Glucocorticoids inhibit IL-1beta-induced GM-CSF expression at multiple levels: roles for the ERK pathway and repression by MKP-1

Abstract

In the present study, IL (interleukin)-1beta increased GM-CSF (granulocyte/macrophage colony-stimulating factor) expression from pulmonary A549 cells and primary HBE (human bronchial epithelial) cells. These responses were repressed by the glucocorticoid dexamethasone, allowing the use of A549 cells as a relevant model. IL-1beta induced GM-CSF release into the culture medium by 6 h and in cell lysates (cytosolic) at 2 h. These effects were profoundly inhibited by dexamethasone, yet IL-1beta-induced GM-CSF mRNA and unspliced nRNA (nuclear RNA; a surrogate of transcription rate) were modestly inhibited by dexamethasone at times up to 2 h. Although this indicates an effect on protein synthesis, actinomycin D chase experiments also indicated post-transcriptional repression by dexamethasone. Dexamethasone-dependent mRNA repression increased with time and was prevented by translational blockade. In addition, dexamethasone and the dissociated steroid RU24858 repressed GM-CSF release in an actinomycin D-sensitive manner, thereby implicating glucocorticoid-induced gene expression. At 2 h, IL-1beta-induced expression of GM-CSF protein, but not mRNA, was sensitive to the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] inhibitors PD098059 and U0126. Although this indicates a role for the MEK/ERK pathway in GM-CSF translation, PD098059 subsequently destabilized GM-CSF mRNA. Dexamethasone and RU24858 both reduced IL-1beta-induced ERK phosphorylation and increased MKP-1 (MAPK phosphatase-1) expression. Inhibition of ERK phosphorylation was reproduced by MKP-1 overexpression and prevented by MKP-1-targeting siRNA (small interfering RNA). Since MKP-1 prevented GM-CSF expression by transcriptional, post-transcriptional and translational processes, we propose that glucocorticoids induce MKP-1 expression to reduce both MEK/ERK activation and GM-CSF protein synthesis. Thus de novo gene expression, particularly of MKP-1, is involved in the repressive effects of glucocorticoids.