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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.Apolipoprotein E (apoE), 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 (1-6). Deficiency in apoE results in atherosclerosis in humans and in animal models (7-12). ApoE knock-out mice are the best-characterized animal models of atherosclerosis (13). ApoE is a ligand for the LDL receptor found in the liver and other tissues and for the LDL receptor-related protein found in hepatocytes and as such it facilitates the clearance of lipoprotein remnants from the circulation (14 -16). Malfunction of the mechanisms of cholesterol clearance leads to the accumulation of remnants in the plasma, a process associated with premature atherosclerosis (8,11,12). ApoE regulates plasma cholesterol levels, also having an important role in cholesterol efflux, as documented by studies in patients and animal models with apoE deficiency or mutated apoE genes (17-24). Recently, antioxidant and anti-inflammatory functions within the atherosclerotic plaque were attributed to apoE (23,24).ApoE is mainly synthesized by the liver and also by various cells and peripheral tissues (25). At the site of atherosclerotic lesion, apoE is provided by macrophages. Transgenic mice expressing apoE only in macrophages are protected against atherosclerosis even th...

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Apolipoprotein CII (apoCII) is a specific activator of lipoprotein lipase and plays an important role in triglyceride metabolism. The aim of our work was to elucidate the regulatory mechanisms involved in apoCII gene modulation in macrophages. Using Chromosome Conformation Capture we demonstrated that multienhancer 2 (ME.2) physically interacts with the apoCII promoter and this interaction facilitates the transcriptional enhancement of the apoCII promoter by the transcription factors bound on ME.2. We revealed that the transcription factor STAT1, previously shown to bind to its specific site on ME.2, is functional for apoCII gene upregulation. We found that siRNA-mediated inhibition of STAT1 gene expression significantly decreased the apoCII levels, while STAT1 overexpression in RAW 264.7 macrophages increased apoCII gene expression. Using transient transfections, DNA pull down and chromatin immunoprecipitation assays, we revealed a novel STAT1 binding site in the −500/−493 region of the apoCII promoter, which mediates apoCII promoter upregulation by STAT1. Interestingly, STAT1 could not exert its upregulatory effect when the RXRα/T3Rβ binding site located on the apoCII promoter was mutated, suggesting physical and functional interactions between these factors. Using GST pull-down and co-immunoprecipitation assays, we demonstrated that STAT1 physically interacts with RXRα. Taken together, these data revealed that STAT1 bound on ME.2 cooperates with RXRα located on apoCII promoter and upregulates apoCII expression only in macrophages, due to the specificity of the long-range interactions between the proximal and distal regulatory elements. Moreover, we showed for the first time that STAT1 and RXRα physically interact to exert their regulatory function.

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