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.

Fig. 1.

Nonenzymatic transformation of the 5S-HETE-derived diendoperoxide. [1-¹⁴C]-5S-HETE was reacted with COX-2 for 5 min (A) or 30 min (B) followed by extraction and RP-HPLC analysis with radiodetection. (C) Normalized UV spectra of 5S-HETE and products 3 and 4. (D) LC-ESI-MS analysis (negative ion mode) of an incubation of 5S-HETE with recombinant COX-2. The extracted ion chromatograms corresponding to 5S-HETE (m/z 319), diHETEs (m/z 335), and the diendoperoxide (m/z 399) are shown.

Fig. 2.

Identification of products 3 and 4. (A) NMR analysis of product 4 (hemiketal D₂). The ¹H NMR spectrum is shown with the H,H-COSY spectrum below. The product is a mixture of two isomers in ≈5∶2 ratio. Signals from the minor isomer with a distinct chemical shift are marked with an asterisk. In the H,H-COSY spectrum only the prominent cross-peaks are visible. Weaker signals like the couplings of H5 to H6 and H14 to H15 were discernible at lesser signal to noise. (B and C) LC-ESI-MS2 spectra of 3 and 4.

Fig. 3.

Transformation of the diendoperoxide by hematopoietic prostaglandin D synthase (H-PGDS) to HKD₂. LC-ESI-MS analyses of reactions of 5S-HETE with recombinant COX-2 in the presence of (A) recombinant human H-PGDS, (B) recombinant murine L-PGDS, (C) in the absence of PGDS. (D) Transformation of exogenous 5S-HETE by LPS-activated RAW264.7 cells. The extracted ion chromatograms for m/z 399 are shown.

Fig. 4.

Isolated human leukocytes form HKE₂ and HKD₂. Human leukocytes were stimulated with LPS for 6 h and then with A23187 for 15 min before extraction and LC-ESI-MS-SRM analysis. (A) Standards of HKE₂ (red trace) and HKD₂ (blue) generated by reaction of recombinant human COX-2 with 5S-HETE (black). (B) HKE₂ and HKD₂ formed by the leukocytes following stimulation with LPS and A23187. (C) Addition of exogenous 5S-HETE together with A23187 to the leukocytes increased formation of HKE₂ and HKD₂. (D) Formation of hemiketals was attenuated by preincubation with the COX-2 inhibitor NS-398.

Fig. 5.

HKD₂ and HKE₂ induce formation of capillary-like structures and migration of endothelial cells. (A) Microvascular endothelial cells were plated onto Matrigel in the presence of 125 or 500 nM of HKD₂ or HKE₂. Representative images of capillary-like structures taken 6 h after plating are shown. (B) Quantification of capillary network formation. Cells incubated with HKD₂ or HKE₂ form tubule-like structures more efficiently than vehicle-treated cells (control). Values are the mean ± SD. calculated for 20 images per treatment. Differences between vehicle versus HKD₂ or HKE₂ treated cells (*), or between 125 versus 500 nM treatment (**) were significant (p < 0.05). (C) Migration of cells toward serum-free medium with or without HKD₂ or HKE₂ or complete medium. Values are fold changes versus serum-free medium from four experiments; (*) p < 0.05 versus control.

Fig. 6.

Biosynthesis of hemiketals and prostaglandins. The 5-LOX metabolite 5S-HETE is converted to the diendoperoxide by COX-2 using three molecules of oxygen. The diendoperoxide is a substrate for H-PGDS opening up both endoperoxide groups into keto/hydroxy moieties to give an open chain intermediate. Hemiketal D₂ results form spontaneous attack of the 8-hydroxyl at the 11-keto group. Nonenzymatic rearrangement of the diendoperoxide gives a mixture of HKE₂ and HKD₂. Oxygenation of arachidonic acid by COX-1 or COX-2 yields the prostaglandin endoperoxide PGH₂ that rearranges to PGE₂ and PGD₂.