Oxygenation by COX-2 (cyclo-oxygenase-2) of 3-HETE (3-hydroxyeicosatetraenoic acid), a fungal mimetic of arachidonic acid, produces a cascade of novel bioactive 3-hydroxyeicosanoids

Abstract

Cyclo-oxygenases-1/2 (COX-1/2) catalyse the oxygenation of AA (arachidonic acid) and related polyunsaturated fatty acids to endoperoxide precursors of prostanoids. COX-1 is referred to as a constitutive enzyme involved in haemostasis, whereas COX-2 is an inducible enzyme expressed in inflammatory diseases and cancer. The fungus Dipodascopsis uninucleata has been shown by us to convert exogenous AA into 3(R)-HETE [3(R)-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid]. 3R-HETE is stereochemically identical with AA, except that a hydroxy group is attached at its C-3 position. Molecular modelling studies with 3-HETE and COX-1/2 revealed a similar enzyme-substrate structure as reported for AA and COX-1/2. Here, we report that 3-HETE is an appropriate substrate for COX-1 and -2, albeit with a lower activity of oxygenation than AA. Oxygenation of 3-HETE by COX-2 produced a novel cascade of 3-hydroxyeicosanoids, as identified with EI (electron impact)-GC-MS, LC-MS-ES (electrospray) and LC-MS-API (atmospheric pressure ionization) methods. Evidence for in vitro production of 3-hydroxy-PGE2 (3-hydroxy-prostaglandin E2) was obtained upon infection of HeLa cells with Candida albicans at an MOI (multiplicity of infection) of 100. Analogous to interaction of AA and aspirin-treated COX-2, 3-HETE was transformed by acetylated COX-2 to 3,15-di-HETE (3,15-dihydroxy-HETE), whereby C-15 showed the (R)-stereochemistry. 3-Hydroxy-PGs are potent biologically active compounds. Thus 3-hydroxy-PGE2 induced interleukin-6 gene expression via the EP3 receptor (PGE2 receptor 3) in A549 cells, and raised cAMP levels via the EP4 receptor in Jurkat cells. Moreover, 3R,15S-di-HETE triggered the opening of the K+ channel in HTM (human trabecular meshwork) cells, as measured by the patch-clamp technique. Since many fatty acid disorders are associated with an 'escape' of 3-hydroxy fatty acids from the b-oxidation cycle, the production of 3-hydroxyeicosanoids may be critical in modulation of effects of endogenously produced eicosanoids.

Bioactive Oxylipins inSaccharomyces cerevisiae

“…Here, arachidonic acid (AA) is released from the infected or inflamed host tissue /cell and then converted by Candida albicans to 3R-hydroxy eicosatetraenoic acid (3R-HETE), which serves as substrate for COX-2 in host tissue /cell to produce proinflammatory 3-OH-PGE 2 . Strikingly, in this investigation a cascade of novel bioactive 3-OH eicosanoids, produced from 3R-HETE via mammalian COX-2, was uncovered 5 . In addition, it was found that C. albicans selectively upregulates COX-2 in mammalian cells 7 .…”

“…The PGs are produced by aspirinsensitive enzymes i.e. cyclooxygenase 1 (COX-1, a constitutive enzyme involved in hemostasis) and cyclooxygenase 2 (COX-2, an inducible enzyme expressed in inflammatory diseases and cancer) 5 .…”

“…Recently, clear-cut evidence for the role of 3-OH eicosanoids in candidiasis was presented [5][6][7] . Here, arachidonic acid (AA) is released from the infected or inflamed host tissue /cell and then converted by Candida albicans to 3R-hydroxy eicosatetraenoic acid (3R-HETE), which serves as substrate for COX-2 in host tissue /cell to produce proinflammatory 3-OH-PGE 2 .…”

Chemical synthesis, pharmacological characterization, and possible formation in unicellular fungi of 3-hydroxy-anandamide

“…First, it will be necessary to assess if the conversion of anandamide into 3-HAEA does occur also during candidiasis, and, if so, to what extent. The production of 3-hydroxyeicosanoids like 3-hydroxyPGE 2 upon infection of HeLa cells with C. albicans supports the hypothesis that also 3-HAEA might be formed during infection with this pathogenic fungus (23). On the other hand, still enough endogenous anandamide to activate CB 1 and CB 2 receptors might remain even if this conversion were to occur in vivo, and this would counteract the effects of 3-HAEA.…”

“…Thus, Candida can chemically modify the metabolites of the host cell to facilitate infection. Notwithstanding the presence of a C-3 hydroxyl, 3(R)-HETE is a substrate of cyclooxygenase-2 (an enzyme up-regulated during candidiasis), thereby generating compounds that, like 3(R)-hydroxy-PGE 2 , are capable to induce interleukin-6 gene expression via the EP 3 receptor (22,23), and to ultimately trigger further inflammation cascades.…”