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Homocysteine Triggers Inflammatory Responses in Macrophages through Inhibiting CSE-H2S Signaling via DNA Hypermethylation of CSE Promoter.

Li JJ, Li Q, Du HP, Wang YL, You SJ, Wang F, Xu XS, Cheng J, Cao YJ, Liu CF, Hu LF - Int J Mol Sci (2015)

Bottom Line: Unfortunately, Hcy-lowering strategies were found to have limited effects in reducing cardiovascular events.DNMT inhibition or knockdown reversed the decrease of CSE transcription induced by Hcy in macrophages.In sum, our findings demonstrate that Hcy may trigger inflammation through inhibiting CSE-H2S signaling, associated with increased promoter DNA methylation and transcriptional repression of cse in macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, the Second Affiliated Hospital of Soochow University, Soochow University, Suzhou 215004, China. dragonrabbit@163.com.

ABSTRACT
Hyperhomocysteinemia (HHcy) is an independent risk factor of atherosclerosis and other cardiovascular diseases. Unfortunately, Hcy-lowering strategies were found to have limited effects in reducing cardiovascular events. The underlying mechanisms remain unclear. Increasing evidence reveals a role of inflammation in the pathogenesis of HHcy. Homocysteine (Hcy) is a precursor of hydrogen sulfide (H2S), which is formed via the transsulfuration pathway catalyzed by cystathionine β-synthase and cystathionine γ-lyase (CSE) and serves as a novel modulator of inflammation. In the present study, we showed that methionine supplementation induced mild HHcy in mice, associated with the elevations of TNF-α and IL-1β in the plasma and reductions of plasma H2S level and CSE expression in the peritoneal macrophages. H2S-releasing compound GYY4137 attenuated the increases of TNF-α and IL-1β in the plasma of HHcy mice and Hcy-treated raw264.7 cells while CSE inhibitor PAG exacerbated it. Moreover, the in vitro study showed that Hcy inhibited CSE expression and H2S production in macrophages, accompanied by the increases of DNA methyltransferase (DNMT) expression and DNA hypermethylation in cse promoter region. DNMT inhibition or knockdown reversed the decrease of CSE transcription induced by Hcy in macrophages. In sum, our findings demonstrate that Hcy may trigger inflammation through inhibiting CSE-H2S signaling, associated with increased promoter DNA methylation and transcriptional repression of cse in macrophages.

No MeSH data available.


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Effects of Hcy on CSE expression and promoter activity in raw264.7 macrophages. (A–D) Hcy down-regulated CSE mRNA and protein expressions in raw264.7 macrophages in dose- and time-dependent manners (E,F). Effect of l-homocystine and l-cysteine on CSE protein expression. CSE protein expression was evaluated by immunoblotting analysis while CSE mRNA level determined by quantitative PCR. GAPDH protein and 18S mRNA served as loading controls. Mean ± SEM, n = 3–8. (G) Reporter assay of a series of different fragments of cse promoter activity in raw264.7 cells that were treated with 100 or 500 µM Hcy for 12 h. Luciferase activities were expressed as fold increases over pGL3-basic vector. n = 7. *p < 0.05, **p < 0.01, ***p < 0.001 compared to its corresponding control group. N.S., not significant.
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ijms-16-12560-f003: Effects of Hcy on CSE expression and promoter activity in raw264.7 macrophages. (A–D) Hcy down-regulated CSE mRNA and protein expressions in raw264.7 macrophages in dose- and time-dependent manners (E,F). Effect of l-homocystine and l-cysteine on CSE protein expression. CSE protein expression was evaluated by immunoblotting analysis while CSE mRNA level determined by quantitative PCR. GAPDH protein and 18S mRNA served as loading controls. Mean ± SEM, n = 3–8. (G) Reporter assay of a series of different fragments of cse promoter activity in raw264.7 cells that were treated with 100 or 500 µM Hcy for 12 h. Luciferase activities were expressed as fold increases over pGL3-basic vector. n = 7. *p < 0.05, **p < 0.01, ***p < 0.001 compared to its corresponding control group. N.S., not significant.

Mentions: We then examined the effect of Hcy on CSE expression in macrophages. It was observed that treatment with increasing concentrations of Hcy (5, 10, 50, 100 and 500 µM) resulted in marked decreases of CSE protein and mRNA expression in a dose-dependent manner (Figure 3A,C). Moreover, Hcy time-dependently reduced CSE protein and mRNA levels in raw264.7 cells (Figure 3B,D). In contrast, neither l-homocystine (the oxidized form of l-homocysteine) nor l-cysteine (a reducing thiol) had any significant effect on CSE protein expression (Figure 3E,F). The promoter activities of different fragments of mouse cse promoter were further evaluated with luciferase assay (Figure 3G). Compared with pGL3-basic vector, the pGL3-CSE construct (−3498/+18) transfection enhanced the luciferase activity by about 10-fold. Deletion from −3498 to −381 resulted in an additional 20-fold increase, indicating a possibility that negative regulatory elements for cse transcription may exist in this region. However, a deletion from −380 to −173 markedly reduced the promoter activity, indicating a basal and essential element for cse transcription may be located between −380 and +18. Compared with controls, treatment with 100 µM Hcy reduced the promoter activities to varying degrees, and 500 µM Hcy resulted in a more pronounced repression. This implies that Hcy treatment may repress CSE transcription in macrophages.


Homocysteine Triggers Inflammatory Responses in Macrophages through Inhibiting CSE-H2S Signaling via DNA Hypermethylation of CSE Promoter.

Li JJ, Li Q, Du HP, Wang YL, You SJ, Wang F, Xu XS, Cheng J, Cao YJ, Liu CF, Hu LF - Int J Mol Sci (2015)

Effects of Hcy on CSE expression and promoter activity in raw264.7 macrophages. (A–D) Hcy down-regulated CSE mRNA and protein expressions in raw264.7 macrophages in dose- and time-dependent manners (E,F). Effect of l-homocystine and l-cysteine on CSE protein expression. CSE protein expression was evaluated by immunoblotting analysis while CSE mRNA level determined by quantitative PCR. GAPDH protein and 18S mRNA served as loading controls. Mean ± SEM, n = 3–8. (G) Reporter assay of a series of different fragments of cse promoter activity in raw264.7 cells that were treated with 100 or 500 µM Hcy for 12 h. Luciferase activities were expressed as fold increases over pGL3-basic vector. n = 7. *p < 0.05, **p < 0.01, ***p < 0.001 compared to its corresponding control group. N.S., not significant.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490461&req=5

ijms-16-12560-f003: Effects of Hcy on CSE expression and promoter activity in raw264.7 macrophages. (A–D) Hcy down-regulated CSE mRNA and protein expressions in raw264.7 macrophages in dose- and time-dependent manners (E,F). Effect of l-homocystine and l-cysteine on CSE protein expression. CSE protein expression was evaluated by immunoblotting analysis while CSE mRNA level determined by quantitative PCR. GAPDH protein and 18S mRNA served as loading controls. Mean ± SEM, n = 3–8. (G) Reporter assay of a series of different fragments of cse promoter activity in raw264.7 cells that were treated with 100 or 500 µM Hcy for 12 h. Luciferase activities were expressed as fold increases over pGL3-basic vector. n = 7. *p < 0.05, **p < 0.01, ***p < 0.001 compared to its corresponding control group. N.S., not significant.
Mentions: We then examined the effect of Hcy on CSE expression in macrophages. It was observed that treatment with increasing concentrations of Hcy (5, 10, 50, 100 and 500 µM) resulted in marked decreases of CSE protein and mRNA expression in a dose-dependent manner (Figure 3A,C). Moreover, Hcy time-dependently reduced CSE protein and mRNA levels in raw264.7 cells (Figure 3B,D). In contrast, neither l-homocystine (the oxidized form of l-homocysteine) nor l-cysteine (a reducing thiol) had any significant effect on CSE protein expression (Figure 3E,F). The promoter activities of different fragments of mouse cse promoter were further evaluated with luciferase assay (Figure 3G). Compared with pGL3-basic vector, the pGL3-CSE construct (−3498/+18) transfection enhanced the luciferase activity by about 10-fold. Deletion from −3498 to −381 resulted in an additional 20-fold increase, indicating a possibility that negative regulatory elements for cse transcription may exist in this region. However, a deletion from −380 to −173 markedly reduced the promoter activity, indicating a basal and essential element for cse transcription may be located between −380 and +18. Compared with controls, treatment with 100 µM Hcy reduced the promoter activities to varying degrees, and 500 µM Hcy resulted in a more pronounced repression. This implies that Hcy treatment may repress CSE transcription in macrophages.

Bottom Line: Unfortunately, Hcy-lowering strategies were found to have limited effects in reducing cardiovascular events.DNMT inhibition or knockdown reversed the decrease of CSE transcription induced by Hcy in macrophages.In sum, our findings demonstrate that Hcy may trigger inflammation through inhibiting CSE-H2S signaling, associated with increased promoter DNA methylation and transcriptional repression of cse in macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, the Second Affiliated Hospital of Soochow University, Soochow University, Suzhou 215004, China. dragonrabbit@163.com.

ABSTRACT
Hyperhomocysteinemia (HHcy) is an independent risk factor of atherosclerosis and other cardiovascular diseases. Unfortunately, Hcy-lowering strategies were found to have limited effects in reducing cardiovascular events. The underlying mechanisms remain unclear. Increasing evidence reveals a role of inflammation in the pathogenesis of HHcy. Homocysteine (Hcy) is a precursor of hydrogen sulfide (H2S), which is formed via the transsulfuration pathway catalyzed by cystathionine β-synthase and cystathionine γ-lyase (CSE) and serves as a novel modulator of inflammation. In the present study, we showed that methionine supplementation induced mild HHcy in mice, associated with the elevations of TNF-α and IL-1β in the plasma and reductions of plasma H2S level and CSE expression in the peritoneal macrophages. H2S-releasing compound GYY4137 attenuated the increases of TNF-α and IL-1β in the plasma of HHcy mice and Hcy-treated raw264.7 cells while CSE inhibitor PAG exacerbated it. Moreover, the in vitro study showed that Hcy inhibited CSE expression and H2S production in macrophages, accompanied by the increases of DNA methyltransferase (DNMT) expression and DNA hypermethylation in cse promoter region. DNMT inhibition or knockdown reversed the decrease of CSE transcription induced by Hcy in macrophages. In sum, our findings demonstrate that Hcy may trigger inflammation through inhibiting CSE-H2S signaling, associated with increased promoter DNA methylation and transcriptional repression of cse in macrophages.

No MeSH data available.


Related in: MedlinePlus