STUDIES ON THE INDUCTION OF CYCLOOXYGENASE ISOZYMES BY VARIOUS PROSTAGLANDINS IN MOUSE OSTEOBLASTIC CELL-LINE WITH REFERENCE TO SIGNAL-TRANSDUCTION PATHWAYS

A mouse osteoblastic cell line MC3T3-E1 has a cyclooxygenase enzyme, a nd produces prostaglandin E(2). When the cells were cultured in the pr esence of iloprost (a stable analogue of prostacyclin) or prostaglandi n E(1) or F-2a, the activity of cyclooxygenase increased in a dose- an d time-dependent manner. The increase of the enzyme activity was attri buted mostly to the cyclooxygenase isoform-2 because immunoprecipitati on using an anti-cyclooxygenase-2 antibody removed the majority of the cyclooxygenase activity from the solubilized enzyme fraction, and the corresponding activity was detected in the immunoprecipitant. In addi tion, there was a marked increase in the cyclooxygenase-2 protein whic h was demonstrated by Western blotting. As analyzed by Northern blotti ng, the cyclooxygenase-2 mRNA increased and reached a maximum 1 and 3 h after the addition of iloprost and prostaglandin F-2a (about 15- and 60-fold increase), respectively, whereas the cyclooxygenase-1 mRNA in creased slowly and only by about 3-fold. Iloprost and prostaglandin E( 1) stimulated the production of cAMP by 60-fold over the basal level, whereas the cAMP level was almost unchanged by prostaglandin F-2a In c ontrast, prostaglandin F-2a stimulated IP3 production more efficiently than iloprost and prostaglandin E(1). These results suggest that the stimulated syntheses prominently of cyclooxygenase-2 and to a lesser e xtent of cyclooxygenase-1 are mediated by at least two distinct signal transduction pathways involving the cAMP-synthesis stimulated by ilop rost and prostaglandin E(1) and the phosphoinositide turnover stimulat ed by prostaglandin F-2a.