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 3

IL-1β alone reduces cytoplasmic GR protein levels without promoting GR nuclear translocation. Cells were pretreated at −32 h and −16 h with vehicle, 5 or 25 ng/mL IL-1β, and then treated the last 2 h with either vehicle or 10⁻⁸ M DEX alone, as indicated in the table: 1, 32 h in vehicle; 2, 16 h veh/16 h 5 ng/mL IL-1β; 3, 16 h veh/16 h 25 ng/mL IL-1β; 4, 32 h 5 ng/mL IL-1β; 5, 32 h 25 ng/mL IL-1β; and 6, 30 h veh/2 h 10⁻⁸ M DEX. The nuclear and cytoplasmic compartments were then fractionated and their lysates studied by Western blot with anti-GR antibody. Anti-α-tubulin (α-tub) and -p53 antibodies are controls for the correct cytoplasmic (a) and nuclear (b) compartment fractionation, respectively, in the representative blots (). Quantitation of cytoplasmic (c) and nuclear (d) GR protein by densitometry. Data are represented as mean ± SEM. ^#, differences in cytoplasmic GR protein content between cells treated with 16 h veh/16 h 5 ng/mL IL-1β (c2), 16 h veh/16 h 25 ng/mL IL-1β (c3), 32 h 5 ng/mL IL-1β (c4), 32 h 25 ng/mL IL-1β (c5), or 30 h veh/2 h DEX (c6), compared with 32 h vehicle-treated cells (c1). , difference in cytoplasmic GR protein levels between cells treated with 5 ng/mL (c2) and 25 ng/mL (c3) IL-1β for the last 16 h. ^#, difference in nuclear GR protein levels between 30 h veh/2 h DEX-treated (d6) and veh-treated cells (d1).

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