Inhibition of phosphatidylinositol 3-kinase and p70S6 kinase blocks osteogenic protein-1 induction of alkaline phosphatase activity in fetal rat calvaria cells

Published studies reveal that Osteogenic Protein-1 (OP-1) and insulin-like growth factor-I (IGF-I) synergistically stimulate alkaline phosphatase (AP) activity and bone nodule formation in fetal rat calvaria (FRC) cells. In the present study, we examined whether there are interactions between the signal transduction pathways activated by these two growth factors. OP-1 did not significantly affect the levels of IRS-1, IRS-2, the p85α subunit of phosphatidylinositol 3-kinase (PI 3-kinase) or the extracellular signal-regulated kinase (ERK)-2, but stimulated ERK-1 protein by twofold. OP-1 also induced phosphorylation of ERK-1 and -2, but not of Akt/protein kinase B (PKB), a protein kinase that is downstream of PI 3-kinase. By comparison, IGF-I increased the levels of the phosphorylated forms of ERK-1 and -2, and Akt/PKB. Inhibition of ERK activation by PD98059 did not significantly alter the stimulation of AP activity by OP-1 or OP-1 in combination with IGF-I. In contrast, inhibition of PI 3-kinase activity by LY294002 blocked the induction of AP activity by OP-1 and OP-1 plus IGF-I. Treatment of cells with rapamycin, an inhibitor of the mammalian target of mTOR, resulted in a 47% and a 53% decrease in the AP activity induced by OP-1 alone and by OP-1 plus IGF-I, respectively. These studies suggest that PI 3-kinase and mTOR contribute to the induction of AP activity by OP-1 and the synergistic effect of OP-1 and IGF-I on AP activity in FRC cells.