Naringin is a major flavonoid found in grapefruit and is an active compound extracted from the Chinese herbal medicine Rhizoma Drynariae. Naringin is a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of naringin involves the Notch signaling pathway. Rat bone marrow stromal cells (BMSCs) were cultured in osteogenic medium containing-naringin, with or without DAPT (an inhibitor of Notch signaling), the effects on ALP activity, calcium deposits, osteogenic genes (ALP, BSP, and cbfa1), adipogenic maker gene PPARγ2 levels, and Notch expression were examined. We found that naringin dose-dependently increased ALP activity and Alizarin red S staining, and treatment at the optimal concentration (50 μg/mL) increased mRNA levels of osteogenic genes and Notch1 expression, while decreasing PPARγ2 mRNA levels. Furthermore, treatment with DAPT partly reversed effects of naringin on BMSCs, as judged by decreases in naringin-induced ALP activity, calcium deposits, and osteogenic genes expression, as well as upregulation of PPARγ2 mRNA levels. These results suggest that the osteogenic effect of naringin partly involves the Notch signaling pathway.
Osteosarcoma is a high-grade bone sarcoma with strong invasive ability. However, treatment with traditional chemotherapeutic drugs is limited by low tolerability and side effects. Resveratrol has been reported previously to have selective antitumor effect on various tumor cells while little is known about its effects and underlying mechanism in osteosarcoma biology. In this study, we found that resveratrol inhibits proliferation and glycolysis, induces apoptosis and reduces the invasiveness of U2-OS cells in vitro. After treatment with resveratrol, the expression of related Wnt/β-catenin signaling pathway target genes, such as β-catenin, c-myc, cyclin D1, MMP-2 and MMP-9, was downregulated and an increased E-cadherin level was observed as well. Additionally, the dual luciferase assay results also indicated that resveratrol suppressed the activity of Wnt/β-catenin signaling pathway. Interestingly, we noticed that the expression of connexin 43 (Cx43) increased with the prolongation of resveratrol treatment time. To further investigate the relationship between Cx43 and the Wnt/β-catenin signaling pathway in osteosarcoma, we used lentiviral-mediated shRNA to knockdown the expression of Cx43. Knockdown of Cx43 activated the Wnt/β-catenin signaling pathway, promoted proliferation and invasion, and inhibited apoptosis of U2-OS cells. Taken together, our results demonstrate that the antitumor activity of resveratrol against U2-OS cells in vitro occurs through up-regulating Cx43 and E-cadherin, and suppressing the Wnt/β-catenin signaling pathway. Moreover, Cx43 expression is negatively related to the activity of the Wnt/β-catenin pathway in U2-OS cells.
Background/Aims: Bone marrow stromal cells (BMSCs) are multipotent precursors that give rise to osteoblasts, and contribute directly to bone formation. Connexin 43 (Cx43) is the most ubiquitous gap junction protein expressed in bone cell types, and plays crucial roles in regulating intercellular signal transmission for bone development, differentiation and pathology. However, the precise role and mechanism of Cx43 in BMSCs are less known. Here, we investigate the function of Cx43 in osteogenic differentiation of BMSCs in vitro. Methods: BMSCs were isolated by whole bone marrow adherent culture. Knock down of Cx43 was performed by using lentiviral transduction of Cx43 shRNA. BMSCs were induced to differentiate by culturing in a-MEM, 10% FBS, 50 µM ascorbic acid, 10 mM beta-glycerophosphate, and 100 nM dexamethasone. Alkaline phosphatase (ALP) activity and alizarin red S staining were used to evaluate osteogenic differentiation in calcium nodules. Target mRNAs and proteins were analyzed by using real-time quantitative PCR (qPCR) and western blotting. Results: Cx43 expression markedly increased during osteogenic differentiation. Osteogenic differentiation was suppressed following lentiviral-mediated knockdown of Cx43 expression, as judged by decreased levels of Runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), osteocalcin (Bglap), Osterix (Osx), alkaline phosphatase (ALP) activity and the number of calcium nodules in response to osteogenic differentiation stimuli. Knock down of Cx43 reduced the level of phosphorylation of GSK-3beta at Ser9 (p-GSK-3beta), resulting in decreased beta-catenin expression and activation. Furthermore, treatment of Cx43-knockdown cells with lithium chloride (LiCl), a GSK-3beta inhibitor, reduced osteogenic differentiation and decreased GSK-3beta levels, as well as partially rescued levels of both total and activated beta-catenin. Conclusion: These findings indicate that Cx43 positively modulates osteogenic differentiation of BMSCs by up-regulating GSK-3beta/beta-catenin signaling pathways, suggesting a potential role for Cx43 in determining bone mass and bone mineral density by modulating osteogenesis.
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