Cystathionine gamma-lyase expression is regulated by exogenous hydrogen peroxide in the mammalian cells

Abstract

Hydrogen sulfide (H2S), as an endogenous signaling molecule in mammals, shows a variety of biological effects. Cystathionine gamma-lyase (CSE)/H2S pathway has been implicated in scavenging reactive oxygen species (ROS) in the mammalian cells. Therefore, we first investigated the regulatory effects of exogenously applied hydrogen peroxide (H2O2) on CSE expression in the mammalian cells. African green monkey kidney fibroblastlike cells (COS-7 cells) or human embryonic kidney 293 cells (HEK 293 cells) were transfected with CSE promoter-luciferase reporter constructs and treated with H2O2 of 1, 5, and 10 microM for 0.5 and 1.5 h at 37 degrees C. The transfected cells were assayed for firefly luciferase activities normalized by Renilla luciferase activity. Human lung adenocarcinoma cells (A549 cells) or human liver cancer cells (SMMC-7721 cells) were treated with H2O2 of 1, 5, and 10 microM for 0.5 and 1.5 h at 37 degrees C, and were then harvested and analyzed by Western blotting and quantitative RT-PCR. Our results showed that the treatment of a medium concentration (5 microM) of H2O2 at a longer time (1.5 h) upregulated CSE expression in the mammalian cells at the levels of the promoter, message RNA, and protein. Collectively, exogenously applied H2O2 can not only markedly affect CSE mRNA and protein expression, but also can affect the CSE promoter activity in the mammalian cells. Our observations indicate that that exogenous H2O2 can upregulate the expression of the CSE gene in the mammalian cells, which will provide the possibility of the scavenging effect of the CSE gene indirectly on ROS in the mammalian cells. However, the regulatory mechanism involved in the effects of exogenously applied H2O2 on CSE expression in the mammalian cells need be further studied.

Figure 1

The effects of exogenous H₂O₂ on CSE gene expression at the promoter level. (A) Both low (1 μM) and high (10 μM) concentration of H₂O₂ decreased the luciferase activity in the transfected HEK 293 cells, while the medium concentration (5 μM) of H₂O₂ had less effect. (B) The upregulation effect caused by the medium concentration (5 μM) of H₂O₂ was more significant in the transfected COS-7 cells at a longer time (1.5 h). *p < 0.01 versus 5 μM + 1.5 h; **p < 0.05 versus 1 μM + 1.5 h; ***p < 0.05 versus 5 μM + 1.5 h; ****p < 0.05 versus 1 μM + 1.5 h.

Figure 2

The effects of exogenous H₂O₂ on CSE gene expression at the mRNA level. The endogenous CSE mRNA level was significantly increased by the treatment of medium concentration (5 μM) of H₂O₂ in both A549 cells (A) and SMMC-7721 cells (B) at 1.5 h posttreatment. *p < 0.01 versus 5 μM + 1.5 h; **p < 0.05 versus 5 μM + 1.5 h; ***p < 0.05 versus 5 μM + 1.5 h; ****p < 0.01 versus 5 μM + 1.5 h.

Figure 3

The effects of exogenous H₂O₂ on CSE gene expression at the protein level. Similar to the effect of H₂O₂ at the transcription level, the CSE protein level was also increased by the treatment of medium concentration (5 μM) of H₂O₂ in both A549 cells (A) and SMMC-7721 cells (B) at a longer time (1.5 h). *p < 0.05 versus 5 μM + 1.5 h; **p < 0.05 versus 1 μM + 1.5 h; ***p < 0.05 versus 1 μM + 1.5 h; ****p < 0.05 versus 5 μM + 1.5 h.