Synergistic effect of p38MAPK and ERK1/2 pathways on regulation of osteoblastic differentiation

Abstract

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 ERK 1/2 was measured. BMSCs were treated with PP2A inhibitor, Okadaic acid (OA), the phosphorylation of ERK1/2 and osteogenic differentiation were measured. lmmunoprecipitation 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 confirmed the association of PP2A with ERK1/2 in BMSCs cultures, which was decreased by SB203580 treatment. Conclusion The present study demonstrates a synergistic effect between p38MAPK and ERK1/2 signaling pathways via PP2A in BMSCs cultures, which may regulate the osteogenic differentiation of BMSCs. Key words: Extracellular signal-regulated MAP kinases; Osteoblasts; Cell differentiation; Phosphoprotein phosphatase