Original Contribution
Shear stress stabilizes NF-E2-related factor 2 and induces antioxidant genes in endothelial cells: Role of reactive oxygen/nitrogen species

https://doi.org/10.1016/j.freeradbiomed.2006.10.043Get rights and content

Abstract

We have previously reported that antioxidant response element (ARE)-regulated genes, such as heme oxygenase 1 (HO-1), sequestosome 1 (SQSTM1), and NAD(P)H quinone oxidoreductase 1 (NQO1), are induced in human umbilical vein endothelial cells (HUVEC) upon exposure to laminar shear stress. In the present study, we have confirmed a critical role for NF-E2-related factor 2 (Nrf2) in the induction of gene expression in HUVEC exposed to laminar shear stress. Although the mRNA levels of Nrf2 were unchanged during exposure to shear stress, the protein levels of Nrf2 were markedly increased. Small interfering RNA (SiRNA) against Nrf2 significantly attenuated the expression of Nrf2-regulated genes such as HO-1, SQSTM1, NQO1, glutamate-cysteine ligase modifier subunit (GCLM), and ferritin heavy chain. Nrf2 was rapidly degraded in cells treated with cycloheximide under static conditions, but shear stress decreased the rate of Nrf2 degradation. Incubation with the thiol antioxidant N-acetylcysteine strongly inhibited both the Nrf2 accumulation and the expression of Nrf2-regulated genes such as HO-1, GCLM, and SQSTM1. Nitric oxide (NO) production was increased with the strength of shear stress but neither the inhibitor of endothelial NO synthase (eNOS) nor the siRNA against eNOS affected the expression of Nrf2-regulated genes. A xanthine oxidase inhibitor oxypurinol and the flavoprotein inhibitor diphenyleneiodonium, which inhibits NAD(P)H oxidase and mitochondrial respiratory chain, markedly suppressed the expression of these genes. Moreover, diphenylpyrenlphosphine, a reducing compound of lipid hydroperoxides, also significantly suppressed Nrf2-regulated gene expression. Taken together, these findings suggest that shear stress stabilizes Nrf2 protein via the lipid peroxidation elicited by xanthine oxidase and flavoprotein mediated generation of superoxide, resulting in gene induction by the Nrf2-ARE signaling pathway.

Introduction

Vascular endothelial cells are continually subjected to shear stress forces due to blood flow. Atherosclerotic lesions are more likely to develop focally at bifurcations and branch points in arteries [1], [2]. It has been reported that the most vulnerable regions are exposed to nonunidirectional, disturbed or oscillatory flow and that atherosclerosis-resistant regions are in contrast exposed to unidirectional laminar flow [3]. We have developed a novel coculture system which exposes vascular cells to combinations of laminar shear stress, oxygen concentration gradient, and low-density lipoprotein (LDL) loading. We evaluated the effects of each factor alone or in combination on gene expression in human umbilical vein endothelial cells (HUVEC) by DNA microarray analysis [4]. It was revealed that many NF-E2-related factor 2 (Nrf2)-regulated genes, such as heme oxygenase 1 (HO-1), sequestosome 1 (SQSTM1/A170), NAD(P)H quinone oxidoreductase 1 (NQO1), solute carrier family 7A No. 11 (SLC7A11/xCT), and the glutamate-cysteine ligase modifier subunit (GCLM) are induced by laminar flow independent of other treatment conditions. Certain genes were specifically affected by exposure to the oxygen gradient and/or high concentration of LDL under shear stress, but the degree of the induction was very low. Upregulation of antioxidant response element (ARE)-regulated genes such as NQO1, HO-1, ferritin, microsomal epoxide hydrolase, glutathione S-transferase, and γ-glutamylcysteine synthase is also induced by steady laminar flow in human aortic endothelial cells (HAEC) [5]. We confirmed the critical contribution of Nrf2 to the gene expression induced by laminar flow by a small interfering RNA (siRNA)-mediated knockdown experiment with HAEC [6]. Thus, it appears that shear stress is the most critical factor affecting gene expression and that Nrf2-regulating proteins may contribute to the protection of endothelial cells against various forms of vascular stress.

Nrf2 plays an essential role in the antioxidant response element (ARE)-mediated expression of a group of genes of phase II detoxification enzymes and antioxidant proteins crucial for protecting cells against electrophile toxicity, oxidative stress, and carcinogenesis [7], [8], [9]. Nrf2 is negatively regulated by Kelch-like ECH-associated protein 1 (Keap1) under basal conditions, preventing Nrf2 from activating target genes [10], [11]. Electrophilic compounds could attack the two cysteine residues in the Keap1 intervening region (IVR), leading to conformational change of the Keap1-Nrf2 association motif and dissociation of Nrf2 from Keap1 [12]. It is reported that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a product of cyclooxygenase-2 (COX2), activates the Nrf2 pathway in HAEC exposed to laminar shear stress through binding to cysteines of Keap1 [6]. Go et al. demonstrated that reactive oxygen/nitrogen species (ROS/RNS) play an important role as signaling molecules in bovine thoracic aortas (BAEC) exposed to laminar shear stress [13], [14]. It was shown that NAD(P)H oxidase-derived superoxide (O2radical dot) production was enhanced in mouse endothelial cells by oscillatory and laminar shear after 1 h, but was decreased after 18 h laminar shear stress [15]. Mitochondrial respiratory chain and xanthine oxidase (XO) are other sources of O2radical dot. McNally et al. showed production of O2radical dot by XO in response to oscillatory shear stress [16]. It has been reported that nitric oxide (NO) is produced by laminar shear stress and is involved in intracellular signaling [17], [18], [19]. Buckley et al. have shown a contribution of exogenous NO to Nrf2 nuclear translocation in vascular endothelium [20].

With the aim of obtaining a better understanding the mechanism for Nrf2/ARE-dependent gene expression by laminar shear stress in human endothelial cells, we investigated the roles of ROS/RNS in the expression of Nrf2-regulated genes in HUVEC exposed to laminar shear stress.

Section snippets

Cell culture

HUVEC (Clonetics, San Diego, CA) were grown in endothelial growth medium (EGM-2) containing 2% FBS (Clonetics) and used within four passages. HUVEC (2 × 105 cells) were cultured on a cell culture insert coated with human fibronectin, having a 0.4 μm pore size filter, (Biocoat, Becton Dickinson, Frankrin Lakes, NJ), and were used for experiments the next day.

Shear stress exposure

The flow system (MK2000, Yamato Scientific. Co., Tokyo, Japan) has been previously described [4]. Briefly, cells seeded on the cell culture

Nrf2 is essential for gene induction by laminar flow

To demonstrate that shear stress induces ARE-containing genes via the Nrf2 pathway, we developed a Nrf2 gene knockdown model in HUVEC by using siRNA transfection. The effect of Nrf2 knockdown was confirmed by quantitative real-time PCR (data not shown) and Western blot analysis. As shown in Fig. 1, Nrf2 protein levels were markedly decreased following siRNA transfection. Transfection with Nrf2-siRNA almost completely diminished the induction levels of GCLM, SQSTM1, NGO1, and ferritin H, and

Discussion

The association of Nrf2 with ARE in the promotor regions of antioxidant genes is a key regulatory step in stress protein expression. Keap1 has been identified as a cytosolic binding protein for Nrf2 which associates with the Kelch domain of Keap1, and is sequestered in association with the actin cytoskeleton under normal physiological conditions, which in turn allows proteasomal degradation of Nrf2. Under oxidative stress or treatment with electrophilic reagents, Nrf2 is released through the

Acknowledgments

This study was supported by the Program of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), by NFAT project of New Energy and Industrial Technology Development Organization (NEDO), and by Special Coordination Fund for Science and Technology and the Academic Frontier Research Project on “New Frontier of Biomedical Engineering Research” of Ministry of Education, Culture, Sports, Science, and Technology. We thank Dr. Tomonori Hosoya and Dr. Richard

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    E.W. and W.T. contributed equally to this work.

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