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

doi:10.1016/j.bbrc.2009.02.132

Biochemical and Biophysical Research Communications

Volume 381, Issue 4, 17 April 2009, Pages 660-665

https://doi.org/10.1016/j.bbrc.2009.02.132 Get rights and content

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.

Introduction

Hyperglycemia-induced production of reactive oxygen species (ROS) plays a crucial role in the development of diabetic vascular diseases [1]. Excessive production of ROS can directly oxidizes biological macromolecules and impairs vascular structure and functions [2], [3].

Several antioxidant systems critically regulate cellular reduction/oxidation (redox) balance. The ubiquitously presented thiol-reducing thioredoxin (Trx) system, including Trx, Trx reductase and NADPH, is an important antioxidative mechanism [4]. Trx system reduces oxidized cysteine groups on proteins through an interaction with the redox-active center of Trx (Cys-Gly-Pro-Cys) to form a disulfide bond, which in turn can be reduced by Trx reductase and NADPH [5]. Trx also regulates cell signaling molecules such as ASK-1 and is involved in the regulation of a wide variety of biological processes [4], [7]. Thioredoxin interacting protein (Txnip), also known as vitamin D3 upregulated protein-1 (VDUP-1) or thioredoxin binding protein-2 (TBP-2) [6], is an endogenous inhibitor of Trx. Txnip directly interacts with the catalytic center of reduced Trx and inhibits its reducing activity [7], [8]. Trx–Txnip interaction, therefore, plays an important role in the redox regulation [7].

Trx has been shown to be important in cardiovascular protection and dysregulation of this system has been implicated in the development of cardiovascular diseases. Increasing Trx level attenuates myocardial damage induced by ischemia–reperfusion injury [9], whereas overexpression of Txnip sensitizes the cardiomyocytes to oxidative stress induced apoptosis [10]. Furthermore, significantly increased Txnip expression and reduced Trx activity has been observed in diabetic animals [11], which may play an important role in the development of diabetic vascular diseases.

In the present study, we examined the activation of the Trx system in glucose-treated endothelial cells. We observed that high glucose upregulated Txnip expression and subsequently inhibited Trx activity. These effects were mediated by activated p38 MAPK and FOXO1 pathway. Our study reports a novel mechanism for glucose-induced increase in intracellular ROS.

Section snippets

Material and methods

Cell culture. Primary human aortic endothelial cells (HAECs, Cell Applications, San Diego, CA) were cultured at 37 °C in 5% CO₂ in EGM-2 medium (Cambrex, East Rutherford, NJ) containing endothelial cell basic medium (EBM), 2% FBS, hydrocortisone, FGF-2, VEGF, IGF-1, EGF, ascorbic acid, GA-1000 and heparin. The cells were transfected with siRNAs, or treated with the p38 MAPK inhibitor PD169316 (100 nM, Calbiochem), or glucose (Sigma) at various concentrations for the time periods indicated in the

High glucose increases the ROS production through p38 MAPK pathway

We first tested whether high glucose increased the intracellular ROS production. HAECs were incubated with increasing amounts of glucose. As shown in Fig. 1A, high glucose significantly increased intracellular levels of ROS in a dose-dependent manner (all P < 0.01), which is consistent with previous reports [11]. We then determined whether p38 MAPK pathway was involved in high glucose-induced increase in ROS level. HAECs were incubated with glucose in the presence or absence of p38 MAPK inhibitor

Discussion

In the present study, we have observed that high glucose-induced Txnip expression and subsequently reduced the Trx activity, which was partially responsible for increased intracellular ROS. The p38 MAPK and FOXO1 pathways mediated high glucose-induced Txnip up-regulation.

Increased ROS level has been consistently reported in diabetes [14], [15] and is involved in vascular dysfunction [15]. Trx is an important antioxidant system and Trx–Txnip interaction is critical in redox regulation [7].

Acknowledgments

This work was supported by AHA-TX 0565134Y (YHS), AHA-0730190 N (YHS) R01-HL071608 (XLW), and the National Basic Research Program (also called 973 Program) 2006CB503803, Shandong Province Bureau of Public Health 1020 Program.

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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

Abstract

Introduction

Section snippets

Material and methods

High glucose increases the ROS production through p38 MAPK pathway

Discussion

Acknowledgments

Free Radic. Biol. Med.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Mol. Cell. Cardiol.

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

J. Biol. Chem.

Cell

Cell

Mol. Cell. Endocrinol.

Rosiglitazone reduces glucose-induced oxidative stress mediated by NAD(P)H oxidase via AMPK-dependent mechanism

Arterioscler. Thromb. Vasc. Biol.

NADPH oxidases, reactive oxygen species, and hypertension: clinical implications and therapeutic possibilities

Diabetes Care