The cell biology of fibroblast cyclooxygenase.
We have prepared polyclonal antisera against sheep seminal vesicles cyclooxygenase (COX) which cross-reacted with human COX. We employed this antisera in studies with human dermal fibroblast cultures to immunoprecipitate selectively the COX enzyme. Labeling of the cells with [35S]-methionine, solubilization of cellular COX followed by its immunoprecipitation, SDS-PAGE electrophoresis and fluorography enabled us to determine directly the synthetic rate of COX protein and its modulation by the monokine interleukin-1 (IL-1). The immunoprecipitated [35S]-labeled COX, as judged from SDS-PAGE electrophoresis, has a molecular size of approximately 73,000 daltons, similar to that of native sheep COX and [3H]-acetyl COX. IL-1 stimulation of enhanced COX synthesis was time and dose dependent; as little as 0.03 units/ml of IL-1 produced significant stimulation of [35S]-labeled COX synthesis. Maximum stimulation was 3-10-fold after preincubation of the cells with IL-1 for 12-16 hours. IL-1 treatment of cells in serum-free media yielded parallel dose response curves for stimulation of PGE2 formation, cellular solubilized COX activity and synthesis of newly formed COX, suggesting that this IL-1 effect is mediated solely via induction of new COX protein synthesis. In contrast, IL-1 effect on cells incubated in the presence of fetal calf serum is more complex. Serum synergistically augments the IL-1 effect on PGE2 synthesis in intact cells but concurrently blunts IL-1 induction of COX synthesis, thus suggesting that a factor (or factors) in serum may stimulate PGE2 production by activating cellular phospholipase(s).
Raz
A
A
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Wyche
A
A
Fagan
D
D
Needleman
P
P
D000818:Animals; D001769:Blood; D001920:Bradykinin / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004731:Endotoxins / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D007106:Immune Sera; D007375:Interleukin-1 / Q000494:pharmacology; D009000:Monocytes / Q000378:metabolism; D011233:Precipitin Tests; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D012756:Sheep; D013997:Time Factors
D004731:Endotoxins; D007106:Immune Sera; D007375:Interleukin-1; D011451:Prostaglandin-Endoperoxide Synthases; D001920:Bradykinin