Long-term effects of prostaglandin E2 on the mineralization of a clonal osteoblastic cell line (MC3T3-E1)

Prostaglandin (PG) E-2 is thought to be a mediator of the effect of mechanical stress on bone formation, but its effects on osteoblasts have not yet been fully described. Here, the effects of the continuous application of PGE(2) and indomethacin, an inhibitor of prostaglandin G/H synthase (cyclo-oxygenase), on the proliferation, differentiation and mineralization of a clonal osteoblastic cell line, MC3T3-E1, were investigated. The cells were cultured in media with either a high (1 mu g/ml) or a low (1 ng/ml) concentration of PGE(2), with indomethacin (1 mu g/ml) and, as a control, with neither agent. The effects of PGE(2) and indomethacin were assessed quantitatively. Indomethacin and a high concentration of PGE(2) increased the total protein compared to the control and low-PGE(2) cultures. 7 days after confluence, alkaline phosphatase (ALP) activity within the cells and extracellular matrices increased. This increase was highest with indomethacin and lowest with a high concentration of PGE(2). ALP activity also increased in the medium, but only 21 days after confluence; the effects of the agents were similar to those on the cells and matrices. The accumulation of calcium, inorganic phosphate and hydroxyproline was highest with indomethacin. PGE(2) production was at its maximum when the cells were at confluence and was inhibited by indomethacin. Specific [H-3]PGE(2) binding to the microsomal fraction of the cell was also measured to examine the expression of the PGE(2) receptor. The amount of [H-3]PGE(2) binding per mg of protein was highest at confluence, then decreased and again increased in the mineralizing stage. These results suggest that indomethacin increases ALP activity and the accumulation of mineralized tissue in MC3T3-E1 cells, presumably by inhibiting the production of PGE(2). PGE(2) could signal the suppression of mineralization as early as confluence. (C) 1999 Elsevier Science Ltd. All rights reserved.