摘要
目的探讨骨髓间质干细胞（bone marrow mesenchymal stem cells，BMSCs）向成骨细胞分化过程中p38MAPK与ERK1／2的协同效应及其机制。方法以成骨细胞分化添加剂诱导小鼠BMSCs向成骨细胞分化，测定碱性磷酸酶活性和钙沉积量。检测磷酸化p38MAPK和磷酸化ERKI／2（p—ERK1／2）的表达水平评估通路的激活状况。以SB203580或PD98059阻断p38MAPK或ERK1／2通路，观察对成骨细胞分化的影响。以SB203580或亚砷酸钠阻断或激活p38MAPK通路，观察p-ERK1／2的变化。以冈田酸抑制蛋白磷酸酯酶2A（protein phosphatases type2A，PP2A）活性，观察p-ERK1／2的变化及对成骨细胞分化的影响。通过免疫共沉淀实验观察PP2A和ERK1／2间的结合及SB203580对结合的影响。结果成骨细胞分化添加剂诱导BMSCs向成骨细胞分化的过程伴有ERK1／2和p38MAPK通路的激活，SB203580剂量依赖性抑制成骨细胞分化，PD98059剂量依赖性增强成骨细胞分化。SB203580使p-ERK1／2表达增加，亚砷酸钠减弱其表达。冈田酸使p-ERK1／2表达增加，并使成骨细胞分化受到抑制。PP2A可直接与ERK1／2结合，SB203580使PP2A与ERK1／2的结合减弱。结论p38MAPK可通过PP2A与ERK1／2产生协同效应，并调节BMSCs向成骨细胞分化。
Objective To study the synergistic effect of p38MAPK and ERK1/2 in bone marrow mesenchymal stem cells （BMSCs）, and to explore their influence on osteogenic differentiation in BMSCs cultures. Methods Mouse BMSCs were cultured in phenol red-free α-MEM containing osteogenic supplements （OS） for inducing osteogenic differentiation. The temporal sequence of osteogenic differentiation in BMSCs cultures was assayed by measuring alkaline phosphatase activity （ALP） and calcium deposition gene expression. The activation of p38MAPK and ERK1/2 was detected by western blotting using phospho-specific MAP kinase antibody. BMSCs were treated with the inhibitor of p38MAPK pathway （SB203580） or ERK1/2 pathway （PD98059）, and osteogenic differentiation was measured. BMSCs were treated with SB203580 or sodium arsenite （ARS）, a strong activator of p38MAPK, and the phosphorylation of ERK1/2 was measured. BMSCs were treated with PP2A inhibitor, Okadaic acid （OA）, the phosphorylation of ERK1/2 and osteogenic differ- entiation were measured. Immunoprecipitation was used to test the binding interaction between PP2A and ERK1/2, and the effect of SB203580 on the interaction. Results Treatment of BMSCs with osteogenic supplements resulted in activation of p38MAPK and ERK1/2 that coincided with osteogenic differentiation. Inhibition of p38MAPK activation by SB203580, blocked the osteogenic differentiation, whereas inhibition of ERK1/2 activation by PD98059, enhanced the osteogenic differentiation in a dose-dependent manner. SB203580 treatment resulted in increased ERK1/2 phosphorylation. By contrast, ARS treatment resulted in decreased ERK1/2 phosphorylation. Inhibition of PP2A by OA resulted in increased ERK1/2 phosphorylation. OS-induced osteogenic differentiation was also attenuated by PP2A inhibition. Immunoprecipitation con- firmed the association of PP2A with ERK1/2 in BMSCs cultures, which was decreased by SB203580 treat- ment. Conclusion The present study demonstrates a synergistic effect between p38MAPK an
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2011年第9期970-975,共6页
Chinese Journal of Orthopaedics
关键词
细胞外信号调节MAP激酶类
成骨细胞
细胞分化
磷蛋白磷酸酶
Extracellular signal-regulated MAP kinases
Osteoblasts
Cell differentiation
Phosphoprotein phosphatase
