Recent studies indicate that the canonical

Recent studies indicate that the canonical Wnt pathway affects BMSC osteogenic differentiation. Wnt signaling can promote osteoblastic precursor differentiation into more differentiated osteoblasts and can serve as a negative regulator of adipogenesis (Glass et al., 2005; Krishnan et al., 2006). This differentiation process is tightly regulated by complex signaling events (Luu et al., 2007). β-Catenin is indeed a key signaling molecule that promotes cell differentiation. Our results showed that Ex-4 dramatically promoted β-catenin nuclear translocation and TCF7L2 expression in BMSCs. These effects were significantly inhibited by the block or knockdown of GLP-1R, suggesting that GLP-1R was involved in crosstalk with the β-catenin signaling pathway. GLP-1R is localized in multiple tissues and cell types (Holst, 2007). GLP-1 agonist binding to GLP-1R activates various different pathways involved in the proliferation, differentiation, and protection from apoptosis. On the basis of our present results we concluded that PKA, a GLP-1R downstream Myriocin Supplier exerting a strong bond to β-catenin and PI3K and induced by Ex-4, played a dual role in its interaction with the Wnt/β-catenin signaling pathway. First, at the molecular level, PKA phosphorylated β-catenin at its Ser675 and promoted β-catenin nuclear translocation, and subsequently recruited LEF/TCF DNA binding factors to initiate the expression of osteoblast differentiation-related gene, driving BMSC differentiation into osteoblasts. It has also been reported that PKA phosphorylates β-catenin at two sites, Ser552 and Ser675, thus preventing its ubiquitination and thereby stabilizing β-catenin (Hino et al., 2005; Taurin et al., 2006). Second, PKA also phosphorylated PI3K, which in turn phosphorylated AKT, leading to GSK3β phosphorylation. GSK3β is a key enzyme that negatively regulates the canonical Wnt/β-catenin signaling pathway, and canonical Wnt signaling activation also requires the inhibition of GSK3β activity (Clevers and Nusse, 2012). The PKA signaling pathway can crosstalk with the PI3K/AKT pathway in endothelial cells (Namkoong et al., 2009), and the PI3K/AKT pathway can communicate with the GSK3β/β-catenin pathway in epithelial cells (Son et al., 2012). Phosphorylated GSK3β dephosphorylates β-catenin and prevents its degradation in the ubiquitin-dependent proteosome pathway. This event also helps to promote β-catenin translocation into the nucleus, leading to BMSC osteoblast differentiation. In summary, our results indicated that BMSC GLP-1R plays a crucial role in the regulation of osteoblast differentiation by interacting with the PKA/β-catenin and PKA/PI3K/AKT/GSK3β/β-catenin signaling pathways, both representing critical events in Ex-4-induced anabolic bone formation (Figure 7).
Experimental Procedures
Author Contributions
Introduction Cancer cells often exhibit similar properties to somatic stem/progenitor cells of the tissue of origin (Reya et al., 2001; Rossi and Weissman, 2006). Considering that progenitor cells at the developmental stage and somatic stem/progenitor cells in some adult tissues have the ability for self-renewal and/or active proliferation, it has been proposed that maintenance of the stem/progenitor cell state could be a driving force for tumor development (Reya et al., 2001). Osteosarcoma is a representative cancer that exhibits shared features with normal stem/progenitor cells (Luo et al., 2008; Thomas et al., 2004). The late markers of osteogenic differentiation are silenced while the early markers are modestly expressed in osteosarcomas (Luo et al., 2008; Thomas et al., 2004). Moreover, more aggressive phenotypes of osteosarcomas are correlated with features of early osteogenic progenitors (He et al., 2010; Luo et al., 2008), suggesting that defects in the osteogenic differentiation program may play a role in osteosarcoma development and progression. However, the causative aberrations that confer stem/progenitor cell properties on osteosarcoma cells are not fully understood.