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First published online February 29, 2008
Journal of Experimental Biology 211, 911-920 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012914
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Homocysteine-induced extracellular superoxide dismutase and its epigenetic mechanisms in monocytes

Yideng Jiang1,*, Jianzhong Jiang2, Jiantuan Xiong1, Jun Cao1, Nan Li1, Guizhong Li and Shuren Wang3

1 Department of Pathophysiology, Ningxia Medical College, Yinchuan, Ningxia 750004, China
2 Department of Pathology, Ningxia Medical College, Yinchuan, Ningxia 750004, China
3 Department of Pathophysiology, West China College of Preclinical and Forensic Medical Sciences, Sichuan University, Chengdu, Sichuan 610041, China


Figure 1
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  		Fig. 1. Sequences of EC-SOD primers and probes. The probes for modified EC-SOD are indicated by shaded boxes, and the polymerase chain reaction (PCR) primers for the EC-SOD sequence are indicated by arrows and bold, italic type. The sequence of EC-SOD after sodium bisulfite modification is also shown. The solid line arrows indicate the extension direction. Methylated and unmethylated PCR have the same primers. Fluorescence detection was carried out by two passages. When PCR was carried out with methylation-modified gDNA, the fluorescence of the methylated probe was detected by the one passage, similar to PCR with unmethylation-modified gDNA. The other kind of fluorescence of unmethylated probes was detected by the other passages. Upper row, original sequence; lower row, bisulfite modified sequence (for display, assume all CpG sites are methylated); +, CpG site; non CpG `C' converted to `T'; >>>, left primer; <<<, right primer.

 

Figure 2
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  		Fig. 2. Percentage of positive oil red O staining cells in groups of different concentrations of Hcy (50, 100, 200 and 500 µmol l–1; A), time of incubation (24, 48 and 72 h; B) and antagonist (C) group (values are means ± s.e.m., N=6). The percentage of positive oil red O-staining cells was evaluated by the semi-quantitative analysis; {dagger}P<0.05, compared with control group; *P<0.05, **P<0.01, compared with the positive group.

 

Figure 3
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  		Fig. 3. Levels of (A) H2O2 and (B) ox-LDL produced by activated monocytes. H2O2 was measured by fluorescence spectrofluorophotometry and the content of ox-LDL was detected by ELISA. See Materials and methods for details.

 

Figure 4
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  		Fig. 4. Relative EC-SOD protein (A) and mRNA (B) levels. Monocytes were treated with 100 µmol l–1 Hcy in control medium for 24, 48 or 72 h. Total RNA was isolated and real-time PCR was carried out to detect mRNA levels. Protein levels of EC-SOD (A) were examined by western blotting (B). Values are means ± s.e.m. from 3 separate experiments with triplicated samples (N=6).*P<0.05, compared with positive group; §P<0.05, compared with 100 µmol l–1 Hcy group (72 h).

 

Figure 5
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  		Fig. 5. The relative mRNA levels of DNMT1, DNMT3, MBD2, MeCP2 in monocytes cultured for different times (A) and with different antagonists (B). Fluorescence was versus the PCR cycle number for both reactions and each sample dilubition is indicated. Each gene RNA level was acquired from the value of the threshold cycle (Ct) of the real-time PCR relative to that of GAPDH using Eqn 1. The final results, expressed as N-fold differences in target gene expression relative to the calibrator, termed `Ntarget', are determined as following: N=2{Delta}Ct(sample)–{Delta}Ct(calibrator) (Eqn 2), where {Delta}Ct values of the calibrator and sample were determined by subtracting the Ct value of the target gene from the Ct value. Values are means ± s.e.m., representative of three separate experiments.

 

Figure 6
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  		Fig. 6. Effects of Hcy on DNMT1, DNMT3, MBD2, MeCP2 protein levels in human monocytes incubated for different times (A) and with different antagonists (B). The monocytes were incubated in the absence or presence of Hcy at various concentrations and immunoblots (top) analyzed by densitometry (below). The results were expressed as percentage of control (100%) ± s.e.m., each performed in duplicate. Relative value was related to that of β-actin through the formula (relative value=experiment densitometry valuex100/β-actin value).

 

Figure 7
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  		Fig. 7. EC-SOD real-time PCR methylated levels (see Materials and methods) *P<0.05, **P<0.001, compared with positive group; {dagger}P<0.05 compared with 100 µmol l–1 Hcy group (72 h).

 

Figure 8
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  		Fig. 8. Relative levels of histone H3 (A) and H4 (B) determined by ChIP assay. The monocytes were treated with 100 µmol l–1 Hcy, folate, AZC and TSA for 24, 48 and 72 h. Nuclear extracts were immunoprecipitated with antibody against AcH3 and AcH4, and relative levels of histones H3 and H4 were analyzed by SDS–PAGE. *P<0.05, compared with positive group; {dagger}P<0.05, compared with 100 µmol l–1 Hcy group (72 h).

 

Figure 9
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  		Fig. 9. Effect of Hcy on homocysteine (HAT) and histone deacetylase (HDAC) levels in cultured monocytes. Levels of HAT and HDAC in supernatant of cultured monocytes were measured by ELISA assays after incubation with 100 µmol l–1 Hcy for the indicated times. Data are expressed as mean ± s.e.m. and were representative of at least 6 independent experiments. *P<0.05 compared with corresponding untreated groups.

 

Figure 10
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  		Fig. 10. Proposed mechanism of homocysteine-induced accumulation of cholesterol and the formation of foam cells.

 




© The Company of Biologists Ltd 2008