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| 1 | High levels of homocysteine downregulate apolipoprotein E expression via nuclear factor kappa B显示文摘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 apo E expression in cells treated with various concentrations(50-500 μmol/L) of Hcy. Calcium phosphatetransient 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 Hcymediated regulation of apoE promoter by using specific inhibitors or a dominant negative form of IKβ. 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(ChI P)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 m RNA levels in HEK-293 cells, while apo E 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 apo E 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. ChI P 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 apo E expression in peripheral tissues may aggravate atherosclerosis, neurodegenerative diseases and renal dysfunctions. | Violeta G Trusca Adina D Mihai Elena V Fuior Ioana M Fenyo Anca V Gafencu | 2016 | World Journal of Biological Chemistry2016，7，1： | 6 |
| 2 | CD105/Ki67 double immunnos- raining expression in liver metastasis from colon carcinoma显示文摘 | Raluca A Anca M Pusa G | 2011 | Rom J Morphol Embryo12011，52，2： | 1 |
| 3 | Investigation of Aeromonas hydrophila presence in trouts and hatchery environment by targeting ah and ahp virulence genes 显示文摘 | Laurentiu T Anca M G Aneta L T | 2013 | Current Opinion in Biotechnology2013，24，1： | 1 |
| 4 | DNA adsorption on a poly-L-lysine-immobilized poly ( 2-hydroxyethyl methacrylate ) membrane显示文摘 | Senel S Bayramoglu G Anca M Y | 2003 | Polymer International2003，52，7： | 1 |
| 5 | Enzymatic Removal of Phenol and p-Chlorophenol in Enzyme Reactor:Horseradish Peroxidase Immobilized on Magnetic Beads显示文摘 | Bayramo G Anca M Y | 2008 | J Hazard Mater2008，156，13： | 1 |
| 6 | Affinity dyeligand poly(hydroxyethyl methacrylate)/chitosan composite membrane for adsorption lysozyme and kinetic properties显示文摘 | Bayramoglu G Yglmaz M Anca MY | | 0，，01： | 1 |
| 7 | Ethylenediamine grafted poly( glycidylmethacrylate-co-methylmethacry- late) adsorbent for removal of chromate anions 显示文摘 | B ayramoglu G Anca M Y | 2005 | Separation and Purification Technology2005，45，3： | 1 |
| 8 | CD105/Ki67 double immunnostaining expression in liver metastasis from colon carcinoma显示文摘 | Raluca A Anca M Pusa G | 2011 | Rom J Morphol Embryol2011，52，2： | 1 |
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