Ceramide binds to the CaLB domain of cytosolic phospholipase A₂and facilitates its membrane docking and arachidonic acid release

Abstract

Excessive production of eicosanoids is characteristic of many inflammatory diseases. In this study we show that ceramide, which is an early messenger of inflammatory cytokine action, exerts a dual effect on the cytosolic phospholipase A2 (cPLA2), the rate-limiting enzyme in arachidonic acid release and subsequent eicosanoid formation. Stimulation of renal mesangial cells with exogenous short-chain ceramide analogs for 30 and 60 min leads to a concentration-dependent increase in arachidonic acid release that is not blocked by specific inhibitors of mitogen-activated protein kinase pathways. This suggests that these established upstream activators of cPLA2 are not involved in ceramide-induced arachidonic acid release. By use of photoactivatable ceramide analogs, D- and L-[125I]3-trifluoromethyl-3-(m-iodophenyl)diazirine-ceramides (TID-ceramides), we observed a direct interaction of ceramide with cPLA2. This interaction was independent of the absolute configuration as D- and L-TID-ceramide were equally effective in binding to cPLA2. Moreover, recombinant CaLB domain of cPLA2 as well as a mutant deficient in the connecting 'hinge' domain of cPLA2, efficiently bound D- and L-TID-ceramides, whereas the catalytic domain did not interact with TID-ceramides. In vitro binding assays reveal that stearoyl-arachidonyl-phosphatidylcholine (SAPC)-liposomes containing increasing mol% of ceramide lead to an increased association of recombinant cPLA2 to the liposomes. Furthermore, measurement of cPLA2 activity in vitro shows that the presence of SAPC-liposomes resulted in only weak cPLA2 activity. However, the activity dramatically increases by addition of ceramide to the liposomes. Furthermore, liposomes containing SAPC and sphingomyelin resulted in no better substrate than SAPC liposomes, unless bacterial sphingomyelinase was added to generate ceramide, which then causes a marked increase in cPLA2 activity. These results demonstrate that ceramide can interact directly with cPLA2 via the CaLB domain and thereby serves as a membrane-docking device that facilitates cPLA2 action in inflammatory diseases.

Essential Ca2+-independent Role of the Group IVA Cytosolic Phospholipase A2 C2 Domain for Interfacial Activity

“…9B. These increases were in the 3-6-fold range, much like those previously reported for PI-4,5-P 2 addition in the small unilamellar vesicle substrate systems (33,55). This 3-6-fold increase is in contrast to the Lyso-PC/Triton X-100 mixed micelle system that gave greater than a 100-fold increase in activity in the presence of 1 mol % PI-4,5-P 2 (Fig.…”

“…Several others reports have shown that PIP n s, including PI-4,5-P 2 , enhance the activity of GIVAPLA 2 by less than 10-fold (21,22,33,55). At first glance, this seems to contradict our reports that PI-4,5-P 2 enhances the activity of GIVAPLA 2 in large unilamellar vesicles composed of PAPC by 20-fold (1 mol %) or 55-fold (3 mol %) and in PAPC/Triton X-100 mixed micelles by up to 120-fold (1 mol %) (23).…”

Omega-3 fatty acids cause dramatic changes in TLR4 and purinergic eicosanoid signaling

“…2B). In this setting, cPLA 2 is translocated to the membrane in a Ca 2+ -independent fashion by increased membrane affinity and enhanced specific activity that has been linked to generation of PIP 2 and conformational changes (5,(31)(32)(33), as well as generation of C1P (34), ceramide (35), and diacylglycerol (36). Additionally, sPLA 2 is reported to significantly contribute to prostaglandin production during late-phase activation, but not during 10-min calcium ionophore-induced metabolism (37).…”

The Role of Sphingolipids and Ceramide in Pulmonary Inflammation in Cystic Fibrosis

“…In addition, ceramide has been shown to directly regulate several molecules including cathepsin D [53], phospholipase A 2 [54], kinase suppressor of Ras [55], ceramide-activated protein serine-threonine phosphatases (CAPP) [56] and protein kinase C isoforms [57, 58]. Further, ceramide has been shown to regulate the activity of the potassium channel Kv1.3 and calcium release-activated calcium (CRAC) channels [59-60].…”