Stimulation of prostaglandin e2 production by interleukin‐1α and transforming growth factor α in osteoblastic MC3T3‐E1 cells

The mechanism by which interleukin-1 (IL-1) and transforming growth factor α (TGF-α) regulate prostaglandin synthesis has been examined in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were grown in DMEM containing 10% fetal calf serum. Prostaglandin E2 (PGE2) production was determined by radioimmunoassay or by prelabeling cells with [H]arachidonic acid, followed by high-performance liquid chromatography (HPLC) analysis of the labeled products released into the medium. Prostaglandin G/H synthase (PGHS) mRNAs were quantified by northern blot analysis using [32P]labeled cDNA probes. By HPLC, PGE2 was the major prostanoid produced under basal or stimulated conditions. No release of thromboxane or 6-keto-PGF1α into the medium was detected. PGE2 production was stimulated approximately 7- to 14-fold by IL-1 (1 ng/ml) and 3- to 8-fold by TGF-α (30 ng/ml) after 24 h. In combination, however, IL-1 and TGF-α caused a synergistic 37- to 71-fold increase in PGE2 accumulation. PGHS-1 mRNA levels were maximally increased approximately 2- to 3-fold by IL-1 and 1.5 to 2.5-fold by TGF-α after 24 h; the combination of IL-1 and TGF-α produced only an additive 3- to 6-fold increase. Western blotting revealed a corresponding 3-fold increase in immunoreactive PGHS-1 protein in response to combined IL-1 and TGF-α. PGHS-2 mRNA was increased 1.4-fold by TGF-α at 1 h, and the combination of IL-1 and TGF-α caused a 1.7-fold increase. After 3.5 h, IL-1 caused a dramatic induction of PGHS-2 mRNA levels but TGF-α alone no longer had an effect. However, the combination of IL-1 and TGF-α produced an increase in PGHS-2 mRNA levels that was twice that of IL-1 alone. The effects of IL-1 and TGF-α on the release of preincorporated [H]arachidonic acid from membrane phospholipid stores were examined at early time points in the presence of indomethacin. After 1 h, arachidonic acid release was enhanced 3-fold by IL-1, 1.5-fold by TGF-α, and 12-fold by IL-1 and TGF-α in combination. In conclusion, the synergistic actions of IL-1 and TGF-α on PGE2 synthesis in MC3T3-E1 cells involve multiple regulatory sites, including stimulation of de novo PGHS-1 and PGHS-2 synthesis and an early mobilization of arachidonic acid from phospholipid stores.