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.

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.

Tyrphostin AG490 reduces NAPDH oxidase activity and expression in the aorta of hypercholesterolemic apolipoprotein E-deficient mice.

Oxidative stress-induced vascular injury represents a major contributor to the pathoetiology of atherosclerosis. Elevated NADPH oxidase (Nox) activity promotes oxidative injury of the cardiovascular cells. Janus-tyrosine-kinase (Jak) family regulate various aspects of the atherosclerotic process e.g., inflammation, cellular growth, proliferation, and migration. Here, we investigated the potential of Jak2 inhibition to counteract Nox-dependent O(2)(•-) formation in atherogenesis in hypercholesterolemic apolipoprotein E-deficient (ApoE(-/-)) mice. Male ApoE(-/-) mice fed a high-fat, cholesterol-rich diet were treated for 5 weeks with either vehicle or tyrphostin AG490 (1 mg/kg), a specific Jak2 inhibitor. Lucigenin-enhanced-chemiluminescence assay, real-time PCR and Western blot analysis revealed that Nox-derived O(2)(•-) generation, Nox1, Nox2, and Nox4 mRNA and protein levels were significantly elevated in the aortas of ApoE(-/-) mice fed a high-fat diet compared to ApoE(-/-) mice fed a normal diet. Treatment with tyrphostin AG490 significantly reduced the up-regulated Nox activity, the expression of each Nox subtype, as well as the protein level of CD68, a macrophage-specific marker. Morphometric analysis showed a marked reduction of atherosclerotic lesions in the aorta of AG490-treated animals. These data provide new insights into the regulation of vascular Nox by tyrphostins in the cardiovascular system. Since Jak2 transduces the signals of various cardiovascular risk factors, pharmacological manipulation of this signaling pathway may represent a novel strategy to reduce oxidative stress in atherosclerosis.