Biosynthesis of hemiketal eicosanoids by cross-over of the 5-lipoxygenase and cyclooxygenase-2 pathways

Abstract

The prostaglandin and leukotriene families of lipid mediators are formed via two distinct biosynthetic pathways that are initiated by the oxygenation of arachidonic acid by either cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LOX), respectively. The 5-LOX product 5S-hydroxyeicosatetraenoic acid, however, can also serve as an efficient substrate for COX-2, forming a bicyclic diendoperoxide with structural similarities to the arachidonic acid-derived prostaglandin endoperoxide PGH 2 [Schneider C, et al. (2006) J Am Chem Soc 128:720-721]. Here we identify two cyclic hemiketal (HK) eicosanoids, HKD 2 and HKE 2 , as the major nonenzymatic rearrangement products of the diendoperoxide using liquid chromatography-mass spectrometry analyses as well as UV and NMR spectroscopy. HKD 2 and HKE 2 are furoketals formed by spontaneous cyclization of their respective 8,9-dioxo-5S,11R,12S,15S-tetrahydroxy-or 11,12-dioxo-5S,8S,9S,15S-tetrahydroxy-eicosadi-6E,13E-enoic acid precursors, resulting from opening of the 9S,11R-and 8S,12S-peroxide rings of the diendoperoxide. Furthermore, the diendoperoxide is an efficient substrate for the hematopoietic type of prostaglandin D synthase resulting in formation of HKD 2 , equivalent to the enzymatic transformation of PGH 2 to PGD 2 . HKD 2 and HKE 2 were formed in human blood leukocytes activated with bacterial lipopolysaccharide and calcium ionophore A23187, and biosynthesis was blocked by inhibitors of 5-LOX or COX-2. HKD 2 and HKE 2 stimulated migration and tubulogenesis of microvascular endothelial cells, implicating a proangiogenic role of the hemiketals in inflammatory sites that involve expression of 5-LOX and COX-2. Identification of the highly oxygenated hemiketal eicosanoids provides evidence for a previously unrecognized biosynthetic cross-over of the 5-LOX and COX-2 pathways.L eukotrienes and prostaglandins are lipid mediators derived from oxidative modification of arachidonic acid. Both families of eicosanoids exert inflammatory and immunomodulatory functions in disease, as well as homeostatic functions in normal physiological processes (1). Atherosclerosis, asthma, and many types of cancer are prototypical inflammatory diseases that are characterized by concomitant formation of both leukotrienes and prostaglandins (2). The formation of leukotrienes and prostaglandins diverges at the point of the initial oxygenation of the common arachidonic acid substrate by either 5-lipoxygenase (5-LOX) or cyclooxygenase-2 (COX-2), respectively. From there, biosynthesis proceeds along separate and distinct pathways, each utilizing specific enzymes that catalyze complex reactions using highly unstable substrates (3, 4).The possibility of a biosynthetic convergence of the 5-LOX and COX-2 pathways was implicated when the 5-LOX product 5S-hydroxyeicosatetraenoic acid (5S-HETE) was identified as an efficient and specific substrate for oxygenation by recombinant COX-2 (5). The COX-2 oxygenation product of 5S-HETE is a bicyclic diendoperoxide with structural similarities to the prostaglandin (PG) e...

Biomimetic synthesis of hemiketal eicosanoids for biological testing

“…All reactions require phenol (0.5 mM) as a co-substrate in the peroxidase active site and hematin (1 μM, freshly prepared) to replenish heme in the POX active site that may have been lost during purification of the enzyme. The hemiketals can be analyzed by their molecular ion using LC-MS in the negative ion mode ( m / z 399) or by characteristic fragment ions in SRM analyses (HKE 2 : m / z 399 -> 151; HKD 2 : m / z 399 -> 183) (7). …”

“…Treatment with redox active metal ions or hematin results in cleavage of the peroxide moieties, yielding the aldehydes 8-oxo-5-hydroxy-6 E -octenoic acid, malondialdehyde, and 4-hydroxy-2 E -nonenal (6). In the absence of a redox catalyst the di-endoperoxide rearranges by opening of the peroxide moieties into keto/hydroxy groups that engage in intramolecular acetal formation yielding the hemiketals HKE 2 and HKD 2 (7). …”

“…Reaction with the lipocalin-type PGDS may result in HKE 2 (7). Activated human leukocytes stimulated with LPS and calcium ionophore A23187 ex vivo release HKE 2 and HKD 2 but it is not known whether these are enzymatic or non-enzymatic rearrangement products of the COX-2 derived di-endoperoxide (7). Stimulation of human leukocytes also gives 5,11- and 5,15-diHETE as products of the 5-LOX/COX-2 biosynthetic cross-over pathway (8).…”

“…The HKs have been shown to stimulate tubulogenesis in microvascular endothelial cells isolated from mouse lungs (7). The endothelial cells also migrated in response to HK treatment.…”

COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

“…While these studies were in progress, we identifi ed two hemiketal eicosanoids as the major transformation products of the unstable di-endoperoxide. The hemiketals are present in activated human leukocytes and induce tubulogenesis of endothelial cells ( 32 ).…”