Research Article

Sustained Interleukin-1β Exposure Modulates Multiple Steps in Glucocorticoid Receptor Signaling, Promoting Split-Resistance to the Transactivation of Prominent Anti-Inflammatory Genes by Glucocorticoids

Figure 4

Sustained IL-1β conditions reduce the levels of DEX-activated cytoplasmic p- and nuclear p-. Cells were pretreated with vehicle or 5 ng/mL IL-1β for 16 h followed by treatment with either 10−8 M DEX (veh/DEX, (gray column)) or 5 ng/mL IL-1β + 10−8 M DEX (IL-1β/IL-1β + DEX, (white column)) for 0, 1, 2, or 4 h. Whole cell lysates were prepared, normalized for total protein concentration, and tested by Western blot with p--, p--, and total GR-specific antibodies. Anti-β-actin was used to confirm that protein load was normalized in each lane before relative quantitation. Blots representative of the p- (a) and p- (b) analyses (). Quantitation of whole cell p- (c), p- (d), and total GR (e) protein by densitometry. Data are represented as mean ± SEM. For each condition, bars represent fold-changes in p-, p-, and at 1, 2, or 4 h compared to veh/DEX-treated cells at 0 h. , differences between sustained IL-1β conditions (IL-1β/IL-1β + DEX, (white column)) and DEX alone-treated cells (veh/DEX, (gray column)). &, differences in p- induction between 2 h and 1 h post-DEX challenge under sustained IL-1β conditions (IL-1β/IL-1β + DEX, (white column)). (f) HCA microscopy analyses of nuclear p- content. Results are expressed as mean ± SEM fold-induction of nuclear p-Ser211-GR at 1, 2, or 4 h compared to veh/DEX-treated cells at 0 h (). IL-1β/IL-1β + DEX-treated versus veh/DEX-treated cells.