RESUMEN
Besides their well-characterized proinflammatory and proatherogenic effects, oxidized phospholipids, such as oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine) have been shown to have beneficial responses in vascular cells via induction of antioxidant enzymes such as heme oxygenase-1. We therefore hypothesized that oxPAPC could evoke a general cytoprotective response via activation of antioxidative transcription factor Nrf2. Here, we show that oxPAPC increases nuclear accumulation of Nrf2. Using the small interfering RNA approach, we demonstrate that Nrf2 is critical in mediating the induction of glutamate-cysteine ligase modifier subunit (GCLM) and NAD(P)H quinone oxidoreductase-1 (NQO1) by oxPAPC in human endothelial cells, whereas the contribution to the induction of heme oxygenase-1 was less significant. The induction of GCLM and NQO1 was attenuated by reduction of electrophilic groups with sodium borohydrate, as well as treatment with thiol antioxidant N-acetylcysteine, suggesting that the thiol reactivity of oxPAPC is largely mediating its effect on Nrf2-responsive genes. Moreover, we show that oxidized phospholipid having a highly electrophilic isoprostane ring in its sn-2 position is a potent inducer of Nrf2 target genes. Finally, we demonstrate that the oxPAPC-inducible expression of heme oxygenase-1, GCLM, and NQO1 is lower in Nrf2-null than wild-type mouse carotid arteries in vivo. We suggest that the activation of Nrf2 by oxidized phospholipids provides a mechanism by which their deleterious effects are limited in the vasculature.
Asunto(s)
Antioxidantes/metabolismo , Arterias Carótidas/enzimología , Núcleo Celular/metabolismo , Células Endoteliales/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Fosfatidilcolinas/farmacocinética , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Animales , Arterias Carótidas/citología , Núcleo Celular/genética , Células Endoteliales/citología , Regulación Enzimológica de la Expresión Génica/fisiología , Glutamato-Cisteína Ligasa/biosíntesis , Glutamato-Cisteína Ligasa/genética , Hemo-Oxigenasa 1/biosíntesis , Hemo-Oxigenasa 1/genética , Ratones , Ratones Mutantes , NAD(P)H Deshidrogenasa (Quinona) , NADPH Deshidrogenasa/biosíntesis , NADPH Deshidrogenasa/genética , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/genética , Oxidación-Reducción/efectos de los fármacos , Fosfatidilcolinas/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
OBJECTIVE: Atheroprotective blood flow induces expression of anti-inflammatory Krüppel-like factor 2 (KLF2) and activates antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in vascular endothelium. Previously, we obtained KLF2-induced gene expression profiles in ECs, containing several Nrf2 target genes. Our aim was to investigate the role of KLF2 in shear stress-mediated activation of Nrf2 in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Expression of Nrf2 and its targets NAD(P)H dehydrogenase quinone 1 (NQO1) and heme oxygenase (HO-1) was elevated by shear and KLF2. KLF2 knockdown showed that shear-induced expression of NQO1 but not Nrf2 was dependent on KLF2. KLF2 overexpression in absence of flow resulted in more efficient activation of Nrf2 by tert-butyl hydroquinone (tBHQ) through enhanced nuclear localization, and promoted expression of a large panel of Nrf2-dependent genes resulting in superior protection against oxidative stress. Comparison of shear-, KLF2-, and Nrf2-induced transcriptomes showed that the majority of shear-modulated gene sets is influenced by KLF2 or Nrf2. CONCLUSIONS: We report that KLF2 substantially enhances antioxidant activity of Nrf2 by increasing its nuclear localization and activation. The synergistic activity of these two transcription factors forms a major contribution to the shear stress-elicited transcriptome in endothelial cells.