RESUMEN
BACKGROUND: Bone morphogenetic protein 9 (BMP9) and Wnt/ß-catenin signaling pathways are able to induce osteogenic differentiation of mesenchymal stem cells (MSCs), but the role of Wnt/ß-catenin signaling pathway in BMP9-induced osteogenic differentiation is not well understood. Thus, our experiment was undertaken to investigate the interaction between BMP9 and Wnt/ß-catenin pathway in inducing osteogenic differentiation of MSCs. METHODS: C3H10T1/2 cells were infected with recombinant adenovirus expressing BMP9, Wnt3a, and BMP9+Wnt3a. ALP, the early osteogenic marker, was detected by quantitative and staining assay. Later osteogenic marker, mineral calcium deposition, was determined by Alizarin Red S staining. The expression of osteopotin (OPN), osteocalcin (OC), and Runx2 was analyzed by Real time PCR and Western blotting. In vivo animal experiment was carried out to further confirm the role of Wnt3a in ectopic bone formation induced by BMP9. RESULTS: The results showed that Wnt3a enhanced the ALP activity induced by BMP9 and increased the expressions of OC and OPN, with increase of mineral calcium deposition in vitro and ectopic bone formation in vivo. Furthermore, we also found that Wnt3a increased the level of Runx2, an important nuclear transcription factor of BMP9. CONCLUSION: Canonical Wnt/ß-catenin signal pathway may play an important role in BMP9-induced osteogenic differentiation of MSCs, and Runx2 may be a linkage between the two signal pathways.
Asunto(s)
Diferenciación Celular/fisiología , Factor 2 de Diferenciación de Crecimiento/metabolismo , Osteogénesis/fisiología , Proteína Wnt3A/metabolismo , Western Blotting , Diferenciación Celular/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Factor 2 de Diferenciación de Crecimiento/genética , Humanos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis/genética , Proteína Wnt3A/genéticaRESUMEN
Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation of mesenchymal stem cells, the molecular mechanism involved remains to be fully elucidated. In this study, we showed that BMP9 simultaneously promotes the activation of Smad1/5/8, p38 and ERK1/2 in C3H10T1/2 cells. Knockdown of Smad4 with RNA interference reduced nuclear translocation of Smad1/5/8, and disrupted BMP9-induced osteogenic differentiation. BMP9-induced osteogenic differentiation was blocked by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. SB203580 decreased BMP9-activated Smads singling, and yet PD98059 stimulated Smads singling in C3H10T1/2 cells. The effects of inhibitor were reproduced with adenovirus expressing siRNA targeted p38 and ERK1/2, respectively. Taken together, our findings revealed that Smads, p38 and ERK1/2 are involved in BMP9-induced osteogenic differentiation. Also, it is noteworthy that p38 and ERK1/2 may play opposing regulatory roles in mediating BMP9-induced osteogenic differentiation of C3H10T1/2 cells.
