Genistein stimulates osteoblastic differentiation via p38 MAPK-Cbfa1 pathway in bone marrow culture
Abstract
Aim: To test the hypothesis that genistein stimulates the osteoblastic differentiation through the p38 mitogen activated protein kinase (MAPK)-core-binding factor 1 (Cbfa1) pathway.
Methods: The activation of p38 MAPK was detected by Western blotting. Alkaline phosphatase (ALP) activity and calcium deposition were assessed for osteoblastic differentiation of bone marrow-derived mesenchymal stem cell (BMSC) cultures. The expression of Cbfa1 was analyzed at both the mRNA and protein levels. The activity of Cbfa1 was detected by electrophoretic mobility shift assay. Bone sialoprotein (BSP), ALP, osteocalcin (OC), and osteopontin (OPN) gene transcription were also evaluated by either RT-PCR or Northern blotting.
Results: Genistein (0.01-1 mumol/L) dose dependently led to the rapid and sustained activation of the p38 MAPK pathway in mouse BMSC cultures. Treatment with genistein (1 mumol/L) resulted in increased ALP activity and calcium deposition of BMSC cultures as a function of time. Genistein also enhanced Cbfa1 DNA binding activity and promoted the expressions of Cbfa1 itself as well as several Cbfa1-regulated genes, including ALP, BSP, OC, and OPN. Concurrent treatment with p38 MAPK inhibitor (SB203580) diminished the genistein-induced osteoblastic maturation and p38 MAPK-Cbfa1 activation in mouse BMSC cultures.
Conclusion: These results indicated that genistein could stimulate the osteoblastic differentiation of BMSC cultures through the p38 MAPK-Cbfa1 pathway.
Keywords:
Methods: The activation of p38 MAPK was detected by Western blotting. Alkaline phosphatase (ALP) activity and calcium deposition were assessed for osteoblastic differentiation of bone marrow-derived mesenchymal stem cell (BMSC) cultures. The expression of Cbfa1 was analyzed at both the mRNA and protein levels. The activity of Cbfa1 was detected by electrophoretic mobility shift assay. Bone sialoprotein (BSP), ALP, osteocalcin (OC), and osteopontin (OPN) gene transcription were also evaluated by either RT-PCR or Northern blotting.
Results: Genistein (0.01-1 mumol/L) dose dependently led to the rapid and sustained activation of the p38 MAPK pathway in mouse BMSC cultures. Treatment with genistein (1 mumol/L) resulted in increased ALP activity and calcium deposition of BMSC cultures as a function of time. Genistein also enhanced Cbfa1 DNA binding activity and promoted the expressions of Cbfa1 itself as well as several Cbfa1-regulated genes, including ALP, BSP, OC, and OPN. Concurrent treatment with p38 MAPK inhibitor (SB203580) diminished the genistein-induced osteoblastic maturation and p38 MAPK-Cbfa1 activation in mouse BMSC cultures.
Conclusion: These results indicated that genistein could stimulate the osteoblastic differentiation of BMSC cultures through the p38 MAPK-Cbfa1 pathway.