Fig. 1.
Dexamethasone increases the phosphorylation of Anx-A1 and PKC in U937 cells within 5 min in a concentration-dependent fashion. (Panel A) In U937 cells, 5 min treatment with dexamethasone in concentrations of 0.02–5.0 nM increases phosphorylation of Anx-A1 on Ser27 (upper panel) by ∼4-fold and, in parallel, PKCα/β phosphorylation (lower panel) by ∼3-fold compared to untreated controls. Whole U937 cell lysates were prepared as detailed. Anx-A1 phosphorylation was detected using a specific anti-Ser27 phospho Anx-A1 antibody as described. Total Anx-A1 (sometimes shown as a 33/37 kDa doublet) and PKC is shown for reference purposes. (Panel B) The dexamethasone (2 nM) effect on Anx-A1 phosphorylation is dependent on GC receptor occupation since the co-administration of 1 μM RU 486, blocks this effect. Note that the amount of phospho Anx-A1 is significantly less in samples treated with RU 486 alone than in vehicle treated samples, suggesting that some residual GCs may be present in the culture media. *P < 0.05 relative to control values; §§P < 0.01 relative to dexamethasone alone. (Panel C) The action of common kinase inhibitors on Anx-A1 phosphorylation. Of these, only PKC 19–31 (5 μM), an inhibitor of PKC, was able to reduce phosphorylation of Anx-A1 when this was stimulated by 2 nM dexamethasone (D). An inhibitor of PI3 kinase inhibitor LY294002 (LY: 10 nM) or a MAP kinase inhibitor PD98059 (PD: 5 μM) were inactive. Insert: a representative Western blot showing effect of the different treatments on Ser27 phospho Anx-A1. ***P < 0.001 relative to dexamethasone alone. (Panel D) Analysis of the PKC isoform activated by GCs. Blots were probed with anti-sera specific for each isoform. No signal was seen with antisera recognising PKC θ (not shown). A faint signal was detected for PKC δ Thr55 (78 kDa) but not for its activated form, Ser643 (not shown). In contrast, activated PKCα/β Thr638/641 (81 kDa), showed a strong signal in response to dexamethasone treatment at 15–30 min. All experiments were performed at least three times and the blots are representatives from one of these experiments. Densitometry was performed as described in the methods and the optical density units normalised by comparison to α-tubulin. Data are expressed as mean ± S.E.M. Statistical differences between groups were established using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test.