Berberine promotes bone marrow-derived mesenchymal stem cells osteogenic differentiation via canonical Wnt/β-catenin signaling pathway

• BBR alone can enhance the osteogenic differentiation of MSCs in a dose-dependent model.
• A sequential combination of BBR and DKK-1 or β-catenin specific siRNA transfection administration inhibits the expression of osteogenic transcriptional factors such as OPN and OCN, and Wnt/β-catenin signaling core factor β-catenin.
• BBR administration triggers osteogenic differentiation of MSCs in vitro though canonical Wnt/β-catenin signaling.

Berberine (BBR) has recently been reported to be extensively used for musculoskeletal disorders such as osteoporosis through enhancing osteogenic differentiation, inhibiting osteoclastogenesis and bone resorption and repressing adipogenesis. Although canonical Wnt signaling plays a crucial role in suppressing bone marrow-derived mesenchymal stem cells (MSCs) commitment to the chondrogenic and adipogenic lineage and enhancing osteogenic differentiation, no previous reports have shown an association between BBR-induced osteogenesis and Wnt/β-catenin signaling pathway. In this study, we aimed to investigate the stimulatory effect and the mechanism of BBR on osteogenic differentiation of human bone marrow-derived MSCs. MSCs were isolated from bone marrow specimens and treated with different concentration of BBR. Cell viability was measured by the WST-8 assay. Effects of BBR on osteogenic differentiation of MSCs were assessed by von Kossa staining, ALP staining and ALP activity. Osteogenic specific genes, chondrogenic and adipogenic related marker genes were determined by quantitative real-time polymerase chain reaction analysis. Western blot and Immunofluorescence staining were performed to analyze OCN and OPN, and β-catenin expression in the presence or absence of BBR combined with DKK-1 or β-catenin siRNA transfection. Increasing concentration of BBR (3, 10 and 30 μM) promoted osteogenic differentiation and osteogenic genes expression after incubation for various days compared with DMSO group, whereas expression levels of chondrogenic and adipogenic related marker genes were dramatically suppressed. After treated with 10 μM BBR for 7 days, β-catenin, OPN and OCN expression were significantly induced, which could be effectively suppressed by the addition of DKK-1 or β-catenin siRNA β-catenin. Interestingly, the expression level of Runx2 gene was also decreased by inhibiting the transduction of Wnt/β-catenin signaling. These findings suggest that BBR can stimulate osteogenic differentiation of MSCs not only by enhancing Runx2 expression but also by activating canonical Wnt/β-catenin signaling pathway, and canonical Wnt/β-catenin signaling pathway is in part responsible for BBR-induced osteogenic differentiation of MSCs in vitro. BBR is a potential pharmaceutical medicine by enhancing osteogenic differentiation for bone disorders, such as osteoporosis.

Illustration of BBR-induced osteogenic differentiation of MSCs. In MSCs, BBR promotes activation of the canonical Wnt pathway resulting in translocation o f β-catenin into the nucleus to form a complex with TCF1 on the Runx2 promoter for its induction, subsequently upregulate Runx2 target genes expression, such as OPN and OCN, and consequently stimulates osteogenesis. Effects of BBR on osteogenesis are potently repressed by specific Wnt signaling specific inhibitor DKK-1 or β-catenin siRNA.Figure optionsDownload as PowerPoint slide