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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 | Causes and predictors of nonresponse to treatment of intensive care unit-acquired pneumonia 显示文摘 | IOANAS M FERRER M CAVALCANTI M | 2004 | Crit Care Med2004，32，4： | 1 |
| 3 | Microbial airway colonization is associated with noninvasive ventilation failure in exacerbation of chronic obstructive pulmonary disease显示文摘 | Ferrer M Ioanas M Arancibia F | 2005 | Crit Care Med2005，33，： | 1 |
| 4 | New antibiotics for the treatment of severe staphylococcal infection in the critically ill patient显示文摘 | Schmidt Ioanas M de Roux A Lode H | 2005 | Curr Opin Crit Care2005，11，5： | 1 |
| 5 | Impact of BAL on the diagnosis and treatment of nosocomial pneumonia in ICU pa- tients显示文摘 | FERRER R IOANAS M AGUSTI C | 2001 | Monaldi Arch Chest Dis2001，56，6： | 1 |
| 6 | Causes and predictors of nonresponse to treatment of intensive care unit-acquired pneumonia显示文摘 | Ioanas M | 2004 | Crit Care Med2004，32，4： | 1 |
| 7 | Integrating keyword search into XML query processing显示文摘 | Daniela F Donnald K Ioana M | 2000 | Computer Network2000，33，6： | 1 |
| 8 | Reduced spontaneous apoptosis in peripheral blood neutrophils during exacerbation of COPD显示文摘 | Pletz MW Ioanas M De Roux A | 2004 | EurRespir J2004，23，4： | 1 |
| 9 | Reduced spontaneous apoptosis in peripheral blood neutrophils during exacerbation of COPD显示文摘 | Pletz M Ioanas M De Roux A | 2004 | Eur Respir J2004，23，11： | 1 |
| 10 | Building a digital model of Michelangelo's Florentine Pieta显示文摘 | Fausto B Holly R Ioana M | 2002 | Comp Graphics Appl2002，22，1： | 1 |
| 11 | Syntheses and structure-activity relationships of novel 3'-difluoromethyl and 3'-trifluoromethyl-taxoids显示文摘 | LARISSA V K ANTONELLA P IOANA M U | 2008 | Journal of Fluorine Chemistry2008，129，9： | 1 |
| 12 | Impact of BAL on the diagnosis and treatment of nosocomial pneumonia in ICU patients显示文摘 | Ferret R Ioanas M Agusti C | 2001 | MonaldiArch Chest Dis2001，56，6： | 1 |
| 13 | VEGF expression in pancreatic cancer and other malignancies:a review of the literature显示文摘 | Costache MI Ioana M Iordache S | 2015 | Rom J Intern Med2015，53，3： | 1 |
| 14 | Treatment of pneumonia in elderly patien- ts显示文摘 | Schmidt Ioanas M Lode H | 2006 | Expert Opin Pharmacother2006，7，5： | 1 |
| 15 | Treatment of pneumonia in: elderly patients显示文摘 | Sehmidt Ioanas M Lode H | 2006 | Expert Opin Pharmaeother2006，7，5： | 1 |
| 16 | Reduced spontaneous apoptosis in peripheral blood neutrophils during exacerbation of COPD显示文摘 | PLETZ MWR IOANAS M DE ROUX A | 2004 | Eur Respir J2004，23，： | 1 |
| 17 | Bronchial bacterial col- onization in patients with resectable lung carcinoma显示文摘 | Ioanas M Angrill J Baldo X | 2002 | Eur Respir J2002，19，2： | 1 |
| 18 | Treatment of pneumonia in elderly pa- tients 显示文摘 | Schmidt - ioanas M Lode H | 2006 | Expert Opin Pharmaeother2006，7，5： | 1 |
| 19 | Impact of BAL on the diagnosis and treatment of nosocomial pneumonia in ICU patients显示文摘 | Ferrer R Ioanas M Agusti C | 2001 | Monaldi Arch Chest Dis2001，56，6： | 1 |
| 20 | Reduced sponta- neous apoptosis in peripheral blood neutrophils during ex- acerbation of COPD 显示文摘 | Pletz MW Ioanas M de Roux A | 2004 | Eur Respir J2004，23，4： | 1 |
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