Regulation of fibroblast cyclooxygenase synthesis by interleukin-1

We have prepared polyclonal antiserum against sheep seminal vesicle prostaglandin H synthase (also termed cyclooxygenase) which cross-reacted with human cyclooxygenase, thereby enabling us to directly determine the synthetic rate of cyclooxygenase protein and its modulation by the monokine interleukin-1 (IL-1). Cultured human dermal fibroblast cells were labeled with [³⁵S]methionine, and the membrane-bound cyclooxygenase was solubilized and immunoprecipitated with the polyclonal antibody. The immunoprecipitated ³⁵S-labeled fibroblast cyclooxygenase migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular size of approximately 73,000 daltons, similar to that of native sheep cyclooxygenase and of cyclooxygenase covalently labeled by [³H]aspirin, i.e. [³H]acetylcyclooxygenase. Additional validation of the immunoprecipitated ³⁵S-labeled cyclooxygenase band indicated that it was specifically displaced by unlabeled sheep cyclooxygenase. N-terminal amino acid radiosequence analysis of [³H]proline-labeled cyclooxygenase revealed [³H]proline residues in positions 3, 6, and 8, consistent with the previously reported N-terminal sequence of sheep cyclooxygenase. Endoglycosidase H treatment of ³⁵S-labeled fibroblast cyclooxygenase caused a decline in apparent molecular size (due to removal of mannose residues) which was similar to that seen with the native sheep cyclooxygenase. [³⁵S]Methionine pulse-chase experiments indicated a half-life of 1 h for fibroblast cyclooxygenase. The monokine interleukin-1 stimulated fibroblast cyclooxygenase synthesis in a time- and dose-dependent fashion; as little as 0.03 unit/ml of IL-1 produced significant stimulation of ³⁵S-labeled cyclooxygenase synthesis. Maximum stimulation was 3-10-fold after preincubation of the cells with 0.3 unit/ml of IL-1 for 12-6 h. IL-1 treatment of cells yielded parallel dose-response curves for stimulation of prostaglandin E₂ formation, increased cellular cyclooxygenase activity, and increased synthetic rate of newly formed cyclooxygenase, suggesting that the IL-1 effect is mediated mainly, if not solely, via induction of cyclooxygenase synthesis.