STIMULATION OF PROSTAGLANDIN E(2) PRODUCTION BY INTERLEUKIN-1-ALPHA AND TRANSFORMING GROWTH-FACTOR-ALPHA IN OSTEOBLASTIC MC3T3-E1 CELLS

The mechanism by which interleukin-1 (IL-1) and transforming growth fa ctor alpha (TGF-alpha) regulate prostaglandin synthesis has been exami ned in the clonal mouse osteoblastic cell line MC3T3-E1. Cells were gr own in DMEM containing 10% fetal calf serum. Prostaglandin E(2) (PGE(2 )) production was determined by radioimmunoassay or by prelabeling cel ls with [H-3]arachidonic acid, followed by high-performance liquid chr omatography (HPLC) analysis of the labeled products released into the medium. Prostaglandin G/H synthase (PGHS) mRNAs were quantified by nor thern blot analysis using [P-32]labeled cDNA probes. By HPLC, PGE, was the major prostanoid produced under basal or stimulated conditions. N o release of thromboxane or 6-keto-PGF(1 alpha) into the medium was de tected. PGE(2) production was stimulated approximately 7- to 14-fold b y IL-1 (1 ng/ml) and 3- to 8-fold by TGF-alpha (30 ng/ml) after 24 h. In combination, however, IL-1 and TGF-alpha caused a synergistic 37- t o 71-fold increase in PGE(2) accumulation. PGHS-1 mRNA levels were max imally increased approximately 2- to 3-fold by IL-1 and 1.5 to 2.5-fol d by TGF-alpha after 24 h; the combination of IL-1 and TGF-alpha produ ced only an additive 3- to 6-fold increase. Western blotting revealed a corresponding 3-fold increase in immunoreactive PGHS-1 protein in re sponse to combined IL-1 and TGF-alpha. PGHS-2 mRNA was increased 1.4-f old by TGF-alpha at 1 h, and the combination of IL-1 and TGF-alpha cau sed a 1.7-fold increase. After 3.5 h, IL-1 caused a dramatic induction of PGHS-2 mRNA levels but TGF-alpha alone no longer had an effect. Ho wever, the combination of IL-1 and TGF-alpha produced an increase in P GHS-2 mRNA levels that was twice that of IL-1 alone. The effects of IL -1 and TGF-alpha on the release of preincorporated [H-3]arachidonic ac id from membrane phospholipid stores were examined at early time point s in the presence of indomethacin. After 1 h, arachidonic acid release was enhanced 3-fold by IL-1, 1.5-fold by TGF-alpha, and 12-fold by IL -1 and TGF-alpha in combination. In conclusion, the synergistic action s of IL-1 and TGF-alpha on PGE(2) 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 p hospholipid stores.