Nrf2 Activation by 5-lipoxygenase Metabolites in Human Umbilical Vascular Endothelial Cells

Abstract

5-hydroxyeicosatetraenoic acid (5-HETE) and 5-hydroxyeicosapentaenoic acid (5-HEPE) are major metabolites produced by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) and eicosapentaenoic acid (EPA). Effects of hydroxides on endothelial cells are unclear, although 5-LOX is known to increase at arteriosclerotic lesions. To investigate the effects of hydroxides on human umbilical vein endothelial cells (HUVECs), the cells were treated with 50 μM each of AA, EPA, 5-HETE, and 5-HEPE. Treatment of HUVECs with 5-HETE and 5-HEPE, rather than with AA and EPA, increased the nuclear translocation of NF-E2 related factor 2 (Nrf2) and upregulated the expression of heme oxygenase-1 and cystine/glutamate transporter regulated by Nrf2. Reactive oxygen species (ROS) generation was markedly elevated in HUVECs after treatment with 5-HETE and 5-HEPE, and the pretreatment with α-tocopherol abrogated ROS levels similar to those in the vehicle control. However, ROS generation was independent of Nrf2 activation induced by 5-HETE and 5-HEPE. 5-HETE was converted to 5-oxo-eicosatetraenoic acid (5-oxo-ETE) in HUVECs, and 5-oxo-ETE increased Nrf2 activation. These results suggest that 5-HETE works as an Nrf2 activator through the metabolite 5-oxo-ETE in HUVECs. Similarly, 5-HEPE works in the same way, because 5-HEPE is metabolized to 5-oxo-eicosapentaenoic acid through the same pathway as that for 5-HETE.

Keywords: 5-hydroxyeicosapentaenoic acid; 5-hydroxyeicosatetraenoic acid; 5-lipoxygenase; 5-oxo-eicosatetraenoic acid; Nrf2; human umbilical vein endothelial cells; reactive oxygen species.

Figure 1

Effects of hydroxy fatty acids on Nrf2 translocation and expression of genes regulated by the Keap1-Nrf2 pathway in HUVECs. Nrf2 translocation was detected by immunostaining (green), and the nuclei were stained with PI (red). Cells were treated for 3 h with: 0.5% MeOH (vehicle) (A); 10 μM tBHQ (B); 50 μM AA (C); 50 μM EPA (D); 50 μM 5-HETE (E); and 50 μM 5-HEPE (F). The intensity of the green fluorescence signal in the nucleus was quantified using Image J (G). Data are expressed as the mean ± standard deviation (SD) (n = 10). Significant differences among the groups are indicated with different letters (one-way ANOVA followed by Tukey’s test, p < 0.05). Gene expression levels of: HMOX1 (H); and SLC7A11 (I) in HUVECs were analyzed and normalized to 18S gene expression levels (as an internal control) after incubation of each sample for 6 h. Data are expressed as mean ± SD (n = 4). Significant differences among the groups are indicated with different letters (one-way ANOVA followed by Tukey’s test, p < 0.05). Abbreviations: Nrf2, NF-E2 related factor 2; Keap1, Kelch-like ECH-associated protein 1; HUVEC, human umbilical vein endothelial cell; PI, propidium iodide; MeOH, methanol; tBHQ, tert-butylhydroquinone; AA, arachidonic acid; EPA, eicosapentaenoic acid; 5-HETE, 5-hydroxyeicosatetraenoic acid; 5-HEPE, 5-hydroxyeicosapentaenoic acid; ANOVA, analysis of variance; and HMOX1, heme oxygenase 1.

Figure 2

Reactive oxygen species (ROS) generation in HUVECs. Cells were incubated with 0.5% MeOH, tertiary butylhydroperoxide (TBHP), AA, EPA, 5-HETE, or 5-HEPE, for 1 h after treatment with or without 400 μM α-tocopherol for 1 h. ROS were detected using CellROX Green and measured with a flow cytometer, and the ratio of ROS positive cells to total live cell was represented as the percentage of ROS positive cells. Histograms of HUVECs treated with 0.5% MeOH (negative control) and 400μM TBHP (positive control) analyzed by flow cytometry are shown above the graph. Data are expressed as mean ± SD (n = 4). * p < 0.05 compared with 0.5% MeOH; † p < 0.05 compared with 400 μM TBHP; # p < 0.05 and § p < 0.05 compared with 50 μM 5-HETE and 50 μM 5-HEPE, respectively. Statistical analysis was performed with one-way ANOVA followed by Tukey’s test.

Figure 3

Effects of antioxidant treatment on nuclear translocation of Nrf2 in HUVECs. Nrf2 translocation was detected by immunostaining (green), and the nuclei were stained with PI (red). HUVECs were pretreated with or without 400μM α-tocopherol for 1 h and followed by incubated for 3 h with each samples: 0.5% MeOH (vehicle) (A); 0.5% MeOH with α-tocopherol (B);10 μM tBHQ (C); 10 μM tBHQ with α-tocopherol (D); 400 μM TBHP (E); 400 μM TBHP with α-tocopherol (F); 50 μM 5-HETE (G); 50 μM 5-HETE with α-tocopherol (H); 50 μM 5-HEPE (I); and 50 μM 5-HEPE with α-tocopherol (J). The intensity of the green fluorescence signal in the nucleus was quantified using Image J (K). Data are expressed as the mean ± SD (n = 8). The intensity of the green fluorescence signal in the nucleus was quantified using Image J (K). Data are expressed as the mean ± SD (n = 8). * p < 0.05 compared with 0.5% MeOH. Statistics analysis was performed with one-way ANOVA followed by Tukey’s test.

Figure 3

Effects of antioxidant treatment on nuclear translocation of Nrf2 in HUVECs. Nrf2 translocation was detected by immunostaining (green), and the nuclei were stained with PI (red). HUVECs were pretreated with or without 400μM α-tocopherol for 1 h and followed by incubated for 3 h with each samples: 0.5% MeOH (vehicle) (A); 0.5% MeOH with α-tocopherol (B);10 μM tBHQ (C); 10 μM tBHQ with α-tocopherol (D); 400 μM TBHP (E); 400 μM TBHP with α-tocopherol (F); 50 μM 5-HETE (G); 50 μM 5-HETE with α-tocopherol (H); 50 μM 5-HEPE (I); and 50 μM 5-HEPE with α-tocopherol (J). The intensity of the green fluorescence signal in the nucleus was quantified using Image J (K). Data are expressed as the mean ± SD (n = 8). The intensity of the green fluorescence signal in the nucleus was quantified using Image J (K). Data are expressed as the mean ± SD (n = 8). * p < 0.05 compared with 0.5% MeOH. Statistics analysis was performed with one-way ANOVA followed by Tukey’s test.

Figure 4

Effects of antioxidant treatment on HMOX1 upregulation by hydroxy fatty acids in HUVECs. Cells were pretreated with 400 μM α-tocopherol for 1 h; subsequently, 0.5% MeOH, 200 μM TBHP, 400 μM TBHP, 50 μM 5-HETE, or 50 μM 5-HEPE was added. After incubation with each sample for 6 h, gene expression of the cells was analyzed. Data are expressed as mean ± SD (n = 3). * p < 0.05 compared with 0.5% MeOH; # p < 0.05 compared with 200 μM TBHP; † p < 0.05 compared with 400 μM TBHP. Statistics analysis was performed with one-way ANOVA followed by Tukey’s test.

Figure 5

Effects of 5-oxo-ETE on Nrf2 activation in HUVECs. Nuclear translocation of Nrf2 was detected by immunostaining. The cells were treated for 3 h with: 0.5% MeOH (vehicle control) (A); and 5 μM 5-oxo-ETE (B); and the pretreatment with 400 μM α-tocopherol for 1 h was performed before treatment of 5-oxo-ETE (C). The intensity of the green fluorescence signal in the nucleus was quantified using Image J (D); Data are expressed as the mean ± SD (n = 8) Significant differences among the groups are indicated with different letters (one-way ANOVA followed by a post hoc Tukey’s test, p < 0.05). Gene expression of HMOX1 in the cells treated with 1, 5, and 10 μM 5-oxo-ETE for 6 h was analyzed (E). Data are expressed as mean ± SD (n = 3). Significant differences among the groups are indicated with different letters (one-way ANOVA followed by a post hoc Tukey’s test, p < 0.05). ROS generation in HUVECs was detected by CellROX Green (F). HUVECs were treated with 400 μM α-tocopherol for 1 h, and subsequently the cells were incubated for 1 h after the addition of 5 μM 5-oxo-ETE. Data are expressed as the mean ± SD (n = 4).