ABSTRACT
Granulocyte colony-stimulating factor (G-CSF) selectively stimulates proliferation and differentiation of neutrophil progenitors which play important roles in host defense against infectious agents. However, persistent G-CSF production often leads to neutrophilia and excessive inflammatory reactions. There is therefore a need to understand the mechanism regulating G-CSF expression. In this study, we showed that U0126, a MEK1/2 inhibitor, decreases lipopolysaccharide (LPS)-stimulated G-CSF promoter activity, mRNA expression and protein secretion. Using short hairpin RNA knockdown, we demonstrated that ERK2, and not ERK1, involves in LPS-induced G-CSF expression, but not LPS-regulated expression of TNF-α. Reporter assays showed that ERK2 and C/EBPß synergistically activate G-CSF promoter activity. Further chromatin immunoprecipitation (ChIP) assays revealed that U0126 inhibits LPS-induced binding of NF-κB (p50/p65) and C/EBPß to the G-CSF promoter, but not their nuclear protein levels. Knockdown of ERK2 inhibits LPS-induced accessibility of the G-CSF promoter region to DNase I, suggesting that chromatin remodeling may occur. These findings clarify that ERK2, rather than ERK1, mediates LPS-induced G-CSF expression in macrophages by remodeling chromatin, and stimulates C/EBPß-dependent activation of the G-CSF promoter. This study provides a potential target for regulating G-CSF expression.
Subject(s)
Gene Expression , Granulocyte Colony-Stimulating Factor/genetics , Lipopolysaccharides/immunology , Macrophages/immunology , Macrophages/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Animals , Butadienes/pharmacology , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cell Line , Deoxyribonucleases/metabolism , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Knockdown Techniques , Macrophages/drug effects , Mice , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/genetics , NF-kappa B/metabolism , Nitriles/pharmacology , Promoter Regions, Genetic , Protein Binding , Protein Kinase Inhibitors/pharmacologyABSTRACT
This article reports an inhibitory effect of rapamycin on the lipopolysaccharide (LPS)-induced expression of both inducible nitric oxide synthase (iNOS) and granulocyte-colony stimulating factor (G-CSF) in macrophages and its underlying mechanism. The study arose from an observation that rapamycin inhibited the LPS-induced increase in octamer-binding factor-2 (Oct-2) protein levels through a mammalian target of rapamycin (mTOR)-dependent pathway in mouse RAW264.7 macrophages. As both iNOS and G-CSF are potential Oct-2 target genes, we tested the effect of rapamycin on their expression and found that it reduced the LPS-induced increase in iNOS and G-CSF mRNA levels and iNOS and G-CSF protein levels. Blocking of mTOR-signaling using a dominant-negative mTOR expression plasmid resulted in inhibition of the LPS-induced increase in iNOS and G-CSF protein levels, supporting the essential role of mTOR. Forced expression of Oct-2 using the pCG-Oct-2 plasmid overcame the inhibitory effect of rapamycin on the LPS-induced increase in iNOS and G-CSF mRNA levels. Chromatin immunoprecipitation assays showed that LPS enhanced the binding of Oct-2 to the iNOS and G-CSF promoters and that this effect was inhibited by pretreatment with rapamycin. Moreover, RNA interference knockdown of Oct-2 reduced iNOS and G-CSF expression in LPS-treated cells. The inhibitory effect of rapamycin on the LPS-induced increase in Oct-2 protein levels and on the iNOS and G-CSF mRNA levels was also detected in human THP-1 monocyte-derived macrophages. This study demonstrates that rapamycin reduces iNOS and G-CSF expression at the transcription level in LPS-treated macrophages by inhibiting Oct-2 expression.
