Coupling between Cyclooxygenase, Terminal Prostanoid Synthase, and Phospholipase A2

Abstract

We have recently shown that two distinct prostaglandin (PG) E 2 synthases show preferential functional coupling with upstream cyclooxygenase (COX)-1 and COX-2 in PGE 2 biosynthesis. To investigate whether other lineage-specific PG synthases also show preferential coupling with either COX isozyme, we introduced these enzymes alone or in combination into 293 cells to reconstitute their functional interrelationship. As did the membrane-bound PGE 2 synthase, the perinuclear enzymes thromboxane synthase and PGI 2 synthase generated their respective products via COX-2 in preference to COX-1 in both the A23187-induced immediate and interleukin-1-induced delayed responses. Hematopoietic PGD 2 synthase preferentially used COX-1 and COX-2 in the A23187-induced immediate and interleukin-1-induced delayed PGD 2 -biosynthetic responses, respectively. This enzyme underwent stimulus-dependent translocation from the cytosol to perinuclear compartments, where COX-1 or COX-2 exists. COX selectivity of these lineage-specific PG synthases was also significantly affected by the concentrations of arachidonate, which was added exogenously to the cells or supplied endogenously by the action of cytosolic or secretory phospholipase A 2 . Collectively, the efficiency of coupling between COXs and specific PG synthases may be crucially influenced by their spatial and temporal compartmentalization and by the amount of arachidonate supplied by PLA 2 s at a moment when PG production takes place.Biosynthesis of prostaglandins (PGs) 1 through the cyclooxygenase (COX) pathway involves oxidation and subsequent isomerization of membrane-derived arachidonic acid (AA) via three sequential enzymatic reactions. The initial step of this metabolic pathway is the stimulus-induced liberation of AA from membrane glycerophospholipids by the action of phospholipase A 2 (PLA 2 ) enzymes, including cytosolic PLA 2 ␣ (cPLA 2 ; group IVA) and several secretory PLA 2 (sPLA 2 ) isozymes (groups IIA, IID, V, and X) (1-10). The released AA is sequentially metabolized to PGG 2 and then to PGH 2 by either COX-1 or COX-2. PGH 2 is then converted to various bioactive PGs (thromboxane (TX) A 2 , PGD 2 , PGE 2 , PGF 2␣ , and PGI 2 ) by the respective terminal PG synthases, which have different structures and exhibit cell-and tissue-specific distributions.Segregated utilization of COX-1 and COX-2 in the PG biosynthetic events has been demonstrated by a number of cell biological, pharmacological, and genetic studies (3,(11)(12)(13)(14)(15)(16)(17). Generally, the constitutive COX-1 is mainly utilized in the immediate PG biosynthesis, which occurs within several minutes after stimulation with Ca 2ϩ mobilizers, whereas the inducible COX-2 is an absolute requirement for delayed PG biosynthesis, which lasts for several hours after proinflammatory stimuli. When cells are first treated with proinflammatory stimuli and subsequently exposed to Ca 2ϩ mobilizers, the induced COX-2 can also promote the immediate response (priming response) (3, 18 -22). However, the precise molecular me...

Cellular Prostaglandin E2 Production by Membrane-bound Prostaglandin E Synthase-2 via Both Cyclooxygenases-1 and -2

“…COX-1 appeared to be distributed rather uniformly throughout the ER membrane, whereas COX-2 was enriched in the perinuclear region. Similar locations of these enzymes were seen in HEK293 cells, as described previously (14,16).…”

Targeted lipidomics reveals mPGES-1-PGE2 as a therapeutic target for multiple sclerosis

“…Previous sketchy studies to focus on individual eicosanoids in EAE or MS patients have obvious limitations, as Ͼ20 different eicosanoids are produced by the concerted actions of cPLA 2 ␣ and downstream enzymes (Fig. S1) (16)(17)(18). To overcome such complexity and some controversy, we evaluated the cascade in the SCs of naive and EAE mice by the nonbiased top-down approach (AA cascade-targeted lipidomics and transcriptomics) and confirmed the results from knockout studies.…”

“…By the AA cascadetargeted lipidomics approach, we found that the PGE 2 pathway is favored and the PGD 2 , PGI 2 , and 5-LO pathways are attenuated. Correlation analysis imply that the AA producing enzyme cPLA 2 ␣ possibly passes more AA to the COX than the 5-LO pathway in EAE, because the sequential actions of eicosanoidsynthesizing enzymes are regulated spatially and temporally in the cells, the so-called functional coupling (16)(17)(18). Likewise, PGH 2 , a common precursor of PGs, appears to be selectively consumed by the PGE 2 pathway rather than the PGI 2 and PGD 2 pathways, thereby producing PGE 2 in the SCs of EAE mice.…”

Selective inhibition of Aβ42 production by NSAID R‐enantiomers

“…In contrast, R-flurbiprofen has similar Ab42 lowering activity to S-flurbiprofen, but much less potent COX inhibitory activity. (see for review Tegeder et al 2001b) Because HEK293 cells produce very low prostaglandin E 2 and have negligible activity of the enzymes involved in this cascade (Ueno et al 2001), the prostaglandin E 2 levels in our HEK293 culture were undetectable, although readily measured in CHO cells (data not shown). While we could not confirm the absence of COX inhibition by R-flurbiprofen in HEK293 cells, previous reports show the IC 50 for human COX-1 and COX-2 by R-flurbiprofen are more than 40 lM and 100 lM, while those of S-flurbiprofen are 0.03 lM and 0.9 lM, respectively (Geisslinger et al 2000; see for review Tegeder et al 2001b).…”