Novel COX-2 products of n-3 polyunsaturated fatty acid-ethanolamine-conjugates identified in RAW264.7 macrophages

Abstract

Supplementary key words cyclooxygenase • fatty acid amides • fatty acid oxidation • high-performance liquid chromatography • inflammation • mass spectrometry • prostaglandins • cyclooxygenase 2 Cyclooxygenase 2 (COX-2) is a nonconstitutional enzyme that is upregulated upon inflammation in order to generate inflammatory regulators (1-3). It is known to convert arachidonic acid (AA) into prostaglandin (PG)H 2 , the precursor of various inflammation-regulating PGs and thromboxanes (1, 2, 4). Although AA is considered the prototypical COX-2 substrate, the enzyme has a broad substrate specificity that includes other PUFAs and their derivatives, such as the endocannabinoid, arachidonoylethanolamide (AEA; also known as anandamide) (2, 5-11). Previous studies revealed that COX-2 converts AEA into PG ethanolamides (EAs), also called prostamides, and potent anti-inflammatory monohydroxylated AEAs (5, 6, 11-14). Although various amide-and ester-bound derivatives of AA are converted by COX-2 (5, 6, 13-17), the interaction between COX-2 and endocannabinoid-like molecules derived from PUFAs other than AA is barely investigated. Previous work revealed that docosahexaenoylethanolamide (DHEA), an n-3 PUFA endocannabinoid-like metabolite, Abstract Cyclooxygenase 2 (COX-2) plays a key role in the regulation of inflammation by catalyzing the oxygenation of PUFAs to prostaglandins (PGs) and hydroperoxides. Next to this, COX-2 can metabolize neutral lipids, including endocannabinoid-like esters and amides. We developed an LC-HRMS-based human recombinant (h)COX-2 screening assay to examine its ability to also convert n-3 PUFA-derived N-acylethanolamines. Our assay yields known hCOX-2-derived products from established PUFAs and anandamide. Subsequently, we proved that eicosapentaenoylethanolamide (EPEA), the N-acylethanolamine derivative of EPA, is converted into PGE 3-ethanolamide (PGE 3-EA), and into 11-, 14-, and 18-hydroxyeicosapentaenoyl-EA (11-, 14-, and 18-HEPE-EA, respectively). Interestingly, we demonstrated that docosahexaenoylethanolamide (DHEA) is converted by hCOX-2 into the previously unknown metabolites, 13-and 16-hydroxy-DHEA (13-and 16-HDHEA, respectively). These products were also produced by lipopolysaccharide-stimulated RAW267.4 macrophages incubated with DHEA. No oxygenated DHEA metabolites were detected when the selective COX-2 inhibitor, celecoxib, was added to the cells, further underlining the role of COX-2 in the formation of the novel hydroxylated products. This work demonstrates for the first time that DHEA and EPEA are converted by COX-2 into previously unknown hydroxylated metabolites and invites future studies toward the biological effects of these metabolites.-de Bus, I., H. Zuilhof, R. Witkamp, M. Balvers, and B. Albada. Novel COX-2 products of n-3 polyunsaturated fatty acid-ethanolamine-conjugates identified in RAW264.7 macrophages.

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“…COX-2 could metabolize neutral lipids, including endocannabinoid-like esters and amides. Knock out of COX-2 could inhibit lipid accumulation in HepG2 cells by promoting mitophagy ( Chen et al, 2019 ; de Bus et al, 2019 ). Cox-2 could also affect the lipid signal transduction by regulating fatty acid synthesis enzyme such as FASN ( Qiu et al, 2019 ).…”

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“…Moreover, RvE1 is a derivative of EPA, which blocks transient receptor potential vanilloid type -1 and TNF-α signaling and relieves inflammation. Taken together, ω-3 PUFAs can down-regulate endotoxin- and cytokine-induced expression of COX-2 and lipid peroxidase, and inhibit phospholipase activity, thereby reducing AA and its induced eicosanoids content of inflammatory mediators ( 54 ).…”

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“…Additionally, COX-2 can oxygenate AA in complex lipids for example; arachidonoylethanolamide, 2-arachidonoylglycerol (2-AG), and N-arachidonoyl-glycine (147)(148)(149). Recently, 2-AA-lysoPL, and ethanolamide derivatives of EPA and DHA were also shown to be COX-2 substrates (88,150). Oxygenation of AA-lysoPL by COX-2 generates eicosanoid-lysoPL, which can be hydrolyzed to release eicosanoids through intracellular lipases (88).…”