Abstract
In atherogenesis, macrophage-derived apolipoprotein E (apoE) has an athero-protective role by a mechanism that is not fully understood. We investigated the regulatory mechanisms involved in the modulation of apoE expression in macrophages. The experiments showed that the promoters of all genes of the apoE/apoCI/apoCIV/apoCII gene cluster are enhanced by multienhancer 2 (ME.2), a regulatory region that is located 15.9 kb downstream of the apoE gene. ME.2 interacts with the apoE promoter in a macrophage-specific manner. Transient transfections in RAW 264.7 macrophages showed that the activity of ME.2 was strongly decreased by deletion of either 87 bp from the 5' end or 131 bp from the 3' end. We determined that the minimal fragment of this promoter that can be activated by ME.2 is the proximal -100/+73 region. The analysis of the deletion mutants of ME.2 revealed the importance of the 5' end of ME.2 in apoE promoter transactivation. Chromatin conformational capture assays demonstrated that both ME.2 and ME.1 physically interacted with the apoE promoter in macrophages. Our data showed that phorbol 12-myristate 13-acetate-induced differentiation of macrophages is accompanied by a robust induction of apoE and STAT1 expression. In macrophages (but not in hepatocytes), STAT1 up-regulated apoE gene expression via ME.2. The STAT1 binding site was located in the 174-182 region of ME.2. In conclusion, the specificity of the interactions between the two multienhancers (ME.1 and ME.2) and the apoE promoter indicates that these distal regulatory elements play an important role in the modulation of apoE gene expression in a cell-specific manner.
Highlights
Apolipoprotein E, a glycoprotein of 35 kDa, is associated with the chylomicron remnants, very low density lipoproteins, low density lipoproteins (LDL), and high density lipoproteins (HDL) and plays an important role in lipid metabolism [1,2,3,4,5,6]
Chemicals—Restriction and modification enzymes (T4 DNA ligase, TaqDNA polymerase, M-MLV reverse transcriptase, calf intestinal alkaline phosphatase) and Lipofectamine 2000 were purchased from Invitrogen or Promega (Madison, WI); Pfu DNA polymerase and FastDigest Restriction Enzymes were from Fermentas; DMEM, RPMI 1640, fetal calf serum, and TRIzol reagent were from Invitrogen; ECL Western blotting kit was from Pierce; phorbol 12-myristate 13-acetate (PMA), LigaFast Rapid DNA Ligation System, and Luciferase assay system were from Promega; Dynabeads M-280 streptavidin was from Invitrogen Dynal (AS, Oslo, Norway), Halt Protease Inhibitor Single-Use mixture EDTA-free was from Thermo Scientific (Rockford, IL); FuGENE 6 Transfection Reagent was from Roche Applied Science; Histopaque-1077 and all other chemicals were from Sigma
Apolipoprotein E is a major component of the lipoprotein transport system playing important roles in lipid metabolism and recognized with an atheroprotective role [1,2,3,4,5,6]
Summary
Chemicals—Restriction and modification enzymes (T4 DNA ligase, TaqDNA polymerase, M-MLV reverse transcriptase, calf intestinal alkaline phosphatase) and Lipofectamine 2000 were purchased from Invitrogen or Promega (Madison, WI); Pfu DNA polymerase and FastDigest Restriction Enzymes were from Fermentas; DMEM, RPMI 1640, fetal calf serum, and TRIzol reagent were from Invitrogen; ECL Western blotting kit was from Pierce; phorbol 12-myristate 13-acetate (PMA), LigaFast Rapid DNA Ligation System, and Luciferase assay system were from Promega; Dynabeads M-280 streptavidin was from Invitrogen Dynal (AS, Oslo, Norway), Halt Protease Inhibitor Single-Use mixture EDTA-free was from Thermo Scientific (Rockford, IL); FuGENE 6 Transfection Reagent was from Roche Applied Science; Histopaque-1077 and all other chemicals were from Sigma. ApoCI and apoCIV proximal promoters were amplified by PCR using the primers described in supplemental Table 1 and cloned at the XhoI/HindIII and KpnI/SacI cloning sites of the pGL3 basic vector, respectively, obtaining the following plasmids: [Ϫ523/ ϩ22]apoCI and [Ϫ650/ϩ19]apoCIV. The sequence of the ME. was amplified by PCR using primers ME. ϩ 19 forward and ME. ϩ619 reverse (described in supplemental Table 1) and was subsequently cloned at the KpnI site of the plasmids [Ϫ500/ϩ73]apoE-luc, [Ϫ523/ϩ22]apoCIluc, [Ϫ650/ϩ19]apoCIV-luc, [Ϫ388/ϩ18]apoCII-luc, [Ϫ500/ ϩ66]p22-luc, and in pGL3 basic vector. To obtain the fragments 87– 619, 165– 619, and 267– 619, the respective plasmids containing the deletion mutants of ME. cloned in pGL3 basic vector were used as templates along with the biotinylated RV3 forward and ME. 619 reverse primers. For p values less than 0.05, the population means are statistically different
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