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J Biol Chem, Vol. 274, Issue 53, 37665-37672, December 31, 1999

Cis-acting DNA Elements of Mouse Granulocyte/Macrophage Colony-stimulating Factor Gene Responsive to Oxidized Low Density Lipoprotein*

Takeshi Matsumura, Masakazu SakaiDagger , Kohji Matsuda, Noboru Furukawa, Kengo Kaneko, and Motoaki Shichiri

From the Department of Metabolic Medicine, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan

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-kappa B 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-kappa B binding site. Supershift assay showed that nuclear proteins bound to NF-kappa B 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-kappa B binding site from -91 to -82 was a negative responsive element in Ox-LDL-induced GM-CSF transcription.

* This work was supported in part by a grant for scientific research from the Ono Memorial Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Metabolic Medicine, Kumamoto University School of Medicine, 1-1-1, Honjo, Kumamoto, 860-5886, Japan. Tel.: 81-96-373-5169; Fax: 81-96-366-8397; E-mail: osakai@gpo.kumamoto-u.ac.jp.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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