Glucocorticoids inhibit IL-1beta-induced GM-CSF expression at multiple levels: roles for the ERK pathway and repression by MKP-1.
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.
Newton
Robert
R
Airways Inflammation Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N4N1. rnewton@ucalgary.ca
King
Elizabeth M
EM
Gong
Wei
W
Rider
Christopher F
CF
Staples
Karl J
KJ
Holden
Neil S
NS
Bergmann
Martin W
MW
D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology*; D054638:Dual Specificity Phosphatase 1 / Q000096:biosynthesis* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004794:Enzyme Repression; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D053583:Interleukin-1beta / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000893:Anti-Inflammatory Agents; D004791:Enzyme Inhibitors; D053583:Interleukin-1beta; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D003609:Dactinomycin; D003907:Dexamethasone; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D003513:Cycloheximide; D048049:Extracellular Signal-Regulated MAP Kinases; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1