Epithelial Wnt/β-catenin signaling regulates palatal shelf fusion through regulation of Tgfβ3 expression

The canonical Wnt/β-catenin signaling plays essential role in development and diseases. Previous studies have implicated the canonical Wnt/β-catenin signaling in the regulation of normal palate development, but functional Wnt/β-catenin signaling and its tissue-specific activities remain to be accurately elucidated. In this study, we show that functional Wnt/β-catenin signaling operates primarily in the palate epithelium, particularly in the medial edge epithelium (MEE) of the developing mouse palatal shelves, consistent with the expression patterns of β-catenin and several Wnt ligands and receptors. Epithelial specific inactivation of β-catenin by the K14-Cre transgenic allele abolishes the canonical Wnt signaling activity in the palatal epithelium and leads to an abnormal persistence of the medial edge seam (MES), ultimately causing a cleft palate formation, a phenotype resembling that in Tgfβ3 mutant mice. Consistent with this phenotype is the down-regulation of Tgfβ3 and suppression of apoptosis in the MEE of the β-catenin mutant palatal shelves. Application of exogenous Tgfβ3 to the mutant palatal shelves in organ culture rescues the midline seam phenotype. On the other hand, expression of stabilized β-catenin in the palatal epithelium also disrupts normal palatogenesis by activating ectopic Tgfβ3 expression in the palatal epithelium and causing an aberrant fusion between the palate shelf and mandible in addition to severely deformed palatal shelves. Collectively, our results demonstrate an essential role for Wnt/β-catenin signaling in the epithelial component at the step of palate fusion during palate development by controlling the expression of Tgfβ3 in the MEE.     

Up-regulation of Axin2 by dexamethasone promotes adipocyte differentiation in ROB-C26 mesenchymal progenitor cells

Dexamethasone (Dex) regulates osteoblastic and adipocytic differentiation in mesenchymal progenitor cells through regulation of Wnt/β-catenin signaling. To elucidate the regulatory mechanisms underlying the effects of Dex, we examine the expression of Axin2, which is an intracellular inhibitor of Wnt/β-catenin signaling, in ROB-C26 clonal mesenchymal progenitor cells (C26). We observed the induction of Axin2 mRNA in C26 cells in response to Dex treatment. Treatment with a glucocorticoid receptor (GR) antagonist, mifepristone, showed that Dex-induced up-regulation of Axin2 is mediated by the GR. In the absence of Dex, gene silencing by using Axin2-targeted short hairpin RNA increased the number of alkaline phosphatase (ALP)-positive and nuclear β-catenin-positive cells and ALP activity. In the presence of Dex, Axin2 knockdown resulted in an increased number of ALP-positive and nuclear β-catenin-positive cells. Furthermore, Axin2 knockdown in Dex-treated cells suppressed adipocyte differentiation (as determined by reduced Oil Red O staining), reduced the number of PPARγ-positive and aP2-positive cells and decreased the mRNA expression of PPARγ2 and aP2. These results suggest that Axin2 plays a key role in adipocyte and osteoblastic differentiation by controlling β-catenin expression.     

A diterpenoid derivative 15-oxospiramilactone inhibits Wnt/β-catenin signaling and colon cancer cell tumorigenesis

The Wnt/β-catenin signaling pathway is a highly conserved pathway in organism evolution and regulates many biological processes. Aberrant activation of the Wnt/β-catenin signaling pathway is closely related to tumorigenesis. In order to identify potent small molecules to treat the over-activated Wnt signaling-mediated cancer, such as colon cancer, we established a mammalian cell line-based reporter gene screening system. The screen revealed a diterpenoid derivative, 15-oxospiramilactone (NC043) that inhibits Wnt3a or LiCl-stimulated Top-flash reporter activity in HEK293T cells and growth of colon cancer cells, SW480 and Caco-2. Treatment of SW480 cells with NC043 led to decreases in the mRNA and/or protein expression of Wnt target genes Axin2, Cyclin D1 and Survivin , as well as decreases in the protein levels of Cdc25c and Cdc2. NC043 did not affect the cytosol-nuclear distribution and protein level of soluble β-catenin, but decreased β-catenin/TCF4 association in SW480 cells. Moreover, NC043 inhibited anchorage-independent growth and xenograft tumorigenesis of SW480 cells. Collectively these results demonstrate that NC043 is a novel small molecule that inhibits canonical Wnt signaling downstream of β-catenin stability and may be a potential compound for treating colorectal cancer.