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Stimulation of prostaglandin e 2 production by interleukin‐1α and transforming growth factor α in osteoblastic MC3T3‐E1 cells
Author(s) -
Harrison J.R.,
Lorenzo J.A.,
Kawaguchi H.,
Raisz L.G.,
Pilbeam C.
Publication year - 1994
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650090607
Subject(s) - stimulation , transforming growth factor , prostaglandin e2 , medicine , endocrinology , interleukin , growth factor , production (economics) , prostaglandin , chemistry , microbiology and biotechnology , cytokine , biology , receptor , economics , macroeconomics
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 E 2 (PGE 2 ) 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 [ 32 P]labeled cDNA probes. By HPLC, PGE 2 was the major prostanoid produced under basal or stimulated conditions. No release of thromboxane or 6‐keto‐PGF 1α into the medium was detected. PGE 2 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 PGE 2 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 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 phospholipid stores.