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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.


Related in: MedlinePlus

Hcy enhanced DNMT expression and activity in macrophages, accompanied with DNA hypermethylation in cse gene promoter. (A,B) One hundred micromoles Hcy treatment for 6 h enhanced DNMTs mRNA expression (A) and activity (B) in raw264.7 cells. n = 3; (C) BSP sequencing revealed DNA hypermethylation of mouse cse promoter that contains 11 CpG sites in Hcy-treated macrophages. Each column indicated the clones from Control (left) and Hcy (100 µM, right)-treated cells. The methylated CpG sites were labeled in black circles and unmethylated CpG sites were labeled in white circles. *p < 0.05, ***p < 0.001 compared to its corresponding control.
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ijms-16-12560-f004: Hcy enhanced DNMT expression and activity in macrophages, accompanied with DNA hypermethylation in cse gene promoter. (A,B) One hundred micromoles Hcy treatment for 6 h enhanced DNMTs mRNA expression (A) and activity (B) in raw264.7 cells. n = 3; (C) BSP sequencing revealed DNA hypermethylation of mouse cse promoter that contains 11 CpG sites in Hcy-treated macrophages. Each column indicated the clones from Control (left) and Hcy (100 µM, right)-treated cells. The methylated CpG sites were labeled in black circles and unmethylated CpG sites were labeled in white circles. *p < 0.05, ***p < 0.001 compared to its corresponding control.

Mentions: Next, we focused on the mechanisms that may underlie the repression by Hcy on cse transcription. Interestingly, it was observed that accompanying with the decrease in CSE expression, 100 µM Hcy treatment markedly increased the DNMT1 mRNA level (Figure 4A) and DNMT activity (Figure 4B) in raw264.7 cells, although DNMT3a and DNMT3b mRNA expression were merely marginally elevated. This prompted us to examine whether DNMT upregulation may drive DNA methylation in cse gene promoter and thus repress its transcription. To achieve this, raw264.7 cells were treated with 100 µM Hcy or vehicle. Genomic DNA samples of vehicle- and Hcy-treated cells were then treated with sodium bisulfite and the 215 bp (−417/−203) fragment containing 11 CpG sites were amplified by PCR, followed by DNA sequencing (Figure 4C). The percentage of DNA methylation in the 11 CpG sites was found to be significantly enhanced in Hcy-treated cells compared to control group.


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)

Hcy enhanced DNMT expression and activity in macrophages, accompanied with DNA hypermethylation in cse gene promoter. (A,B) One hundred micromoles Hcy treatment for 6 h enhanced DNMTs mRNA expression (A) and activity (B) in raw264.7 cells. n = 3; (C) BSP sequencing revealed DNA hypermethylation of mouse cse promoter that contains 11 CpG sites in Hcy-treated macrophages. Each column indicated the clones from Control (left) and Hcy (100 µM, right)-treated cells. The methylated CpG sites were labeled in black circles and unmethylated CpG sites were labeled in white circles. *p < 0.05, ***p < 0.001 compared to its corresponding control.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-12560-f004: Hcy enhanced DNMT expression and activity in macrophages, accompanied with DNA hypermethylation in cse gene promoter. (A,B) One hundred micromoles Hcy treatment for 6 h enhanced DNMTs mRNA expression (A) and activity (B) in raw264.7 cells. n = 3; (C) BSP sequencing revealed DNA hypermethylation of mouse cse promoter that contains 11 CpG sites in Hcy-treated macrophages. Each column indicated the clones from Control (left) and Hcy (100 µM, right)-treated cells. The methylated CpG sites were labeled in black circles and unmethylated CpG sites were labeled in white circles. *p < 0.05, ***p < 0.001 compared to its corresponding control.
Mentions: Next, we focused on the mechanisms that may underlie the repression by Hcy on cse transcription. Interestingly, it was observed that accompanying with the decrease in CSE expression, 100 µM Hcy treatment markedly increased the DNMT1 mRNA level (Figure 4A) and DNMT activity (Figure 4B) in raw264.7 cells, although DNMT3a and DNMT3b mRNA expression were merely marginally elevated. This prompted us to examine whether DNMT upregulation may drive DNA methylation in cse gene promoter and thus repress its transcription. To achieve this, raw264.7 cells were treated with 100 µM Hcy or vehicle. Genomic DNA samples of vehicle- and Hcy-treated cells were then treated with sodium bisulfite and the 215 bp (−417/−203) fragment containing 11 CpG sites were amplified by PCR, followed by DNA sequencing (Figure 4C). The percentage of DNA methylation in the 11 CpG sites was found to be significantly enhanced in Hcy-treated cells compared to control group.

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