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.
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/EBPβ 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/EBPβ 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/EBPβ-dependent activation of the G-CSF promoter. This study provides a potential target for regulating G-CSF expression.
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