The Mechanism of Bisphenol A Atherogenicity Involves Apolipoprotein A-I Downregulation through NF-κB Activation.

Apolipoprotein A-I (apoA-I) is the major protein component of high-density lipoproteins (HDL), mediating many of its atheroprotective properties. Increasing data reveal the pro-atherogenic effects of bisphenol A (BPA), one of the most prevalent environmental chemicals. In this study, we investigated the mechanisms by which BPA exerts pro-atherogenic effects. For this, LDLR-/- mice were fed with a high-fat diet and treated with 50 µg BPA/kg body weight by gavage. After two months of treatment, the area of atherosclerotic lesions in the aorta, triglycerides and total cholesterol levels were significantly increased, while HDL-cholesterol was decreased in BPA-treated LDLR-/- mice as compared to control mice. Real-Time PCR data showed that BPA treatment decreased hepatic apoA-I expression. BPA downregulated the activity of the apoA-I promoter in a dose-dependent manner. This inhibitory effect was mediated by MEKK1/NF-κB signaling pathways. Transfection experiments using apoA-I promoter deletion mutants, chromatin immunoprecipitation, and protein-DNA interaction assays demonstrated that treatment of hepatocytes with BPA induced NF-κB signaling and thus the recruitment of p65/50 proteins to the multiple NF-κB binding sites located in the apoA-I promoter. In conclusion, BPA exerts pro-atherogenic effects downregulating apoA-I by MEKK1 signaling and NF-κB activation in hepatocytes.

The Opposite Effect of c-Jun Transcription Factor on Apolipoprotein E Gene Regulation in Hepatocytes and Macrophages.

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.

High levels of homocysteine downregulate apolipoprotein E expression via nuclear factor kappa B.

AIM: To investigate the effect of high homocysteine (Hcy) levels on apolipoprotein E (apoE) expression and the signaling pathways involved in this gene regulation. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to assess apoE expression in cells treated with various concentrations (50-500 µmol/L) of Hcy. Calcium phosphate-transient transfections were performed in HEK-293 and RAW 264.7 cells to evaluate the effect of Hcy on apoE regulatory elements [promoter and distal multienhancer 2 (ME2)]. To this aim, plasmids containing the proximal apoE promoter [(-500/+73)apoE construct] alone or in the presence of ME2 [ME2/(-500/+73)apoE construct] to drive the expression of the reporter luciferase gene were used. Co-transfection experiments were carried out to investigate the downstream effectors of Hcy-mediated regulation of apoE promoter by using specific inhibitors or a dominant negative form of IKß. In other co-transfections, the luciferase reporter was under the control of synthetic promoters containing multiple specific binding sites for nuclear factor kappa B (NF-κB), activator protein-1 (AP-1) or nuclear factor of activated T cells (NFAT). Chromatin immunoprecipitation (ChIP) assay was accomplished to detect the binding of NF-κB p65 subunit to the apoE promoter in HEK-293 treated with 500 µmol/L Hcy. As control, cells were incubated with similar concentration of cysteine. NF-κB p65 proteins bound to DNA were immunoprecipitated with anti-p65 antibodies and DNA was identified by PCR using primers amplifying the region -100/+4 of the apoE gene. RESULTS: RT-PCR revealed that high levels of Hcy (250-750 µmol/L) induced a 2-3 fold decrease in apoE mRNA levels in HEK-293 cells, while apoE gene expression was not significantly affected by treatment with lower concentrations of Hcy (100 µmol/L). Immunoblotting data provided additional evidence for the negative role of Hcy in apoE expression. Hcy decreased apoE promoter activity, in the presence or absence of ME2, in a dose dependent manner, in both RAW 264.7 and HEK-293 cells, as revealed by transient transfection experiments. The downstream effectors of the signaling pathways of Hcy were also investigated. The inhibitory effect of Hcy on the apoE promoter activity was counteracted by MAPK/ERK kinase 1/2 (MEK1/2) inhibitor U0126, suggesting that MEK1/2 is involved in the downregulation of apoE promoter activity by Hcy. Our data demonstrated that Hcy-induced inhibition of apoE took place through activation of NF-κB. Moreover, we demonstrated that Hcy activated a synthetic promoter containing three NF-κB binding sites, but did not affect promoters containing AP-1 or NFAT binding sites. ChIP experiments revealed that NF-κB p65 subunit is recruited to the apoE promoter following Hcy treatment of cells. CONCLUSION: Hcy-induced stress negatively modulates apoE expression via MEK1/2 and NF-κB activation. The decreased apoE expression in peripheral tissues may aggravate atherosclerosis, neurodegenerative diseases and renal dysfunctions.

Thyroid hormones upregulate apolipoprotein E gene expression in astrocytes.

Apolipoprotein E (apoE), a protein mainly involved in lipid metabolism, is associated with several neurodegenerative disorders including Alzheimer's disease. Despite numerous attempts to elucidate apoE gene regulation in the brain, the exact mechanism is still uncovered. The mechanism of apoE gene regulation in the brain involves the proximal promoter and multienhancers ME.1 and ME.2, which evolved by gene duplication. Herein we questioned whether thyroid hormones and their nuclear receptors have a role in apoE gene regulation in astrocytes. Our data showed that thyroid hormones increase apoE gene expression in HTB14 astrocytes in a dose-dependent manner. This effect can be intermediated by the thyroid receptor ß (TRß) which is expressed in these cells. In the presence of triiodothyronine (T3) and 9-cis retinoic acid, in astrocytes transfected to overexpress TRß and retinoid X receptor α (RXRα), apoE promoter was indirectly activated through the interaction with ME.2. To determine the location of TRß/RXRα binding site on ME.2, we performed DNA pull down assays and found that TRß/RXRα complex bound to the region 341-488 of ME.2. This result was confirmed by transient transfection experiments in which a series of 5'- and 3'-deletion mutants of ME.2 were used. These data support the existence of a biologically active TRß binding site starting at 409 in ME.2. In conclusion, our data revealed that ligand-activated TRß/RXRα heterodimers bind with high efficiency on tissue-specific distal regulatory element ME.2 and thus modulate apoE gene expression in the brain.

Metformin reduces the endotoxin-induced down-regulation of apolipoprotein E gene expression in macrophages.

The atheroprotective role of macrophage-derived apolipoprotein E (apoE) is well known. Our previous reports demonstrated that inflammatory stress down-regulates apoE expression in macrophages, aggravating atherogenesis. Metformin, extensively used as an anti-diabetic drug, has also anti-inflammatory properties, and thus confers vascular protection. In this study, we questioned whether metformin could have an effect on apoE expression in macrophages in normal conditions or under lipopolysaccharide (LPS)-induced stress. The results showed that metformin slightly increases the apoE expression only at high doses (5-10 mM). Low doses of metformin (1-3 mM) significantly reduce the LPS down-regulatory effect on apoE expression in macrophages. Our experiments demonstrated that LPS-induced NF-κB binds to the macrophage-specific distal regulatory element of apoE gene, namely to the multienhancer 2 (ME.2) and its 5'-deletion fragments. The NF-κB binding on ME.2 and apoE promoter has a down-regulatory effect. In addition, data revealed that metformin impairs NF-κB nuclear translocation, and thus, improves the apoE levels in macrophages under inflammatory stress. The positive effect of metformin in the inflammatory states, its clinical safety and low cost, make this drug a potential adjuvant in the therapeutic strategies for atherosclerosis.

STAT1 interacts with RXRα to upregulate ApoCII gene expression in macrophages.

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/T3Rß 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.