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 reg...

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Veröffentlicht in:The Journal of biological chemistry 1999-12, Vol.274 (53), p.37665-37672
Hauptverfasser: Matsumura, T, Sakai, M, Matsuda, K, Furukawa, N, Kaneko, K, Shichiri, M
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container_end_page 37672
container_issue 53
container_start_page 37665
container_title The Journal of biological chemistry
container_volume 274
creator Matsumura, T
Sakai, M
Matsuda, K
Furukawa, N
Kaneko, K
Shichiri, M
description 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-κB 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-κB binding site. Supershift assay showed that nuclear proteins bound to NF-κB 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-κB binding site from −91 to −82 was a negative responsive element in Ox-LDL-induced GM-CSF transcription.
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subjects Animals
Base Sequence
Cell Line
DNA - genetics
DNA Primers
Granulocyte-Macrophage Colony-Stimulating Factor - genetics
Humans
Lipoproteins, LDL - metabolism
Mice
Nuclear Proteins - metabolism
Promoter Regions, Genetic
Protein Binding
Protein Kinase C - metabolism
Reverse Transcriptase Polymerase Chain Reaction
RNA, Messenger - genetics
Transcription, Genetic
title Cis-acting DNA Elements of Mouse Granulocyte/Macrophage Colony-stimulating Factor Gene Responsive to Oxidized Low Density Lipoprotein
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