Cis-acting DNA elements of mouse granulocyte/macrophage colony-stimulating factor gene responsive to oxidized low density lipoprotein.
We previously demonstrated that the induction of granulocyte/macrophage colony-stimulating factor (GM-CSF) played an important role in oxidized low density lipoprotein (Ox-LDL)-induced macrophage growth as a growth priming factor. The present study was undertaken to elucidate the transcriptional regulation of the GM-CSF gene using Raw 264.7 cells, a mouse macrophage cell line. Transient transfection into Raw 264.7 cells of several 5'-flanking regions of GM-CSF gene-luciferase fusion plasmids revealed the presence of two positive regulatory sites in regions spanning from -97 to -59 and from -59 to -37 and one negative regulatory site from -120 to -97 in unstimulated cells. When cells were stimulated by Ox-LDL, there was one positive responsive site from -225 to -120 and one negative responsive site from -97 to -59, which contained the NF-kappaB binding site. Computer analysis revealed the presence of a putative AP-2 binding site from -169 to -160. Mutagenesis of a putative AP-2 binding site and tandem repeat of this site in plasmid resulted in a complete loss and increased responsiveness to Ox-LDL, respectively. Electrophoretic mobility shift assay showed that Ox-LDL increased the binding of certain nuclear protein(s) to a putative AP-2 binding site but decreased their binding to NF-kappaB binding site. Supershift assay showed that nuclear proteins bound to NF-kappaB binding site contained, at least, p50 and p65 but could not demonstrate nuclear protein(s) bound to a putative AP-2 binding site. Our results suggested that a putative AP-2 binding site from -169 to -160 was a positive responsive element to Ox-LDL and that the NF-kappaB binding site from -91 to -82 was a negative responsive element in Ox-LDL-induced GM-CSF transcription.
Matsumura
T
T
Department of Metabolic Medicine, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan.
Sakai
M
M
Matsuda
K
K
Furukawa
N
N
Kaneko
K
K
Shichiri
M
M
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D017931:DNA Primers; D008077:Lipoproteins, LDL; D009687:Nuclear Proteins; D012333:RNA, Messenger; C099252:oxidized low density lipoprotein; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D004247:DNA; D011493:Protein Kinase C