Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions

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.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...

Regulation of HDL Genes: Transcriptional, Posttranscriptional, and Posttranslational

“…These enhancers are located at 3.3 and 15.9 kb downstream of the apoE gene, respectively. We demonstrated by chromosome conformational capture (3C) and transient transfections that both ME.1 and ME.2 can interact with the apoE promoter only in phorbol 12-myristate 13-acetate (PMA)-differentiated macrophages, but not in undifferentiated monocytes (Trusca et al 2011). The results showed that the interactions take place in antisense orientation of the promoter and ME.1/2.…”

Apolipoprotein C-II: New findings related to genetics, biochemistry, and role in triglyceride metabolism

“…3A) 70 . Both ME.1 and ME.2 are required for APOE gene expression in macrophages, adipose tissue, and brain 71, 72 .…”

Differential action of glucocorticoids on apolipoprotein E gene expression in macrophages and hepatocytes

“…The apoE proximal promoter [-500/+73]apoE-luc and its deletion mutants: [-200/+73]apoE-luc, [-100/+73]apoE-luc and [-55/+73]apoE-luc, previously described [16, 17], were cloned in KpnI/XhoI sites in pGL4 vector (Promega). GR_synthRE (S900015) reporter construct (which contains a synthetic glucocorticoid response element) was purchased from SwitchGear Genomics (Belgium), and the pcDNA3-hGRα plasmid was kindly provided by Dr. Russcher (University Medical Center, Rotterdam).…”

“…Subsequently, two multienhancer regions, namely ME.1 and ME.2, were found to regulate apoE expression in macrophages and adipose tissue [14]. Previous studies showed that apoE gene transcription increased upon monocyte differentiation into macrophages [15, 16]. On the other hand, the inflammatory conditions (such as those induced by lipopolysaccharides) lead to a decrease in apoE expression in macrophages [17], a process with negative consequences on cholesterol efflux from the atherosclerotic plaque.…”