lines transduced with lentiviral 7xTCF-GFP and 7xTCF-luciferase reporters, respectively. qRT-PCR was used to confirm transcription of the Wnt target gene AXIN2. Our results show that exogenous WNT3A induces β-catenin nuclear localization in ES cells and is associated with induction of TCF-mediated transcriptional activity and AXIN2 expression. Furthermore, Wnt/β-catenin activation is potentiated in the presence of Rspo2, and ES cells lines with the highest expression of LGRs show the strongest potentiation. Interestingly, however, exogenous WNT3A and Rspo2 did not induce Wnt activity in all cells. Together these data confirm that in the presence of exogenous ligand, Wnt/β-catenin signaling can be robustly activated in a subpopulation of ES cells and that signaling is potently enhanced in the presence of Rspo2 and LGRs. To our knowledge, this is the first evidence of R-spondins influencing Wnt activity in a non-epithelial cancer. These studies support the concept that R-spondin expression in the tumor microenvironment may contribute to ES pathogenesis by modulating Wnt/β-catenin signaling. Citation Format: Elisabeth A. Pedersen, Christopher A. Scannell, Elizabeth R. Lawlor. R-spondin potentiates Wnt/β-catenin signaling in Ewing sarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5031. doi:10.1158/1538-7445.AM2013-5031