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. 2009 Apr 17;381(4):660-5.
doi: 10.1016/j.bbrc.2009.02.132. Epub 2009 Feb 28.

Up-regulation of thioredoxin interacting protein (Txnip) by p38 MAPK and FOXO1 contributes to the impaired thioredoxin activity and increased ROS in glucose-treated endothelial cells

Xiaonan Li  1 , Yuanyuan Rong, Mei Zhang, Xing Li Wang, Scott A LeMaire, Joseph S Coselli, Yun Zhang, Ying H Shen
Affiliations

Up-regulation of thioredoxin interacting protein (Txnip) by p38 MAPK and FOXO1 contributes to the impaired thioredoxin activity and increased ROS in glucose-treated endothelial cells

Xiaonan Li et al. Biochem Biophys Res Commun. .

Abstract

Oxidative stress induced by hyperglycemia is a key factor in the development of cardiovascular diseases in diabetes. Thioredoxin (Trx) system, a major thiol antioxidant system, regulates the reduction of intracellular reactive oxygen species (ROS). In this study, we demonstrated that high glucose significantly increased intracellular ROS levels in human aortic endothelial cells (HAECs). Additionally, high glucose reduced the antioxidant activity of thioredoxin. To investigate the mechanisms involved, we found that glucose enhanced the expression of thioredoxin interacting protein (Txnip), a Trx inhibitory protein, through p38 mitogen-activated protein kinase (MAPK). We also showed that glucose regulated Txnip at transcription level and p38 MAPK and forkhead box O1 transcriptional factor (FOXO1) were involved in the process. Taken together, upregulation of Txnip and subsequent impairment of thioredoxin antioxidative system through p38 MAPK and FOXO1 may represent a novel mechanism for glucose-induced increase in intracellular ROS.

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Figures

Fig. 1
Fig. 1. High glucose increased the ROS production through p38 MAPK pathway
(A) High glucose increased the intracellular ROS level. HAECs were treated with glucose for 24 hours. Treated cells were incubated with oxidant-sensitive fluorogenic probe CM-H2DCFDA. Fluorescence was detected by a fluorescent microscope. Representative microscopic scans from three experiments are shown. Representative microscopic scan from three experiments and quantitative analysis of fluorescent intensity randomly counted in 5 fields per coverslip are shown. Data represent the mean ±SEM (N=3), **P < 0.01, versus control. Glucose significantly increased intracellular levels of ROS in a dose-dependent manner. (B) High glucose increased the ROS production through p38 MAPK pathway. HAECs were treated with glucose in the presence or absence of the p38 MAPK inhibitor PD169316 for 24 hours. Fluorescence was detected by a fluorescent microscope. Data represent the mean ±SEM (N=3), *P < 0.05, **P < 0.01, ***P < 0.001 versus control. Glucose significantly increased intracellular levels of ROS through the p38 MAPK pathway.
Fig. 2
Fig. 2. High glucose increased the expression of Txnip via p38 MAPK pathway
(A) The effects of glucose on Txnip protein expression. HAECs were treated with glucose in the absence or presence of the p38 MAPK inhibitor PD169316 for 24 hours. Txnip was measured by Western blot. Glucose increased the Txnip expression through p38 MAPK. (B) The effects of glucose on Txnip mRNA level. HAECs were treated with glucose in the absence or presence of the p38 MAPK inhibitor PD169316 for 24 hours, and Txnip mRNA levels were examined by RT-PCR. Glucose upregulated the Txnip mRNA via p38 MAPK. Representative blots from more then 3 independent experiments are shown. Data represent the mean ±SEM (N=3). *P < 0.05, **P < 0.01, ***P < 0.001 versus control.
Fig. 3
Fig. 3. Txnip was responsible for glucose induced reduction of trx activity and increase in ROS levels
(A) The effect of Txnip siRNA on expression of Txnip. HAECs were treated with Txnip siRNA or scrambled siRNA for 24 hours. The expression of Txnip was examined by anti-Txnip antibody. Gene silencing of Txnip reduced Txnip protein. (B) Txnip siRNA prevented the glucose-induced reduction of Trx activity. HAECs were transfected with Txnip siRNA followed by treatment with glucose for 24 hours. Silencing the Txnip gene by siRNA enhanced the activity of Trx. Trx activity was assessed (n=12 per data point). (C) Txnip was involved in the glucose induced increase in ROS level. HAECs were transfected with Txnip siRNA followed by treatment with glucose for 24 hours. Intracellular ROS levels were detected by CM-H2DCFDA. Representative microscopic scans from 3 experiments and the quantitative analysis of fluorescent intensity randomly counted in 5 fields per coverslip are shown. Knockdown of Txnip by siRNA decreased basal and glucose-induced ROS levels. Representative blots from more then 3 independent experiments are shown. Data represent the mean ±SEM (N=3). *P < 0.05, **P < 0.01, ***P < 0.001 versus control.
Fig. 4
Fig. 4. FOXO 1 was responsible for glucose-induced induction of Txnip
(A) Glucose increased the binding of FOXO1 to the Txnip promoter via p38 MAPK pathway. HAECs were treated with glucose in the presence or absence of p38 inhibitor. The binding of FOXO1 to the Txnip promoter was examined by Chip assay as described in the methods. (B) The effect of FOXO1 siRNA on the expression of FOXO1. HAECs were treated with FOXO1 siRNA or scrambled siRNA for 24 hours. The expression of FOXO1 was examined by anti-FOXO1 antibody. Gene silencing of FOXO1 reduces protein levels of FOXO1. (C). FOXO1 was responsible for glucose-induced upregulation of Txnip. HAECs were transfected with FOXO1 siRNA followed by treatment with glucose for 24 hours. Txnip was measured by Western blot. (D) FOXO1 was involved in glucose-induced induction of Txnip mRNA. HAECs were transfected with FOXO1 siRNA for 24 hours. Txnip mRNA was examined by RT-PCR. FOXO1 siRNA decreased basal Txnip and prevented glucose-induced induction of Txnip mRNA. Representative blots from more then 3 independent experiments are shown. Data represent the mean ±SEM (N=3). *P < 0.05, ***P < 0.001 versus glucose with scrambled siRNA. (E) Schematic diagram of possible mechanisms for high glucose induced increase in intracellular ROS. High glucose, by activating p38 MAPK and FOXO1, induces Txnip expression and subsequently reduces Trx activity that leads to increased intracellular ROS levels.
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