Abstract
Apolipoprotein E (apoE) is mainly secreted by hepatocytes and incorporated into most plasma lipoproteins. Macrophages, which accumulate cholesterol and are critical for the development of the atherosclerotic plaque, are also an important, albeit smaller, apoE source. Distal regulatory elements control cell-specific activity of the apoE promoter: multienhancers (ME.1/2) in macrophages and hepatic control regions (HCR-1/2) in hepatocytes. A member of AP-1 cell growth regulator, c-Jun regulates the transcription of various apolipoproteins and proinflammatory molecules implicated in atherosclerosis. We aimed to investigate the effect of c-Jun on apoE expression in macrophages versus hepatocytes and to reveal the underlying molecular mechanisms. Herein we show that c-Jun had an opposite, cell-specific effect on apoE expression: downregulation in macrophages but upregulation in hepatocytes. Transient transfections using ME.2 deletion mutants and DNA pull-down (DNAP) assays showed that the inhibitory effect of c-Jun on the apoE promoter in macrophages was mediated by a functional c-Jun binding site located at 301/311 on ME.2. In hepatocytes, c-Jun overexpression strongly increased apoE expression, and this effect was due to c-Jun binding at the canonical site located at −94/−84 on the apoE proximal promoter, identified by transient transfections using apoE deletion mutants, DNAP, and chromatin immunoprecipitation assays. Overall, the dual effect of c-Jun on apoE gene expression led to decreased cholesterol efflux in macrophages resident in the atherosclerotic plaque synergized with an increased level of systemic apoE secreted by the liver to exacerbate atherogenesis.
Highlights
Apolipoprotein E is a glycoprotein with a molecular mass of 35 kDa, comprising 299 amino acids [1,2]
We assessed Apolipoprotein E (apoE) gene expression in hepatocytes and macrophages upon overexpression of AP-1 transcription factors. Both cell types were transfected with a c-Jun expression vector, and apoE gene expression was evaluated by real-time PCR
The results showed that c-Jun overexpression in RAW 264.7 macrophages caused a reduction in apoE mRNA level down to ~40% of the basal apoE level (p < 0.01) compared to control cells (Figure 1A)
Summary
Apolipoprotein E (apoE) is a glycoprotein with a molecular mass of 35 kDa, comprising 299 amino acids [1,2]. It functions as a ligand in receptor-mediated endocytosis of lipoprotein particles, mediating lipid transfer between the circulating lipoproteins and tissues through its functional domains: the lipid-binding region and the receptor-binding region [3,4]. Studies in transgenic mice have shown that selective apoE expression only in macrophages inhibits atherosclerosis without affecting plasma lipid levels [11]. Macrophage-secreted apoE contributes to reverse cholesterol transport in vivo by redirecting the excess cholesterol produced by peripheral tissues to the liver for removal [12]
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