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).