Inhibition of thromboxane A synthesis in U937 cells by glucocorticoids. Lack of evidence for lipocortin 1 as the second messenger.
The mechanism of inhibition of eicosanoid synthesis by glucocorticoids has been investigated using differentiated U937 cells as a model. These cells synthesize thromboxane A2 (TXA2) in response to a variety of agonists, and synthesis of TXA2 initiated by certain stimuli was inhibited by pretreatment of the cells with glucocorticoids. The inhibitory response was specific for glucocorticoid steroids and required receptor occupancy based on both the rate of onset of the inhibitory activity and the correlation between potency and receptor affinity of various analogs. The inhibitory response was also specific for the agonist used to initiate TXA2 synthesis. Both lipopolysaccharide- and zymosan-induced TXA2 synthesis were inhibited by increasing concentrations of dexamethasone (greater than 80%, IC50 10 nM), while synthesis initiated by addition of either exogenous arachidonic acid or the Ca2+ ionophore A23187 was unaffected over the same concentration range. The latter result indicates that the dexamethasone block is upstream from release of esterified arachidonic acid. Attempts to localize the block more accurately showed that although dexamethasone was not acting as a generalized inhibitor of transcription or translation, its ability to inhibit TXA2 synthesis was mimicked by the activity of actinomycin D and cycloheximide. The role of the purported phospholipase inhibitor protein lipocortin 1 in mediating the dexamethasone inhibition of TXA2 synthesis was studied by examining the effect of dexamethasone on lipocortin 1 metabolism. Under conditions which gave maximal inhibition of lipopolysaccharide- or zymosan-stimulated TXA2 synthesis, dexamethasone had no effect on the steady state level of lipocortin 1 mRNA or protein, indicating that lipocortin 1 induction by dexamethasone is not responsible for the observed inhibition. Furthermore, lipocortin 1 was not secreted from the cells under any conditions examined, and the intracellular form had a relatively long half-life (greater than 21 h). The lack of induction of lipocortin 1 by dexamethasone and the fact that it is not released from the cells are both inconsistent with the properties previously described for lipocortin-like activities and indicate that lipocortin 1 is not a glucocorticoid second messenger in this experimental model. Although the data are consistent with a mechanism involving inhibition of a factor that activates TXA2 synthesis, we cannot rule out a mechanism involving glucocorticoid induction of a phospholipase inhibitor protein distinct from lipocortin 1.
Bienkowski
M J
MJ
Department of Cell Biology, Upjohn Company, Kalamazoo, Michigan 49007.
Petro
M A
MA
Robinson
L J
LJ
D000450:Aldosterone / Q000494:pharmacology; D017302:Annexins; D002454:Cell Differentiation / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005938:Glucocorticoids / Q000378:metabolism / Q000494:pharmacology*; D006023:Glycoproteins / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D013739:Testosterone / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013928:Thromboxane A2 / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured; D015054:Zymosan / Q000494:pharmacology
D017302:Annexins; D005938:Glucocorticoids; D006023:Glycoproteins; D008070:Lipopolysaccharides; D003609:Dactinomycin; D013739:Testosterone; D000450:Aldosterone; D004958:Estradiol; D013928:Thromboxane A2; D015054:Zymosan; D003513:Cycloheximide; D013755:Tetradecanoylphorbol Acetate