Abstract
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.
Highlights
Atherosclerosis is an inflammatory fibroproliferative process involving a complex set of interconnected events, including endothelial cell injury; smooth muscle cell migration and phenotypic changes; accumulation of monocytes, macrophages, and T lymphocytes; and formation of lipid-laden foam cells [1]
To confirm whether Raw 264.7 cells respond to oxidized low density lipoprotein (Ox-LDL), we investigated the Ox-LDL-induced granulocyte/macrophage colony-stimulating factor (GM-colony-stimulating factor (CSF)) production in Raw 264.7 cells at protein and mRNA levels by using Enzyme-linked Immunosorbent Assay (ELISA) and RT-PCR, respectively
RT-PCR analysis showed that GM-CSF mRNA was increased by Ox-LDL with the peak level occurring at 3 h (Fig. 1B), whereas both LDL and acetyl-LDL had no effect on GMCSF mRNA expression in these cells
Summary
Atherosclerosis is an inflammatory fibroproliferative process involving a complex set of interconnected events, including endothelial cell injury; smooth muscle cell migration and phenotypic changes; accumulation of monocytes, macrophages, and T lymphocytes; and formation of lipid-laden foam cells [1]. It is reasonable to speculate that induction of GM-CSF by Ox-LDL is regulated by the activation of certain cis-acting DNA element(s) and nuclear transcription factor(s) after PKC activation in macrophages.
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