Regulation of prostaglandin H synthase mRNA levels and prostaglandin biosynthesis by platelet-derived growth factor.
Stimulation of serum-starved NIH-3T3 cells with 20 ng/ml recombinant platelet-derived growth factor BB (rPDGF) results in the synthesis of prostaglandin E2 (PGE2) that is detectable within 10 min and which peaks after 2 h. Inhibition of translation with 36 microM cycloheximide inhibits rPDGF-stimulated PGE2 synthesis (greater than 90%), suggesting that de novo synthesis of prostaglandin H synthase (PGHS) is required for growth factor stimulation of PGE2 synthesis. Paradoxically, the addition of 10 microM exogenous arachidonate to the serum-starved cells resulted in 2-fold more PGE2 synthesis, and maximal synthesis occurred at 30 min compared to 2 h following rPDGF treatment. These data suggest that the serum-starved cells have ample biosynthetic capacity to support maximal rPDGF-stimulated PGE2 synthesis without de novo enzyme synthesis. Kinetic analysis of PGHS mRNA levels showed that although rPDGF did elevate the steady-state level of PGHS mRNA, the increase occurred after maximal PGE2 synthesis was achieved. Quantification of PGHS levels by immunoblot showed that there was no significant increase in enzyme levels following rPDGF stimulation even under conditions where PGHS mRNA levels were elevated. Irreversible inactivation of PGHS by treatment with aspirin and subsequent stimulation with rPDGF synchronized the maximal elevation of PGHS mRNA levels and PGE2 synthesis; both peaked at 3 h. Interestingly, aspirin pretreatment increased both the rate and amplitude of rPDGF-stimulated PGHS mRNA induction. The enhanced induction was not simply the result of a loss in PGE2 production since treatment with exogenous PGE2 also induced PGHS mRNA levels. Since rPDGF-stimulated PGE2 synthesis is blocked by both transcription and translation inhibitors but arachidonate-stimulated PGE2 synthesis is unaffected, the requirement for protein synthesis does not appear to involve de novo PGHS synthesis. The synthesis of a protein required for coupling of the rPDGF receptor to phospholipase activation and arachidonic acid mobilization is a more likely interpretation. In summary, our data suggest that rPDGF can induce PGHS mRNA levels, but that rPDGF-stimulated PGE2 synthesis is not dependent upon de novo PGHS synthesis.
Lin
A H
AH
Department of Cell Biology, Upjohn Company, Kalamazoo, Michigan 49008.
Bienkowski
M J
MJ
Gorman
R R
RR
D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D001241:Aspirin / Q000494:pharmacology; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D015232:Dinoprostone / Q000096:biosynthesis*; D005786:Gene Expression Regulation / Q000502:physiology*; D007700:Kinetics; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics*; D011994:Recombinant Proteins
D001095:Arachidonic Acids; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D003513:Cycloheximide; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone; D001241:Aspirin