Original ContributionShear stress stabilizes NF-E2-related factor 2 and induces antioxidant genes in endothelial cells: Role of reactive oxygen/nitrogen species
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 (O2−) 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 O2−. McNally et al. showed production of O2− 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.