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-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. 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. colony-stimulating factor granulocyte/macrophage colony-stimulating factor oxidized low density lipoprotein protein kinase C electrophoretic mobility shift assay enzyme-linked immunosorbent assay polymerase chain reaction reverse transcriptase-PCR 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 (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9989) Google Scholar). In the early stages of atherosclerotic lesions, monocytes/macrophages are the major source of foam cells, which play an essential role in the development and progression of atherosclerotic lesions via production of various active molecules (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9989) Google Scholar). Thus, to elucidate the pathogenesis of atherosclerosis, it seems reasonable to investigate the mechanisms of macrophage activation, proliferation, and survival processes that are regulated by the colony-stimulating factor (CSF)1 family, such as macrophage colony-stimulating factor (2Stanley E.R. Cifone M. Heard P.M. Defendi V. J. Exp. Med. 1976; 143: 631-647Crossref PubMed Scopus (152) Google Scholar, 3Stewart C.C. Lin H.-S. J. Reticuloendothel. Soc. 1978; 23: 269-285PubMed Google Scholar), interleukin-3 (4Chen B.D. Clark C.R. J. Immunol. 1986; 137: 563-570PubMed Google Scholar), and granulocyte/macrophage colony-stimulating factor (GM-CSF) (5Chen B.D. Clark C.R. 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Shichiri M. Horiuchi S. J. Biol. Chem. 1998; 273: 28305-28313Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). GM-CSF is a glycoprotein produced by many cells including lymphocytes (18Wrong G.G. Witek J.S. Temple P.A. Wilkens K.M. Leary A.C. Luxenberg D.P. Jones S.S. Brown E.L Kay R.M. Orr E.C. Shoemaker C. Golde D.W. Kaufman R.J Hewick R.M. Wang E.A. Clark S.C. Science. 1985; 228: 810-815Crossref PubMed Scopus (762) Google Scholar), fibroblasts (19Munker R. Gasson J. Ogawa M. Koeffler H.P. Nature. 1986; 323: 79-82Crossref PubMed Scopus (365) Google Scholar), vascular endothelial cells (20Broudy V.C. Kaushansky K. Harlan J.M. Adamson J.W. J. Immunol. 1987; 139: 464-468PubMed Google Scholar), eosinophils (21Kita H. Ohnishi T. Okubo Y Weiler D. Abrams J.S. Gleich G.J. J. Exp. Med. 1991; 174: 745-748Crossref PubMed Scopus (280) Google Scholar), keratinocytes (22Kupper T.S. Lee F. Birchall N. Clark S. Dower S. J. Clin. 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Immunol. 1991; Scopus (30) Google demonstrated that two the GM-CSF were for the induction of GM-CSF by and calcium Moreover, al. A. M. A. K. N. 1994; PubMed Scopus Google demonstrated that and was for the induction of which was located downstream of and pathways in T lymphocytes. al. B. P. J. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus (84) Google that Ox-LDL could the activation of in smooth muscle cells. it is reasonable to that induction of GM-CSF by Ox-LDL is regulated by the activation of certain and nuclear PKC activation in the of GM-CSF production by Ox-LDL in macrophages at In this the of GM-CSF in Ox-LDL-induced GM-CSF production by C was from and in The of were in the which not cell and cell growth. was from for NF-κB p50 and and were from were of the from was by from the of H. Miyazaki A. Sakai M. Y. Y. Horiuchi S. Arterioscler. Thromb. 1994; PubMed Scopus Google Scholar). was and was by of A. Sakai M. Y. Hakamata H. Y. Horiuchi S. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). Ox-LDL was by of for at by the of and M. Miyazaki A. Y. Shichiri M. Horiuchi S. PubMed Scopus Google Scholar, T. K. Horiuchi S. Y. Matsuda 1989; PubMed Scopus Google Scholar). The of proteins was by protein assay using as a A. S. Y. Horiuchi S. 1991; PubMed Scopus Google Scholar). were protein by a Moreover, growth and of Raw 264.7 cells were not by at a of in our The for electrophoretic mobility shift assay contained the one is the element of the from to of the mouse GM-CSF and the from to of the mouse GM-CSF to a putative AP-2 and NF-κB binding sites were from their to a putative AP-2 and NF-κB binding sites were from their Raw 264.7 cells were in at a density of to in and the cells were in or at a density of in cell were in a in at When Raw 264.7 cells in in cells were and for or of was as described previously by al. R.M. PubMed Scopus Google Scholar). were by the were as or were using and nuclear was as described previously K. R. T. N. K. H. K. H. Shichiri M. 1997; PubMed Scopus Google Scholar). plasmids the regions of the GM-CSF gene was into Raw 264.7 cells by the using a plasmid a cells were for in the presence or of plasmid the of the GM-CSF gene was as a negative cells were by and for at The in the protein was using the The results were as by to in transfection Raw 264.7 cells in were in of or for of the were at various and at for to The was at of the were to GM-CSF in the The of GM-CSF protein was to the by the of the using GM-CSF as a (12Biwa T. Hakamata H. Sakai M. Miyazaki A. Suzuki H. Kodama T. Shichiri M. Horiuchi S. J. Biol. Chem. 1998; 273: 28305-28313Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). were J. T. Scholar). of Raw 264.7 cells in a in or Ox-LDL for was The first of was for of GM-CSF and were the of GM-CSF S. T. T. Lee T. K. J. 1988; Scopus Google and S. A. Y. M. J. 1986; 23: PubMed Scopus Google as for was of GM-CSF and reverse was T of GM-CSF S. T. T. Lee T. K. J. 1988; Scopus Google for was of and reverse was of S. A. Y. M. J. 1986; 23: PubMed Scopus Google Scholar). The of of GM-CSF and were to and respectively. The in the of a first of for by of at for at for and at for a at for were by that the were the of GM-CSF and were into and by using (12Biwa T. Hakamata H. Sakai M. Miyazaki A. Suzuki H. Kodama T. Shichiri M. Horiuchi S. J. Biol. Chem. 1998; 273: 28305-28313Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). were as groups were for using the presence of a We previously demonstrated that mouse macrophages could GM-CSF in to Ox-LDL (12Biwa T. Hakamata H. Sakai M. Miyazaki A. Suzuki H. Kodama T. Shichiri M. Horiuchi S. J. Biol. Chem. 1998; 273: 28305-28313Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 16Sakai M. Biwa T. Matsumura T. Takemura T. Matsuda H. Anami Y. Sasahara T. Kobori S. Shichiri M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1726-1733Crossref PubMed Scopus (30) Google Scholar). Raw 264.7 cells to Ox-LDL, the Ox-LDL-induced GM-CSF production in Raw 264.7 cells at protein and mRNA by using and respectively. that or not GM-CSF into the the of Ox-LDL to Raw 264.7 cells GM-CSF into the the at the of Ox-LDL analysis showed that GM-CSF mRNA was increased by Ox-LDL the level at and GM-CSF mRNA expression in cells not These results demonstrated that Ox-LDL GM-CSF expression in Raw 264.7 cells. studies demonstrated that several nuclear factor binding sites in GM-CSF gene 5′-flanking from to which regulated GM-CSF expression in to PKC activation in T lymphocytes (27Sugimoto K. Tsuboi A. Miyake S. K. N. Immunol. PubMed Scopus Google Scholar, A. Sugimoto K. J. S. K. N. Immunol. 1991; Scopus (30) Google Scholar, A. M. A. K. N. 1994; PubMed Scopus Google Scholar). Thus, the GM-CSF gene 5′-flanking from −225 to was and into the plasmid and into Raw 264.7 cells. Ox-LDL increased and a level at In and These results suggested that Ox-LDL-induced GM-CSF mRNA expression at in by the transcriptional activation of GM-CSF from −225 to the regulatory in the mouse GM-CSF a of plasmids of GM-CSF to the gene in in the unstimulated in cells was to that in cells. to which the resulted in increased In to −59 and to −37 resulted in a of These results suggested that a negative regulatory site in the from −120 to −97 and that two positive regulatory sites in the regions from −97 to −59 and from −59 to −37 in the unstimulated this two plasmids in a from −120 to −97 and a from −97 to −59 showed that in or cells were or in cells, that a from −120 to −97 was a negative regulatory site and that a from −97 to −59 was a positive regulatory site for GM-CSF expression unstimulated the other cells were Ox-LDL, in cells was not by These results suggested that the from −225 to −120 contained a positive responsive site for Ox-LDL In was decreased by Ox-LDL in cells, which contained a positive regulatory site unstimulated that a negative responsive site for Ox-LDL in the 5′-flanking from −97 to We a analysis of the from −225 to which showed the presence of a putative AP-2 binding site from −169 to −160 in the mouse GM-CSF Moreover, the NF-κB binding site was to from −91 to −82 R. P.A. Cell. Biol. PubMed Google Scholar). We a analysis using a plasmid in a putative AP-2 binding site from −169 to −160 in in the putative AP-2 binding site by and Ox-LDL-induced that the putative AP-2 binding site is a positive responsive site for Ox-LDL which contained the spanning of GM-CSF and tandem which two of the putative AP-2 binding site plasmid expression of in to Ox-LDL, to unstimulated cells of the tandem plasmid increased level at and Ox-LDL-induced at to unstimulated cells These results demonstrated that Ox-LDL-induced GM-CSF expression was for a putative AP-2 binding in a putative AP-2 binding site transcriptional activation by of the GM-CSF that was from by the using The of the in a putative AP-2 binding site is and were into Raw 264.7 cells by a cells were Ox-LDL and was as described the of unstimulated unstimulated repeat of AP-2 binding site the transcriptional activation of GM-CSF by of the GM-CSF which has two of putative AP-2 binding site and tandem plasmids were into Raw 264.7 cells by a cells were Ox-LDL and was as described the of unstimulated unstimulated unstimulated elucidate nuclear protein(s) to the of GM-CSF the nuclear protein for binding to from to a putative AP-2 binding site and from to an NF-κB binding site were by in using a putative AP-2 binding site as a the nuclear proteins from unstimulated cells produced two which by These were by but not by AP-2 Moreover, analysis using and not the and density of the not These results suggested that the binding of nuclear to a putative AP-2 binding site was from or but a nuclear protein to the AP-2 in the NF-κB binding element was as a a was in unstimulated cells. this was decreased by Ox-LDL for NF-κB this NF-κB to for nuclear protein binding to B. Moreover, the density of this was by and but not by that this contained, at least, NF-κB p50 and of NF-κB to a NF-κB binding site of mouse GM-CSF for electrophoretic mobility shift assay are The of the NF-κB binding site is by a and and the of NF-κB is electrophoretic mobility shift assay was by using as a from Raw 264.7 cells or cells Ox-LDL for nuclear were from Raw 264.7 cells Ox-LDL for were of for or and was as described Our study demonstrated that PKC activation by Ox-LDL increased GM-CSF mRNA level in macrophages (12Biwa T. Hakamata H. Sakai M. Miyazaki A. Suzuki H. Kodama T. Shichiri M. Horiuchi S. J. Biol. Chem. 1998; 273: 28305-28313Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We the of PKC activation by Ox-LDL in GM-CSF expression using a PKC C. of cells C resulted in of Ox-LDL-induced GM-CSF mRNA expression to the level of unstimulated cells Moreover, activation of by Ox-LDL using plasmid was by by a of C and Ox-LDL-induced nuclear binding to was by C These results suggested that Ox-LDL-induced nuclear activation by PKC induction in macrophages is an important process in the development and progression of the early stages of atherosclerotic lesions (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9989) Google Scholar). and growth are produced from macrophages in atherosclerotic lesions (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9989) Google Scholar). have demonstrated that GM-CSF an important role in Ox-LDL-induced macrophage growth (12Biwa T. Hakamata H. Sakai M. Miyazaki A. Suzuki H. Kodama T. Shichiri M. Horiuchi S. J. Biol. Chem. 1998; 273: 28305-28313Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 16Sakai M. Biwa T. Matsumura T. Takemura T. Matsuda H. Anami Y. Sasahara T. Kobori S. Shichiri M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1726-1733Crossref PubMed Scopus (30) Google Scholar). The of GM-CSF induction has been in T lymphocytes (27Sugimoto K. Tsuboi A. Miyake S. K. N. Immunol. PubMed Scopus Google Scholar, A. Sugimoto K. J. S. K. N. Immunol. 1991; Scopus (30) Google Scholar, A. M. A. K. N. 1994; PubMed Scopus Google Scholar), such mechanisms are in In there are studies the 5′-flanking of GM-CSF gene that are for the to Ox-LDL in We in the present study the in the mouse GM-CSF gene using plasmids various regions of the mouse GM-CSF Our results demonstrated the presence of two positive and a negative element in the unstimulated showed a positive responsive a putative AP-2 binding site from −169 to and a negative responsive NF-κB binding site from −91 to for Ox-LDL The results showed that GM-CSF expression regulated by the binding of certain nuclear proteins to Ox-LDL increased the binding of certain nuclear protein(s) to a putative AP-2 binding site but decreased the binding of NF-κB p50 and p65 to NF-κB binding site. the plasmids and plasmids demonstrated the presence of two positive in unstimulated −97 and −59 the element contained the and the element −59 and −37 contained the In there was a negative element −120 and −97 the These results suggested that and important for transcriptional regulation of GM-CSF expression in unstimulated cells. studies demonstrated that the from to could as a N. J. N. K. T. Immunol. 1993; PubMed Scopus Google Scholar). In NF-κB and binding sites to and to are for induction of and binding of nuclear to their site was increased T cell activation by (22Kupper T.S. Lee F. Birchall N. Clark S. Dower S. J. Clin. Invest. 1988; 82: 1787-1792Crossref PubMed Scopus (116) Google Scholar, M. J. M. S. A. 1988; PubMed Scopus Google Scholar, D. 1993; PubMed Scopus Google Scholar). Our results are of unstimulated that the from −225 to −120 important for transcriptional activation of GM-CSF in Raw 264.7 cells Computer analysis using the J. T. Scholar, H. Tsuboi A. J. P. K. N. Cell. Biol. 1993; 13: PubMed Scopus Google showed the presence of a putative AP-2 binding site −169 to in the spanning −225 to −120 of the mouse GM-CSF our results of transfection of plasmids a of a putative AP-2 binding site and the two of a putative AP-2 binding site this putative AP-2 binding site to Ox-LDL-induced GM-CSF induction in Raw 264.7 cells. such as and analysis elucidate the of to the binding of certain nuclear of proteins of binding to the GM-CSF have been including NF-κB R. P.A. Cell. Biol. PubMed Google Scholar), and M. J. M. S. A. 1988; PubMed Scopus Google Scholar), A. Sugimoto K. J. Miyake S. K. N. Immunol. 1991; PubMed Scopus (30) Google Scholar), the R. J.M. Cell. Biol. 1994; PubMed Scopus Google Scholar, B. J.M. Science. 1993; PubMed Scopus Google Scholar), and complex H. Tsuboi A. J. P. K. N. Cell. 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Our results demonstrated that the binding of nuclear protein to a putative AP-2 binding site was but by AP-2 the nuclear bound to the putative AP-2 binding site was the assay using and nuclear binding to the was not by Moreover, the and proteins were bound to the nuclear proteins from cells not These results suggested that the nuclear bound to other AP-2 or other protein(s) to AP-2 are to elucidate nuclear In the present demonstrated that the from −97 to −59 was a positive regulatory site for GM-CSF expression in unstimulated cells but in cells unstimulated cells that Ox-LDL the transcriptional activation via the NF-κB is to to R. P.A. Cell. Biol. PubMed Google Scholar, A. Sugimoto K. J. Miyake S. K. N. Immunol. 1991; PubMed Scopus (30) Google Scholar). The binding of the nuclear factor to was by NF-κB but not by NF-κB Moreover, density of complex of and nuclear proteins was by and but not by These results demonstrated that the nuclear factor bound to contained, at least, NF-κB p50 and The nuclear binding the was not by of Ox-LDL for the factor complex was by of Ox-LDL These results suggested that the of GM-CSF by Ox-LDL via by a in NF-κB This is by the results of et al. K. T. M. S. M. A. C. P.A. D. Arterioscler. Thromb. Vasc. Biol. 1997; 17: PubMed Scopus Google Scholar), demonstrated that to Ox-LDL NF-κB activation and expression in cells and that this was of Ox-LDL-induced GM-CSF production was increased but to level The binding of nuclear to a putative AP-2 binding positive responsive element for Ox-LDL, was increased by Ox-LDL level at by a In the binding of the nuclear protein to the NF-κB element not at and decreased by Ox-LDL Thus, it is to that activation of GM-CSF by the increased binding of nuclear proteins to a putative AP-2 binding and a in the binding of nuclear proteins to a putative AP-2 binding element and NF-κB element GM-CSF in Our studies demonstrated that Ox-LDL-induced GM-CSF production at mRNA level was by the activation of PKC in mouse macrophages (12Biwa T. Hakamata H. Sakai M. Miyazaki A. Suzuki H. Kodama T. Shichiri M. Horiuchi S. J. Biol. Chem. 1998; 273: 28305-28313Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, T. Sakai M. Kobori S. Biwa T. Takemura T. Matsuda H. Hakamata H. Horiuchi S. Shichiri M. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3013-3020Crossref PubMed Scopus (93) Google Scholar). in the present PKC is in the signaling for GM-CSF production by of Raw 264.7 cells a PKC induction of GM-CSF mRNA C Ox-LDL-induced in and the binding of certain nuclear proteins to a putative AP-2 binding site These results suggested that Ox-LDL-induced GM-CSF production by PKC via transcriptional activation a putative AP-2 binding site. In a from et al. J.S. Reiner N.E Herrera-Velit P. Steinbrecher U.P. J. Biol. Chem. 1998; 273: 4915-4920Abstract Full Text Full Text PDF PubMed Scopus (78) Google demonstrated that in Ox-LDL-induced macrophage growth. Thus, studies are to elucidate the of in signaling pathway for GM-CSF expression in We are to Horiuchi and Hakamata and Miyazaki of of for