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, and produces prostaglandin E2. When the cells were cultured in the presence of iloprost (a stable analogue of prostacyclin) or prostaglandin E1 or F2 alpha, the activity of cyclooxygenase increased in a dose- and time-dependent manner. The increase of the enzyme activity was attributed mostly to the cyclooxygenase isoform-2 because immunoprecipitation 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 addition, there was a marked increase in the cyclooxygenase-2 protein which was demonstrated by Western blotting. As analyzed by Northern blotting, the cyclooxygenase-2 mRNA increased and reached a maximum 1 and 3 h after the addition of iloprost and prostaglandin F2 alpha (about 15- and 60-fold increase), respectively, whereas the cyclooxygenase-1 mRNA increased slowly and only by about 3-fold. Iloprost and prostaglandin E1 stimulated the production of cAMP by 60-fold over the basal level, whereas the cAMP level was almost unchanged by prostaglandin F2 alpha. In contrast, prostaglandin F2 alpha stimulated IP3 production more efficiently than iloprost and prostaglandin E1. These results suggest that the stimulated syntheses prominently of cyclooxygenase-2 and to a lesser extent of cyclooxygenase-1 are mediated by at least two distinct signal transduction pathways involving the cAMP-synthesis stimulated by iloprost and prostaglandin E1 and the phosphoinositide turnover stimulated by prostaglandin F2 alpha.