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Ceramide binds to the CaLB domain of cytosolic phospholipase A 2 and facilitates its membrane docking and arachidonic acid release
Author(s) -
Huwiler Andrea,
Johansen Berit,
Skarstad Anita,
Pfeilschifter Josef
Publication year - 2001
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.00-0370fje
Subject(s) - ceramide , arachidonic acid , lipid signaling , chemistry , phospholipase a2 , biochemistry , sphingomyelin , second messenger system , enzyme , membrane , apoptosis
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 A 2 (cPLA 2 ), 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 cPLA 2 are not involved in ceramide‐induced arachidonic acid release. By use of photoactivatable ceramide analogs, D‐ and L‐ [ 125 I]3‐trifluoromethyl‐3‐( m ‐iodophenyl)diazirineceramides (TID‐ceramides), we observed a direct interaction of ceramide with cPLA 2 . This interaction was independent of the absolute configuration as D‐ and L‐TID‐ceramide were equally effective in binding to cPLA 2 . Moreover, recombinant CaLB domain of cPLA 2 as well as a mutant deficient in the connecting ‘hinge’ domain of cPLA 2 , 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 cPLA 2 to the liposomes. Furthermore, measurement of cPLA 2 activity in vitro shows that the presence of SAPC‐liposomes resulted in only weak cPLA 2 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 cPLA 2 activity. These results demonstrate that ceramide can interact directly with cPLA 2 via the CaLB domain and thereby serves as a membrane‐docking device that facilitates cPLA 2 action in inflammatory diseases.