15-Oxoeicosatetraenoic acid is a 15-hydroxyprostaglandin dehydrogenase-derived electrophilic mediator of inflammatory signaling pathways

Abstract

Bioactive lipids govern cellular homeostasis and pathogenic inflammatory processes. Current dogma holds that bioactive lipids, such as prostaglandins and lipoxins, are inactivated by 15-hydroxyprostaglandin dehydrogenase (15PGDH). In contrast, the present results reveal that catabolic "inactivation" of hydroxylated polyunsaturated fatty acids (PUFAs) yields electrophilic α,β-unsaturated ketone derivatives. These endogenously produced species are chemically reactive signaling mediators that induce tissue protective events. Electrophilic fatty acids diversify the proteome through post-translational alkylation of nucleophilic cysteines in key transcriptional regulatory proteins and enzymes that govern cellular metabolic and inflammatory homeostasis. 15PGDH regulates these processes as it is responsible for the formation of numerous electrophilic fatty acids including the arachidonic acid metabolite, 15-oxoeicosatetraenoic acid (15-oxoETE). Herein, the role of 15-oxoETE in regulating signaling responses is reported. In cell cultures, 15-oxoETE activates Nrf2-regulated antioxidant responses (AR) and inhibits NF-κB-mediated pro-inflammatory responses via IKKβ inhibition. Inhibition of glutathione S-transferases using ethacrynic acid incrementally increased the signaling capacity of 15-oxoETE by decreasing 15-oxoETE-GSH adduct formation. This work demonstrates that 15PGDH plays a role in the regulation of cell and tissue homeostasis via the production of electrophilic fatty acid signaling mediators.

Keywords: 15-KETE; 15-oxoETE; 15PGDH; Bioactive lipid; Electrophile.

Fig 1

Structures of electrophilic fatty acids and 15-oxoETE formation. (A) Chemical structures of 15-oxoETE, 15-ketoPGE₂ and 15d-PGJ₂ provided for comparison. (B) The currently elucidated COX-2/15-LOX and 15PGDH dependent pathway for generation of 15-oxoETE. Arachidonic acid (AA) is converted by cyclooxygenase-2 (COX) or 15-lipoxygenase (LOX) to 15-hydroperoxyeicosatetraenoic acid (15-HpETE) which is reduced by a peroxidase to 15-HETE. 15PGDH oxidizes 15-HETE to 15-oxoETE, which contains an α,β-unsaturated ketone that confers 15-oxoETE with electrophilic properties.

Fig 1

Structures of electrophilic fatty acids and 15-oxoETE formation. (A) Chemical structures of 15-oxoETE, 15-ketoPGE₂ and 15d-PGJ₂ provided for comparison. (B) The currently elucidated COX-2/15-LOX and 15PGDH dependent pathway for generation of 15-oxoETE. Arachidonic acid (AA) is converted by cyclooxygenase-2 (COX) or 15-lipoxygenase (LOX) to 15-hydroperoxyeicosatetraenoic acid (15-HpETE) which is reduced by a peroxidase to 15-HETE. 15PGDH oxidizes 15-HETE to 15-oxoETE, which contains an α,β-unsaturated ketone that confers 15-oxoETE with electrophilic properties.

Fig. 2

15-oxoETE up-regulates hemeoxygense-1. (A) THP-1 cells were incubated with increasing concentrations of 15-oxoETE for 6 hr (B) THP-1 cells were incubated with 25 µM 15-oxoETE from 3 to 24 hr. HO-1 expression was used as a marker of activation of the Nrf2-mediated antioxidant response. One way ANOVA with a Bonferroni post-test was used for statistical analysis.

Fig. 2

15-oxoETE up-regulates hemeoxygense-1. (A) THP-1 cells were incubated with increasing concentrations of 15-oxoETE for 6 hr (B) THP-1 cells were incubated with 25 µM 15-oxoETE from 3 to 24 hr. HO-1 expression was used as a marker of activation of the Nrf2-mediated antioxidant response. One way ANOVA with a Bonferroni post-test was used for statistical analysis.

Fig. 3

15-oxoETE up-regulates the Nrf2-mediated anti-oxidant response. THP-1 cells were incubated with 25 µM 15-oxoETE for 12 hr (A) and 18 hr (B). Student's t-test was used for statistical analysis.

Fig. 4

Intracellular 15-oxoETE levels are low. 15-oxoETE (A) and 15-oxoETE-GSH adduct (B) levels were measured in media and cell lysate after a 12 hr incubation with 25 µM 15-oxoETE. Pre-treatment of THP-1 cells with 100 µM ethacrynic acid before 25 µM 15-oxoETE treatment for 1.5 hr resulted in an incremental increase in free intracellular 15-oxoETE (C) and a decrease in 15-oxoETE-GSH adduct formation (D). (E) Product ion spectrum of 15-oxoETE-GSH.

Fig. 4

Intracellular 15-oxoETE levels are low. 15-oxoETE (A) and 15-oxoETE-GSH adduct (B) levels were measured in media and cell lysate after a 12 hr incubation with 25 µM 15-oxoETE. Pre-treatment of THP-1 cells with 100 µM ethacrynic acid before 25 µM 15-oxoETE treatment for 1.5 hr resulted in an incremental increase in free intracellular 15-oxoETE (C) and a decrease in 15-oxoETE-GSH adduct formation (D). (E) Product ion spectrum of 15-oxoETE-GSH.

Fig. 4

Intracellular 15-oxoETE levels are low. 15-oxoETE (A) and 15-oxoETE-GSH adduct (B) levels were measured in media and cell lysate after a 12 hr incubation with 25 µM 15-oxoETE. Pre-treatment of THP-1 cells with 100 µM ethacrynic acid before 25 µM 15-oxoETE treatment for 1.5 hr resulted in an incremental increase in free intracellular 15-oxoETE (C) and a decrease in 15-oxoETE-GSH adduct formation (D). (E) Product ion spectrum of 15-oxoETE-GSH.

Fig. 5

15-oxoETE inhibits NF-κB activity. (A) HEK293T cells stably expressing a 5x-κB driven firefly luciferase reporter were pre-treated with or without 100 µM of the GST inhibitor ethacrynic acid for 1 hr then treated with 15-oxoETE for 5 min at increasing doses followed by TNFα (40 ng/mL) for 6 hr. Luciferase activity was determined in a luminometer then normalized to the vehicle (0.25% DMSO) treated control. (B) Recombinant human IKKβ was incubated with increasing concentrations of 15-oxoETE or wedelolactone (100 µM), followed by addition of biotin tagged substrate (IκBα) and ATP.

Fig. 6

15-oxoETE inhibits NF-κB cytokines. THP-1 cells were treated with 15-oxoETE (25 µM), LPS (100 ng/mL) and LPS + 15-oxoETE for 6 hr. Relative expression of TNFα, IL-6 and IL-1β mRNA was determined. One way ANOVA with a Bonferroni post-test was used for statistical analysis.

Scheme 1

An illustration of 15-oxoETE-mediated modulation of inflammation via Nrf2 and NF-κB signaling pathways.